Receptor Tyrosine Kinase Tie2 Research Articles

Abstract 2047: Molecular photoacoustic imaging and serum diagnostics rapidly detect response to angiopoietin 1 and 2 blockade in ovarian cancer

Abstract Introduction: There is an urgent clinical need to develop noninvasive biomarkers that early detect tumor responses to anti-angiogenic therapy. Treatment response in ovarian cancer patients is assessed with transvaginal ultrasound imaging of tumor size and CA125 screening. We hypothesized that combining photoacoustic (PA) imaging to non-invasively visualize tumor vascular architecture, with serum diagnostics using endothelial biomarkers, would yield a sensitive and readily translatable approach for monitoring response to anti-angiogenic therapy. Methods: Ovarian tumors were established in nude mice by orthotopic injection of OV2008 cells at day 0. At day 10 mice were randomized into groups of at least n = 10. Baseline PA imaging and submandibular blood draw were performed, then repeated 24h after dosing with a peptibody that prevents the interaction of secreted angiopoietin 1 (Ang1; agonist) and 2 (Ang2; antagonist) with the receptor tyrosine kinase Tie2, or vehicle, on days 13, 16 and 19. Mice were then sacrificed and tumors excised for histology. Results: PA imaging uses endogenous hemoglobin light absorption to generate contrast. At 797nm, we found a 1.5-fold increase in hemoglobin-weighted signal in vehicle mice by day 19, while treated mice remained close to the baseline (n=9; p=0.008). The oxyhemoglobin-weighted PA signal (837nm/797nm) was elevated by 20% in treated mice but decreased 15% in vehicle mice (n=9; p<0.01). This indicated treated mice had significantly lower tumor vessel density but improved function of residual vessels. To gain insight into the mechanism of drug action, we measured host serum angiopoietins over the treatment time course. Ang1 in vehicle mice increased by 1.5-fold (n=10; p<0.0001) and was significantly higher than treated mice by day 19 (n=10; p=0.03). Ang2 was identical between treatment groups and healthy control mice (n=10; p=0.65). Elevated Ang1 in the circulation of vehicle mice is consistent with excess Ang1 promoting vascular remodeling and plasticity in tumors, while normalization in treated mice allows vessel stabilization. Immunofluorescence (IF) confirmed this by indicating that CD31 positive vessel density was 30% lower in treated mice but their tumor vessel coverage of desmin-positive pericytes was 2.5-fold higher. Conclusions: We have shown for the first time that PA imaging and serum angiopoietin levels measure complementary parameters that indicate response to angiopoietin blockade in ovarian cancer. PA imaging noninvasively detected drug-induced vessel regression and normalization, giving a functional readout of response. This modality can be readily combined with transvaginal ultrasound. The drug action was mediated by normalization of mAng1 levels, indicated by serum measures and IF. Importantly, this work supports the combined use of in vitro and in vivo diagnostics to monitor response to anti-angiogenic therapy in clinical trials. Citation Format: Sarah E. Bohndiek, Laura Sasportas, Steven Machtaler, Jesse V. Jokerst, Sharon Hori, Sanjiv S. Gambhir. Molecular photoacoustic imaging and serum diagnostics rapidly detect response to angiopoietin 1 and 2 blockade in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2047. doi:10.1158/1538-7445.AM2014-2047

Abstract 4492: Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models

Abstract Angiopoietin-1 (Ang1) and -2 (Ang2) regulate angiogenesis via the endothelial cell-specific receptor tyrosine kinase Tie2. Blocking Ang2 binding to Tie2 decreases vessel density and inhibits tumor growth in various human xenograft models. However, not all tumors respond to Ang2 blockade, which could be due to high Ang1 levels acting as another ligand for Tie2, to Ang2 activities independent of Tie2, or to a weak role for Ang-2/Tie2 signaling in these tumors. To elucidate if Ang1 plays an integral role in tumor angiogenesis and growth, we compared the effects of blocking Ang2 or Tie2 in various tumor xenografts with differing levels of Ang1 expression. We found that in six out of seven tumor models, antibodies that specifically bind Ang2 (REGN910, 10 mg/kg 2x/wk) or that bind Tie2 and block binding of both Ang1 and Ang2 (REGN1376, 10 mg/kg 2x/wk) were equally efficacious at inhibiting tumor growth and decreasing vessel density. In only one tumor model (Calu-6), blockade of Tie2 with REGN1376 was more effective than blockade of Ang2 (REGN910) at reducing tumor growth and decreasing tumor vessel density. Although Calu-6 tumors express high Ang1 levels, other tumors tested (Lox, LS174T) have comparable or higher Ang1 levels and respond similarly to Ang2 and Tie2 blockade, thus the levels of Ang1 do not account for the differential response of Calu-6 tumors. Taken together, our data suggest that Ang2 is the dominant ligand for Tie2 in the microenvironment of most tumors, and further, that the effects of Ang2 are mediated primarily via Tie2. These findings support the approach to specifically block the binding of Ang2 to Tie2 as an anti-angiogenic therapy. Citation Format: Alexander P. Adler, Christopher Daly, Asma A. Parveen, Thomas Nevins, Jing Shan, Jeanette Fairhurst, Tammy Huang, Joel Martin, Nicholas Papadopoulos, George D. Yancopoulos, Gavin Thurston, Gavin Thurston, Alexandra Eichten. Blockade of angiopoietin-2 or Tie2 is equally effective at inhibiting tumor growth and reducing tumor vessel density in most human tumor xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4492. doi:10.1158/1538-7445.AM2014-4492

