Tie2 Activation Research Articles

Rigosertib as a selective anti-tumor agent can ameliorate multiple dysregulated signaling transduction pathways in high-grade myelodysplastic syndrome.

Rigosertib has demonstrated therapeutic activity for patients with high-risk myelodysplastic syndrome (MDS) in clinical trials. However, the role of rigosertib in MDS has not been thoroughly characterized. In this study, we found out that rigosertib induced apoptosis, blocked the cell cycle at the G2/M phase and subsequently inhibited the proliferation of CD34+ cells from MDS, while it minimally affected the normal CD34+ cells. Further studies showed that rigosertib acted via the activation of the P53 signaling pathway. Bioinformatics analysis based on gene expression profile and flow cytometry analysis revealed the abnormal activation of the Akt-PI3K, Jak-STAT and Wnt pathways in high-grade MDS, while the p38 MAPK, SAPK/JNK and P53 pathways were abnormally activated in low-grade MDS. Rigosertib could markedly inhibit the activation of the Akt-PI3K and Wnt pathways, whereas it activated the SAPK/JNK and P53 pathways in high-grade MDS. A receptor tyrosine kinase phosphorylation array demonstrated that rigosertib could increase the activation of RET and PDGFR-β while reducing the activation of Tie2 and VEGFR2 in MDS cells. Taken together, these data indicate that rigosertib is a selective and promising anti-tumor agent that could ameliorate multiple dysregulated signaling transduction pathways in high-grade MDS.

Noncanonical PAR3 activation by factor Xa identifies a novel pathway for Tie2 activation and stabilization of vascular integrity

AbstractEndothelial barrier protective effects of activated protein C (APC) require the endothelial protein C receptor (EPCR), protease-activated receptor (PAR) 1, and PAR3. In contrast, PAR1 and PAR3 activation by thrombin results in barrier disruption. Noncanonical PAR1 and PAR3 activation by APC vs canonical activation by thrombin provides an explanation for the functional selectivity of these proteases. Here we found that factor Xa (FXa) activated PAR1 at canonical Arg41 similar to thrombin but cleaved PAR3 at noncanonical Arg41 similar to APC. This unique PAR1-PAR3 activation profile permitted the identification of noncanonical PAR3 activation as a novel activation pathway for barrier protective tunica intima endothelial receptor tyrosine kinase 2 (Tie2). APC, FXa, and the noncanonical PAR3 tethered-ligand peptide induced prolonged activation of Tie2, whereas thrombin and the canonical PAR3 tethered-ligand peptide did not. Tie2 activation by FXa required PAR3 and EPCR. FXa and the noncanonical PAR3 tethered-ligand peptide induced Tie2- and PAR3-dependent upregulation of tight-junction-associated protein zona occludens 1 (ZO-1), translocation of ZO-1 to cell-cell borders, and the formation of typical ZO-1 honeycomb patterns that are indicative of tight-junction stabilization. These data provide intriguing novel insights into the diversification of functional selectivity of protease signaling achievable by canonical and noncanonical PAR activation, such as the activation of vascular-protective Tie2 by noncanonical PAR3 activation.

Treatment of Diabetic Macular Edema with an Inhibitor of Vascular Endothelial-Protein Tyrosine Phosphatase That Activates Tie2

AKB-9778 is a small-molecule competitive inhibitor of vascular endothelial-protein tyrosine phosphatase (VE-PTP) that promotes Tie2 activation and reduces vascular leakage and neovascularization in mouse models. The purpose of this study was to test the safety, tolerability, pharmacokinetics, and biological activity of AKB-9778 in patients with diabetic macular edema (DME). Open-label, dose-escalation clinical trial. Four dose cohorts of 6 patients with DME self-administered subcutaneous injections of 5 mg, 15 mg, 22.5 mg, or 30 mg AKB-9778 twice daily for 4 weeks. Patients were seen weekly during a 4-week treatment period for safety assessments, best-corrected visual acuity (BCVA) assessment by Early Treatment Diabetic Retinopathy Study protocol, and measurement of central subfield thickness (CST) by spectral-domain optical coherence tomography. Additional safety assessments were performed at 6, 8, and 12 weeks. Safety assessments, change from baseline BCVA, and change from baseline CST. All doses were well tolerated. A modest, transient reduction in blood pressure and adverse events consistent with vasodilatory activity of AKB-9778 emerged at doses of 22.5 mg or more twice daily. At the week 4 primary end point, BCVA improved 5 letters or more from baseline in 13 of the 18 patients receiving 15 mg or more twice daily; 1 patient improved by 10 to 15 letters, and 2 patients improved by more than 15 letters. Among 18 patients receiving 15 mg or more twice daily, CST decreased by more than 100 μm in 5 patients and by 50 to 100 μm in 2 patients. There was a significant correlation between reduction in CST and improvement in BCVA. No safety concerns were identified after systemic administration of AKB-9778 for 4 weeks in patients with DME, and doses of 15 mg or more twice daily reduced macular edema and improved vision in some patients. This is a preliminary demonstration of clinical safety and efficacy of a VE-PTP inhibitor and Tie2 activator.

Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature

Retinal and choroidal neovascularization (NV) and vascular leakage contribute to visual impairment in several common ocular diseases. The angiopoietin/TIE2 (ANG/TIE2) pathway maintains vascular integrity, and negative regulators of this pathway are potential therapeutic targets for these diseases. Here, we demonstrated that vascular endothelial-protein tyrosine phosphatase (VE-PTP), which negatively regulates TIE2 activation, is upregulated in hypoxic vascular endothelial cells, particularly in retinal NV. Intraocular injection of an anti-VE-PTP antibody previously shown to activate TIE2 suppressed ocular NV. Furthermore, a small-molecule inhibitor of VE-PTP catalytic activity (AKB-9778) activated TIE2, enhanced ANG1-induced TIE2 activation, and stimulated phosphorylation of signaling molecules in the TIE2 pathway, including AKT, eNOS, and ERK. In mouse models of neovascular age-related macular degeneration, AKB-9778 induced phosphorylation of TIE2 and strongly suppressed NV. Ischemia-induced retinal NV, which is relevant to diabetic retinopathy, was accentuated by the induction of ANG2 but inhibited by AKB-9778, even in the presence of high levels of ANG2. AKB-9778 also blocked VEGF-induced leakage from dermal and retinal vessels and prevented exudative retinal detachments in double-transgenic mice with high expression of VEGF in photoreceptors. These data support targeting VE-PTP to stabilize retinal and choroidal blood vessels and suggest that this strategy has potential for patients with a wide variety of retinal and choroidal vascular diseases.

Abstract W P201: Angiopoietin-1 Mimetic Peptide Treatment of Stroke Regulates MicroRNA Expression and Promotes Neuroprotection in Type One Diabetic Rats

Introduction: Angiopoietin-1 (Ang-1) mediates vascular remodeling. Diabetes decreases Ang1 and disrupts Ang1/Tie2 signaling, impairs vascular maturation and regulates immune response. MicroRNAs have been implicated in vascular diseases and inflammation. Vasculotide (VT) is an Ang1 mimetic peptide that promotes Tie2 activity. We hypothesize that treating type one diabetic rats (T1DM) with VT after stroke regulates miRNA expression decreases pro-inflammatory factors and thereby produces a neuroprotective effect. Methods: T1DM was induced in male Wistar rats with streptozotocin followed by 2h transient middle cerebral artery occlusion (MCAo). These rats were then treated with: 1) PBS control; 2) VT (3 ug/kg, ip injection) just prior to stroke and at 8h and 24h after MCAo. Functional tests were conducted 48h after MCAo. Afterwards, brain hemorrhage, BBB leakage, miRNA expression, RT-PCR, immunostaining and Western blot assays were done. Results: VT treatment did not decrease brain hemorrhage, but significantly decreased Evans Blue leakage (30.6±5.8 ng/mg vs. 17.9±2.0 ng/mg) lesion volume (35.1±3.5% vs 25.8±5.3%) and improved functional outcome after stroke when compared to control (p<0.05). To elucidate the mechanism, miRNA was measured. VT treatment significantly increased miR-451 (81.6 folds), miR-155 (2.4 fold), miR-98 (1.9 fold), and miR-126 (1.5 fold) expression, but decreased miR-200b (3.8 fold). Immunostaining showed that VT significantly decreased the number of apoptotic (45.5±3.7 vs 30.8±2.6) and cleaved-caspase-3 positive cells (45.2±3.5 vs 33.1±4.3), as well as monocyte chemotactic protein-1 (MCP1, 5.02±0.4% vs 1.8±0.2%) and tumor necrosis factor (TNF)-alpha (3.1±0.5% vs 1.5±0.2%). Western blot showed that VT significantly decreased receptor for advanced glycation end products (RAGE), MCP1 and TNFa (3-5 fold) in the ischemic border area compared to T1DM control. Conclusion: VT promotes neuroprotection after stroke in T1DM rats. Regulation of miRNA and neuroinflammatory factor expression and decreased BBB leakage may contribute to the VT induced neuroprotective effects observed after stroke in T1DM rats.