Abstract 1026: Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in mice

Abstract The receptor tyrosine kinase Tie2 is predominantly expressed in the endothelium but has also been identified on primitive hematopoietic stem cells, monocyte and macrophage subclasses, as well as on glioma or hematological tumor cells. Based on its expression in many patient-derived leukemic blasts inhibition of the Tie2 pathway may provide an attractive opportunity for therapeutic intervention in leukemias. In this study we report the pharmacological profile of a novel, highly potent and orally available Tie2 inhibitor (BAY-Tie2). The discovery and design process leading to BAY-Tie2 was performed with the goal of sparing other angiogenic RTKs, such as VEGFRs, FGFRs or PDGFRs. BAY-Tie2 is based on a novel imidazopyrazole core, combined with a SF5-substituted phenyl ring that fills the deep DFG-out pocket. BAY-Tie2 binds to Tie2 with a Kd value of 1.6 nM and is selective against VEGFR2 (Kd of 1600 nM), FGFR1 (<30% inhibition at 1 µM), FGFR2/3/4 (<10% inhibition at 1 µM) and PDGFRα/β (<30% inhibition at 100 nM). BAY-Tie2 potently inhibits Tie2 autophosphorylation in recombinant CHO-Tie2 and primary human umbilical vein endothelial cells (HUVEC) with IC50 values of 0.7 and 1.3 nM. Consistently, BAY-Tie2 was shown to inhibit Tie2 phosphorylation in vivo by analyzing angiopoietin-1 induced Tie2 phosphorylation status in extracts of murine lungs from BAY-Tie2-treated mice. In subcutaneous xenograft models of highly angiogenic tumors, BAY-Tie2 reduced tumor growth and showed evidence for potential combination benefit with anti-VEGF therapy. In order to explore the potential of a Tie2 inhibitor beyond affecting angiogenesis, we established disseminated leukemia models, using Tie2-expressing cell lines, such as the CML cell lines MEG-01 and EM-2. Both cell lines engrafted predominantly in bone marrow and spleen. Treatment started 3 days after i.v. cell implantation with either BAY-Tie2 or cytarabine and was well tolerated. Efficacy was monitored by a) inhibition of disease progression, b) weekly fluorescence-based in vivo imaging (IVI) using an Alexa750-labeled anti-human CD33 antibody, and c) q-RT-PCR specific for BCR-ABL and hCD45 in murine peripheral blood. BAY-Tie2 inhibited disease progression comparable to cytarabine. Tumor load measured by IVI was reduced in BAY-Tie2 treated groups by 45% in the MEG-01 and by 65% in the EM-2 model compared with the untreated control, very similar to the cytotoxic treatment with cytarabine. Quantitative RT-PCR on peripheral blood revealed that BAY-Tie2 and cytarabine delayed the appearance of circulating tumor cells in both CML models. These data demonstrate that BAY-Tie2 is an orally active Tie2 inhibitor that may have therapeutic benefit not only in angiogenic tumors but also in hematological, Tie2-expressing malignancies. Citation Format: Sylvia Gruenewald, Julia Schueler, Michael Haerter, Frank Suessmeier, Kerstin Klingner, Ulf Boemer, Stefan Kaulfuss, Alexander Walter, Mario Lobell, Ingo V. Hartung, Bernd Buchmann, Dieter Heldmann, Holger Hess-Stumpp, Karl Ziegelbauer. Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1026. doi:10.1158/1538-7445.AM2014-1026

Abstract 3944: Caveolin-mediated Tie2 nuclear translocation results in enhanced NHEJ repair and glioma radioresistance

Abstract Glioblastoma is the most frequent subtype of primary brain tumor in adults and are resistant to current strategies of surgery, irradiation, and chemotherapy. Patients suffering from this disease exhibit a median survival that ranges from 9 to 15 months. Glioblastomas invariably recur after therapy due to the presence of cells exhibiting a multitherapy-resistance phenotype. To decipher the aberrant DNA repair pathways that enable tumor cells to survive DNA damage upon chemotherapy and radiotherapy should guide us to provide our patients with a more efficacious therapy. The abnormal function of tyrosine kinase receptors (TKRs) is a hallmark of malignant gliomas. Our group reported the expression of the TKR Tie2 in in brain tumor stem cells (BTSCs) and in human surgical glioma specimens in relation to malignancy. In in vivo experiments, consisting of ionizing irradiation (IR) of mice bearing intracranial BTSCs-derived xenografts showed unexpected Tie2 nuclear localization. We observed that Tie2 traffics from the cellular membrane to the nucleus upon IR stimuli, as assessed by immunoflurorescence studies using confocal microscope and subcellular fractionation followed by Western blots. This phenomenon is ligand dependent, as increased levels of Tie2 natural ligand, Angiopoietin1 (Ang1), were observed after ionizing radiation, and nuclear Tie2 levels were decreased using soluble Tie2, which blocks the Ang1/Tie2 interaction. Of clinical significance, the presence of Tie2 in the nucleus is significantly associated with radioresistance, as observed by cell viability and clonogenic assays. Tie2 trafficking was associated to Caveolin-1, and this functional complex was disturbed when Caveolin-1 inhibitor or siCaveolin-1 were used, resulting in decreased Tie2 nuclear levels. In addition, we observed that upon IR treatment, nuclear Tie2 bound to DNA/protein complexes, and specifically to the key DNA repair protein γH2AX, as a component of the DNA-repair foci. Based on these results, we hypothesized that Tie2 was involved in DNA damage repair and focused on one of the main pathways involved in double strand break (IR induced), the non-homologous end-joining (NHEJ) repair mechanism. By using a fluorescent reporter construct in which a functional GFP gene is reconstituted following an NHEJ event (gift from Dr. Gorbunova, University of Rochester), we observed that Tie2-expressing cells displayed a more efficient NHEJ repair than Tie2 negative counterparts. Mutation of a Tie2 nuclear localization signal significantly decreased NHEJ efficiency repair and radiosensitization of BTSCs, suggesting a role of nuclear Tie2 in DNA damage repair, specifically in the NHEJ repair mechanism. Collectively, our results should propel the development of preclinical studies of the combination of nuclear Tie2-targeting strategies with radiotherapy for patients with glioblastomas. Citation Format: Mohammad B. Hossain, Nahir Cortes-Santiago, Xuejun Fan, Konrad Gabrusiewicz, Joy Gumin, Erik P. Sulman, Frederick Lang, Raymond Sawaya, W.K.Alfred Yung, Juan Fueyo, Candelaria Gomez-Manzano. Caveolin-mediated Tie2 nuclear translocation results in enhanced NHEJ repair and glioma radioresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3944. doi:10.1158/1538-7445.AM2014-3944

The angiopoietin1–Akt pathway regulates barrier function of the cultured spinal cord microvascular endothelial cells through Eps8

In mammalian central nervous system (CNS), the integrity of the blood–spinal cord barrier (BSCB), formed by tight junctions (TJs) between adjacent microvascular endothelial cells near the basement membrane of capillaries and the accessory structures, is important for relatively independent activities of the cellular constituents inside the spinal cord. The barrier function of the BSCB are tightly regulated and coordinated by a variety of physiological or pathological factors, similar with but not quite the same as its counterpart, the blood–brain barrier (BBB). Herein, angiopoietin 1 (Ang1), an identified ligand of the endothelium-specific tyrosine kinase receptor Tie-2, was verified to regulate barrier functions, including permeability, junction protein interactions and F-actin organization, in cultured spinal cord microvascular endothelial cells (SCMEC) of rat through the activity of Akt. Besides, these roles of Ang1 in the BSCB in vitro were found to be accompanied with an increasing expression of epidermal growth factor receptor pathway substrate 8 (Eps8), an F-actin bundling protein. Furthermore, the silencing of Eps8 by lentiviral shRNA resulted in an antagonistic effect vs. Ang1 on the endothelial barrier function of SCMEC. In summary, the Ang1–Akt pathway serves as a regulator in the barrier function modulation of SCMEC via the actin-binding protein Eps8.