Tie2 Signaling Cooperates with TNF to Promote the Pro-Inflammatory Activation of Human Macrophages Independently of Macrophage Functional Phenotype

Angiopoietin (Ang) -1 and -2 and their receptor Tie2 play critical roles in regulating angiogenic processes during development, homeostasis, tumorigenesis, inflammation and tissue repair. Tie2 signaling is best characterized in endothelial cells, but a subset of human and murine circulating monocytes/macrophages essential to solid tumor formation express Tie2 and display immunosuppressive properties consistent with M2 macrophage polarization. However, we have recently shown that Tie2 is strongly activated in pro-inflammatory macrophages present in rheumatoid arthritis patient synovial tissue. Here we examined the relationship between Tie2 expression and function during human macrophage polarization. Tie2 expression was observed under all polarization conditions, but was highest in IFN-γ and IL-10 –differentiated macrophages. While TNF enhanced expression of a common restricted set of genes involved in angiogenesis and inflammation in GM-CSF, IFN-γ and IL-10 –differentiated macrophages, expression of multiple chemokines and cytokines, including CXCL3, CXCL5, CXCL8, IL6, and IL12B was further augmented in the presence of Ang-1 and Ang-2, via Tie2 activation of JAK/STAT signaling. Conditioned medium from macrophages stimulated with Ang-1 or Ang-2 in combination with TNF, sustained monocyte recruitment. Our findings suggest a general role for Tie2 in cooperatively promoting the inflammatory activation of macrophages, independently of polarization conditions.

Local Synovial Engagement of Angiogenic TIE‐2 Is Associated With the Development of Persistent Erosive Rheumatoid Arthritis in Patients With Early Arthritis

To examine the role of vascular endothelial growth factor (VEGF) and angiopoietin signaling in the diagnosis and disease outcome of patients with early arthritis. Fifty patients with early arthritis (disease duration <1 year) who had not been treated with disease-modifying antirheumatic drugs (DMARDs) were monitored prospectively and were classified at baseline and after 2 years as having undifferentiated arthritis (UA), rheumatoid arthritis (RA), or spondyloarthritis (SpA). All patients underwent arthroscopic synovial biopsy at baseline. Synovial expression of VEGF, VEGF receptor, angiopoietin 1 (Ang-1), Ang-2, TIE-2, and activated p-TIE-2 was evaluated by immunohistochemistry. Serum levels of VEGF, Ang-1, and Ang-2 were measured by enzyme-linked immunosorbent assay. Secreted products of macrophages stimulated with Ang-1 and Ang-2 were measured using a multiplex system. Expression of Ang-1 was comparable between the patients with RA at baseline and patients with UA who fulfilled the criteria for RA over time (UA/RA), and it was significantly higher in patients with RA (P < 0.05) or UA/RA (P < 0.005) than in patients with SpA. TIE-2 and p-TIE-2 were more highly expressed in patients with RA (P < 0.005) or UA/RA (P < 0.05) than in patients with SpA. Ang-1 significantly enhanced the tumor necrosis factor-dependent macrophage production of cytokines and chemokines that are known to be elevated in the synovial fluid of patients with early RA. In RA, relative TIE-2 activation predicted the development of erosive disease (R(2) = 0.35, P < 0.05). Local engagement of synovial TIE-2 is observed during the earliest phases of RA, suggesting that TIE-2 signaling may contribute to disease development and progression or may indicate an attempt to protect against these processes. Early therapeutic targeting of TIE-2 signaling may be useful in improving outcome in arthritis.