The zebrafish Tie2 signaling controls tip cell behaviors and acts synergistically with Vegf pathway in developmental angiogenesis.

Angiogenesis process in development is temporally accurate, and involves sprouting, subsequent endothelial cell proliferation, and migration. Tip cells, sensing the extracellular cues, play an important role in this process. Although it is known that several pathways including vascular endothelial growth factor (VEGF) and Notch control tip cell behaviors, the signaling process is largely unknown. Here we showed that an endothelial tyrosine kinase receptor-Tie2 was required for intersegmental vessel (ISV) growth and essential for the sprouting, migration, and proliferation of tip cells with morpholino knockdown strategy in zebrafish. Knockdown of vegf effectively reduced tie2 mRNA level, and tie2 knockdown efficiently blocked the vegf over-expression induced tyrosine kinase receptor-VEGFR1 (flk1) expression, which suggested that the function of Tie2 may be linked to the downstream of VEGF signaling pathway. Furthermore, we found that the embryos displayed normal ISV growth when injected with tie2 or vegf morpholino alone at a low dose, while co-knockdown of them resulted in a severe ISV defect, indicating a synergistic role in ISV formation. These observations demonstrate that Tie2 is an important regulator of tip cell behaviors. Moreover, these findings provide in vivo evidence that Tie2 acts coordinately with Vegf signaling to control angiogenesis.

PTH-138 Tnf-a Dependent Angiopoeitin Mediated Angiogenesis In Sporadic Small Bowel Angiodysplasia; Novel Pathophysiology And Potential Clinical Marker

IntroductionAngiodysplasias account for over 50% of small bowel causes of obscure gastrointestinal bleeding. Angiodysplasias are thought to develop as a result of an imbalance in the angiogenic cascade, although the...

Vascular Niche Controls Organ Regeneration

Endothelial Cell-Derived Angiopoietin-2 Controls Liver Regeneration as a Spatiotemporal Rheostat Hu et al Science. 2014;343:416–419. Vessels provide the conduits that deliver metabolites and oxygen to the tissue and export waste products. After ischemia or tissue injury, endothelial cells migrate and proliferate to re-establish the capillary network in a process termed angiogenesis to maintain oxygen supply. Besides these essential and well-established functions in oxygen delivery, recent studies suggest that endothelial cells contribute to the multicellular-crosstalk that balances regeneration and dysfunctional or maladaptive healing. Thus, endothelial cells not only seem important for oxygen delivery but act as paracrine source for signals that determine tissue regeneration versus fibrosis. The interaction of endothelial-derived signals with hepatocyte functions has already been shown in the development of model organisms such as zebrafish1 and more recently during regeneration of lung and liver in adult mice.2,3 Two recent studies provide novel important insights into the regulation and nature of the endothelial-derived signals that control liver regeneration.4,5 The liver is a highly regenerative organ and as such well suited to defining the signals that control proliferation of hepatocytes to restore liver function and prevent maladaptive responses that cause liver fibrosis and finally liver failure. In a recent elegant study in Science , Hu et al5 report a critical role of angiopoietin-2 (Ang2) in liver regeneration. Ang2 acts as contextual antagonist of the vascular receptor tyrosine kinase Tie2 and was identified by Hu et al5 among the most profoundly downregulated genes during liver regeneration. To explore the function of Ang2 in liver regeneration, Ang2−/− mice were partially hepatectomized and liver regeneration was time-dependently analyzed. Within the first days, Ang2−/− mice showed significantly increased …

The RNA binding protein hnRNP-K mediates post-transcriptional regulation of uncoupling protein-2 by angiopoietin-1

Angiopoietin-1 (Ang1) is a ligand for the receptor tyrosine kinase Tie2 and has key roles in the development of the vascular system and vascular protection. In a screen to define signalling pathways regulated by Ang1 in endothelial cells we found the RNA-binding protein hnRNP-K to be phosphorylated in response to Ang1. The ligand stimulated both tyrosine phosphorylation of hnRNP-K and recruitment of the tyrosine kinase Src to the RNA-binding protein. In endothelial cells hnRNP-K was found bound to mRNA encoding the mitochondrial protein uncoupling protein-2 (UCP2). Ang1 stimulation of cells resulted in the release of UCP2 mRNA from hnRNP-K. Using in vitro assays we confirmed direct binding between hnRNP-K and UCP2 mRNA. Furthermore Src induced phosphorylation of purified hnRNP-K and prevented UCP2 mRNA binding. Tyrosine 458 in the RNA-binding protein was found to be required for suppression of UCP2 mRNA binding by Src phosphorylation. In addition to releasing UCP2 mRNA from hnRNP-K, Ang1 induced an increase in UCP2 protein expression in endothelial cells without affecting total UCP2 mRNA levels. Consistent with the known effects of UCP2 to suppress generation of reactive oxygen species, Ang1 limited ROS production in endothelium stimulated with tumour necrosis factor-α. Taken together these data suggest that UCP2 mRNA is present in endothelial cells bound to hnRNP-K, which holds it in a translationally inactive state, and that Ang1 stimulates Src interaction with hnRNP-K, phosphorylation of the RNA-binding protein, release of these transcripts and upregulation of UCP2 protein expression. This study demonstrates a new mechanism for post-transcriptional regulation of UCP2 by the vascular protective ligand Ang1. The ability to rapidly upregulate UCP2 protein expression may be important in protecting endothelial cells from excessive generation of potentially damaging reactive oxygen species.

Disturbed Balance between Serum Levels of Receptor Tyrosine Kinases Tie-1, Tie-2 and Angiopoietins in Systemic Sclerosis

Objective: The aim of this study was to determine the characteristic factors for vascular development and maintenance levels as well as correlation between Tie-1 receptors, Tie-2 receptors and the corresponding ligands - angiopoietins - in systemic sclerosis (SSc) patients. Materials and Methods: Serum levels of Tie-1, Tie-2, Ang-1 and Ang-2 were measured in 25 SSc patients and healthy controls. Results: There was a statistically significant difference in serum Tie-1 (p = 0.009) and Ang-2 (p = 0.001) levels in SSc patients compared with healthy controls. Significant correlations between Tie-1 and Tie-2 (ρ = 0.70, p = 0.0001) and between Tie-1 and Ang-2 (ρ = -0.92, p = 0.002) were found in the SSc group. Serum levels of Tie-2 were positively associated with esophagus changes (U = 2.03, p = 0.041) and Ang-1 was negatively correlated with duration of Raynaud's phenomenon (ρ = -0.75, p = 0.00008). Conclusion: The increase in serum concentration of Tie-1 and Ang-2 in patients with SSc may confirm a molecular imbalance between receptor tyrosine kinases Tie and their ligands.

Tazarotene-induced gene 3 may affect inflammatory angiogenesis in psoriasis by downregulating placental growth factor expression.

Tazarotene-induced gene 3 may affect inflammatory angiogenesis in psoriasis by downregulating placental growth factor expression.

Abstract C1: Functional normalization of the breast cancer vasculature through activation of Tie2 using a vascular-endothelial protein tyrosine phosphatase inhibitor.

Abstract Background: The solid tumor microvasculature is characterized by structural immaturity and functional abnormality, and mediates several deleterious aspects of tumor behavior. The vascular endothelial protein tyrosine phosphatase (VE-PTP) attenuates the activity of the endothelial cell (EC) Tie-2 receptor tyrosine kinase, a key mediator of vessel maturation. Here we determine the role of VE-PTP in the vasculature of primary and metastatic mammary carcinomas. Methods: AKB-9778 is a first-in-class pharmacologic VE-PTP inhibitor. We systematically examined its effects in vitro and in vivo. First we studied the effects of AKB-9778 on Tie-2 signaling in two endothelial cell lines in vitro, and also measured its impact on Tie-2 activation in normal and tumor ECs in mice. We next used embryonic zebrafish assays to determine the effects of AKB-9778 on embryonic angiogenesis. We also studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models (4T1, E0771, P0008, MMTV-PyMT). Finally, we used endothelial nitric oxide synthase (eNOS) deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. Results: AKB-9778 induced ligand-independent Tie-2 activation in ECs in vitro and in vivo, and impaired embryonic zebrafish angiogenesis. In mouse models of breast cancer, AKB-9778 (i) delayed the early phase of mammary tumor growth by maintaining vascular maturity; (ii) slowed the growth of spontaneous micrometastases by preventing extravasation of tumor cells into distant organ parenchyma (prolonging survival above adjuvant chemotherapy alone); and (iii) matured established primary tumor blood vessels (increased pericyte coverage, reduced permeability) in turn enhancing tumor perfusion, reducing hypoxia, and improving radiation response. Experiments using eNOS knockout mice showed that the effects of AKB-9778 on tumor vessels were mediated in part by eNOS activation. Conclusions: Phosphatase inhibition is a relatively unexplored field in cancer, and this is to our knowledge the first demonstration of the effects of VE-PTP inhibition in any disease. Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments. Furthermore we provide genetic evidence for the causal role of eNOS in mediating the beneficial effects of VEPTP inhibition in established tumors. By using models of adjuvant therapy (combined with chemotherapy) and radiation treatment, our results indicate possible avenues for further studying the clinical translatability of VE-PTP inhibition in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C1. Citation Format: Shom Goel, Nisha Gupta, Brian P. Walcott, Matija Snuderl, Cristina T. Kesler, Benjamin J. Vakoc, Randall T. Peterson, Timothy P. Padera, Dan G. Duda, Dai Fukumura, Rakesh K. Jain. Functional normalization of the breast cancer vasculature through activation of Tie2 using a vascular-endothelial protein tyrosine phosphatase inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C1.

Cardioprotective effects of adipokine apelin on myocardial infarction

Angiogenesis plays an important role in myocardial infarction. Apelin and its natural receptor (angiotensin II receptor-like 1, AGTRL-1 or APLNR) induce sprouting of endothelial cells in an autocrine or paracrine manner. The aim of this study is to investigate whether apelin can improve the cardiac function after myocardial infarction by increasing angiogenesis in infarcted myocardium. Left ventricular end-diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP), left ventricular developed pressure (LVDP), maximal left ventricular pressure development (±LVdp/dtmax), infarct size, and angiogenesis were evaluated to analyze the cardioprotective effects of apelin on ischemic myocardium. Assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2'-deoxyuridine incorporation, wound healing, transwells, and tube formation were used to detect the effects of apelin on proliferation, migration, and chemotaxis of cardiac microvascular endothelial cells. Fluorescein isothiocyanate-labeled bovine serum albumin penetrating through monolayered cardiac microvascular endothelial cells was measured to evaluate the effects of apelin on permeability of microvascular endothelial cells. In vivo results showed that apelin increased ±LV dp/dtmax and LVESP values, decreased LVEDP values (all p < 0.05), and promoted angiogenesis in rat heart after ligation of the left anterior descending coronary artery. In vitro results showed that apelin dose-dependently enhanced proliferation, migration, chemotaxis, and tube formation, but not permeability of cardiac microvascular endothelial cells. Apelin also increased the expression of vascular endothelial growth factor receptors-2 (VEGFR2) and the endothelium-specific receptor tyrosine kinase (Tie-2) in cardiac microvascular endothelial cells. These results indicated that apelin played a protective role in myocardial infarction through promoting angiogenesis and decreasing permeability of microvascular endothelial cells via upregulating the expression of VEGFR2 and Tie-2 in cardiac microvascular endothelial cells.

Abstract 375: A New Mouse Model to Characterize the Mechanisms of Endothelin-1-Induced Vascular Injury

Objective: To develop a model permitting the study of the mechanisms of endothelin (ET-1)-induced vascular injury, we generated an inducible and endothelial cell (EC)-restricted human ET-1 ( EDN1 ) cDNA overexpressing transgenic mouse, which would accordingly be devoid of developmental effects. Methods and Results: A transgene was engineered that expresses chloramphenicol acetyltransferase ( cat ) cDNA prior to Cre-mediated excision, and EDN1 cDNA after Cre-mediated excision, under the control of cytomegalovirus immediate early enhancer/chicken β-actin promoter (CAG). Co-transfection of Cre expression vector and pCAG- cat - EDN1 caused a 30-fold increase in ET-1 production evaluated by ELISA. A DNA fragment containing the CAG- cat - EDN1 transgene was used to generate transgenic mice. PCR genotyping demonstrated that 14/132 mice were transgenic. Two of 7 transgenic lines were selected based on cardiac cat expression level, which were twice higher in line C-134 than C-170. Inducible EC-restricted EDN1 mice were generated crossing CAG- cat - EDN1 mice with mice expressing Cre recombinase fused to a tamoxifen (TAM)-inducible modified estrogen receptor ligand binding domain ( Cre ER T2 ) under control of EC-specific receptor tyrosine kinase ( Tie2 ) promoter. To investigate the extent of CreER T2 activation by TAM and tissue specificity, Tie2 -CreER T2 mice were crossed with ROSA mT-mG/mT-mG reporter mice expressing a membrane-targeted tandem dimer tomato (mT) before Cre-mediated excision, and membrane-targeted enhanced green fluorescent protein (mG) after excision. Tie2 -CreER T2 / cat - EDN1 and Tie2 -CreER T2 / ROSA mT-mG/+ mice were treated with 1 mg TAM/d SC for 5 d and sacrificed 14-16 d later. mG expression was detected in 22±3% of mesenteric artery EC of Tie2 -CreER T2 / ROSA mT-mG/+ mice. Plasma ET-1 levels were similar in vehicle-treated Tie2 -CreER T2 / cat - EDN1 (1.2±0.1 pg/mL, n=4) and wild-type mice (1.1±0.1 pg/mL, n=2), demonstrating no leaky expression. TAM induced 8-fold increase in plasma ET-1 levels in Tie2 -CreER T2 / cat - EDN1 mice (9.1 ± 0.3 pg/mL, n = 4). Conclusion: Tie2 -CreER T2 / cat - EDN1 -inducible EC-restricted EDN1 overexpressing mice allow the study of ET-1 vascular effects in adult independently of developmental effects.

Effects of Vascular-Endothelial Protein Tyrosine Phosphatase Inhibition on Breast Cancer Vasculature and Metastatic Progression

The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels. AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)-deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided. AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ(2) test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel-Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation. Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments.

The role of the angiopoietin/Tie system in regulating hematopoietic stem cell maintenance and recruitment

The Angiopoietin/Tie system controls angiogenesis and vessel maturation. However, signalling through the receptor tyrosine kinase Tie2 and its ligand Angiopoietin-1 (Ang-1) has recently been identified to play a crucial role in controlling quiescence and maintenance of hematopoietic stem cells in the bone marrow niche. Pericytes, smooth muscle cells and fibroblasts express Ang-1 in a paracrine manner. Recently, bone marrow osteoblasts have also been identified to express Ang-1. The receptor tyrosine kinases Tie1 and Tie2 are both expressed by hematopoietic stem cells.

A phase I trial and pharmacokinetic study of trebananib (AMG386) in children with recurrent or refractory solid tumors: A Children’s Oncology Group Phase 1 Consortium report.

2538 Background: Trebananib is a first-in-class peptibody (peptide-Fc fusion protein) that selectively inhibits Angiopoietin 1 and Angiopoietin 2 to inhibit interaction with the Tie2 receptor tyrosine kinase and prevent angiogenesis by a VEGF independent mechanism. A pediatric phase 1 trial was performed to define the dose limiting toxicities (DLT), maximum tolerated dose (MTD) and pharmacokinetics (PK) of trebananib. Methods: Trebananib was administered as a weekly 30 - 60 minute IV infusion. Three dose levels (10, 15 or 30 mg/kg/dose) were evaluated using a rolling-six design. PK sampling and analysis of peripheral blood biomarkers was performed during the first 4 weeks of therapy. Results: Fifteen eligible patients (14 evaluable for toxicity) with a median age of 14 yrs (range, 3 to 20) and diagnoses of neuroblastoma (n=4), rhabdomyosarcoma (n=3), Ewing sarcoma (n=3), osteosarcoma (n=2), other soft tissue sarcoma (n=2), or nasopharyngeal carcinoma (n=1) have been enrolled. There were no DLTs observed at either the 10 mg/kg (n=6 pts) or 15 mg/kg (n=3 pts) dose. 1/6 pts receiving 30 mg/kg/dose developed DLT (venous thrombosis at a central line site). Non-dose limiting grade 3 or 4 toxicities included lymphopenia (n=2) hypertension (n=1), and neutropenia (n=1). Response in evaluable patients after eight weeks of therapy included stable disease (n=6 pts) and progressive disease (n=7 pts). PK were linear over the 3 dose levels, with t1/2 and Clpvalues of 69±18 h and 1.6±0.5 ml/h/kg, respectively. Conclusions: Trebananib is well tolerated in pediatric patients with recurrent or refractory solid tumors with recommended Phase 2 dose of 30 mg/kg. Correlative biology studies will be presented. Further study is planned to evaluate tolerability and changes in vascular permeability in patients with primary CNS tumors. Clinical trial information: NCT01538095.

Venous malformation-causative TIE2 mutations mediate an AKT-dependent decrease in PDGFB

Mutations in the endothelial cell (EC) tyrosine kinase receptor TIE2 cause inherited and sporadic forms of venous malformation. The recurrent somatic mutation L914F and common germline mutation R849W differ in terms of phosphorylation level, as well as sub-cellular localization and trafficking of the receptor. Previous studies have shed light on certain pathogenic properties of R849W, but the mechanisms of action of L914F are unknown. We used global gene expression profiling to study the effects of L914F on ECs. We found that L914F strongly dysregulates genes involved in vascular development, cell migration and extracellular matrix processing, while R849W has weak effects. We also demonstrate, for the first time, that TIE2-mutant ECs are deficient in the production of PDGFB, both in vitro and ex vivo in patient tissues. This defect is mediated by the chronic, ligand-independent activation of AKT by the mutant receptors. Inadequate secretion of the major mural cell attractant likely plays an important role in the development of abnormal vascular channels, contributing to the characteristic paucity of surrounding vascular smooth muscle cells.

To study the mode and mechanism of interaction of Angiopoietin II with receptor tyrosine kinase Tie-2 using molecular mechanics and molecular dynamics approach

BACKGROUD &amp; OBJECTIVE: Angiopoietins are protein growth factors which play key role in Angiogenesis.Angiogenesis is the process of forming blood vessels from pre-existing ones. Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) have been identified as ligands of the endothelial receptor tyrosine kinase Tie-2. ANG-2 is a key regulator ofangiogenesis that exerts context-dependent effects on endothelial cell (ECs). ANG-2 binds the endothelial-specificreceptor TIE2 and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling theresponsiveness of ECs to exogenous cytokines. The transmembrane tyrosine kinase TIE-2 and the receptor forangiopoietins have been shown to be involved in angiogenic processes. They are also known to play a role in tumorangiogenesis. However, the mode of interactions between ANG-2 and TIE2 receptor is not known because of theabsence of high resolution co-crystal structure. Therefore in this study attempts were made to investigate the mode andmechanism of molecular interactions between Tie2 with Ang2 using molecular modeling and molecular dynamicsstudies. METHODOLOGY: In the present study, both Tie2 (PDB Id: 2GY5) and Angiopoietins (PDB Id: 2GY7) werefirst prepared using protein preparation wizard (Schrodinger package). Protein-protein interaction between both theproteins was studied using ZDock followed by refinement using Rdock. The best docked pose was then subjected toMolecular dynamics (MD) simulations to study the precise interaction between TIE2 (Receptor) and Angiopoietin-2(Ligand) over a specific time span using AMBER 11.0. The obtained MD trajectories were further used to estimate thebinding free energy of the complex using the molecular mechanics/Poisson Boltzmann surface area (MM-PBSA)method. RESULTS: The binding energy (∆Gbinding) between both the proteins, Tie2 and Ang2 was predicted to be -28.77kcal/mol using Rdock. The other energy parameters between Tie2 and APC interactions such as electrostatic (Eelec), vander Waals (Evdw) and desolvation (Esol) energy are -44.68 kcal/mol, -99.83 kcal/mol and 6.10 kacal/mol respectively,demonstrating modest interactions between them. The interacting surface area between Tie2 and Ang2 is 842:858Å2.CONCLUSION: Results obtained from this study revealed that both Ang2 and Tie2 bind with high affinity with modestinteracting surface area. Further the results guided us in designing specific experiments for biological evaluations.

The activity of the Ang/Tie-2 system in the brain that suffered acute carbon monoxide poisoning

Acute carbon monoxide poisoning (ACMP) leads to significant toxicity of the central nervous system and heart, and even death, following it, some patients suffered delayed encephalopathy. Until now, no theory had explained it exactly. It was reported that neovascularization was found in acute ischemic brains and also that angiopoietins (Ang) play important roles in the process of angiogenesis, for example, the members of Ang family, Ang-1 and Ang-2 may promote angiogenesis by combining with endothelial-specific cell surface tyrosine kinase receptor Tie-2. Interestingly, some studies suggested that small vascular injury may play an important role in the pathogenesis of delayed encephalopathy after carbon monoxide poisoning. Does neovascularization also occur in the brains after ACMP? Do Ang also take part in the pathologic processes in the brains that suffered ACMP? People know little about it. In the present study, we showed that neovascularization also occurred in the brains that suffered ACMP, and there are two expression peaks of Ang-1, Ang-2 and Tie-2, respectively, in the mice brains on the 3rd day and the 7th day following ACMP, and draw a conclusion that the Ang/Tie-2 system takes part in the pathologic processes in the brains that suffered ACMP by participating in neovascularization.