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.

Abstract C5: T11TS arrests angiogenic signaling in malignant glioma by attenuating brain endothelial cell angiopoietin-1/Tie2 expression and inducing apoptosis in glioma associated brain endothelial cells.

Abstract Malignant gliomas are highly aggressive primary brain tumors resistant to conventional treatments mainly because of sustained uncontrolled angiogenesis. Limited efficacies of currently available treatments necessitate development of multi-level targeted anti-angiogenic therapy for treatment of malignant glioma. Previously we reported that sheep erythrocyte membrane glycopeptide T11 target structure (T11TS) therapy in a rodent glioma model inhibits brain endothelial cell VEGF signaling and downstream PI3K/Akt/eNOS pathway. The angiopoietin-1/Tie2 signaling operative in brain endothelial cells is implicated in glioma angiogenesis but the role of T11TS therapy on pro-angiogenic Ang-1/Tie2 axis has not been determined yet. Induction of endothelial cell apoptosis is a common mechanism of action of many anti-angiogenic agents that restrict tumor progression. The purpose of the present study is to investigate the effect of T11TS therapy in regulation of angiopoietin-1/Tie2 signaling in glioma associated brain endothelial cells and to identify whether T11TS therapy triggers apoptosis in glioma associated brain endothelial cells. Additionally, the study evaluated the apoptotic pathways and proteins involved in T11TS induced apoptosis of brain endothelial cells. Primary brain endothelial cells isolated from the cerebral cortices of glioma induced rats before and after T11TS therapy were used for experiments. Expression of angiopoietin-1, Tie2, phospho-Tie2, and apoptotic pathway regulator proteins and caspases in brain endothelial cells were determined by flowcytometry, immunoblotting, immunofluorescent imaging, and immunohistochemical staining of rat cerebral sections. Flow cytometric Annexin V-PI assay and JC-1 assay were employed for detection of apoptosis in glioma associated brain endothelial cells. Our findings revealed that T11TS therapy significantly downregulates angiopoietin-1 and Tie2 receptor expression and activation in glioma associated brain endothelial cells. T11TS therapy induced apoptotic death of glioma associated brain endothelial cells as evidenced from membrane phosphatidylserine translocation and reduction of mitochondrial membrane potential. T11TS therapy stimulated both mitochondrial and extrinsic pathways of apoptosis by activating caspases 9, 3, and 8, triggering cleavage of Bid and modulating expression of apoptotic regulator proteins like p53, Bax, Bcl-2, Fas, and FasL in brain endothelial cells. Blockage of Ang-1/Tie2 signaling in brain endothelial cells and induction of intrinsic and extrinsic pathway mediated apoptosis in glioma associated brain endothelial cells are the underlying mechanistic events contributing to the anti-angiogenic action of T11TS therapy in malignant glioma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C5. Citation Format: Debanjan Bhattacharya, Swapna Chaudhuri. T11TS arrests angiogenic signaling in malignant glioma by attenuating brain endothelial cell angiopoietin-1/Tie2 expression and inducing apoptosis in glioma associated brain endothelial cells. [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 C5.

Effect of penehyclidine hydrochloride on level of angiopoietin-1 and tyrosine kinase receptor-2 during endotoxin-induced acute lung injury in rats

Objective To evaluate the effect of penehyclidine hydrochloride (PHC) on the level of angiopoietin-1 (Ang-1) and tyrosine kinase receptor-2 (Tie-2) during endotoxin-induced acute lung injury (ALI) in rats.Methods Forty adult male Sprague-Dawley rats,weighing 180-220 g,were randomly divided into 4 groups using a random number table (n =10 each):control group (group C),ALI group,low-dose PHC group (group L-PHC) and high-dose PHC group (group H-PHC).ALI was induced with iv injection of lipopolysaccharide 5.0 mg/kg via the tail vein.In L-PHC and H-PHC groups,PHC 0.6 and 2 mg/kg were injected,respectively,via the tail vein at 1 and 24 h after lipopolysaccharide injection.The rats were sacrificed at 48 h after the initial injection of PHC to measure the lung water content,protein concentration in bronchoalveolar lavage fluid (BALF),and the expression of Ang-1,Tie-2 and phosphorylated Tie-2 in lung tissues.The morphological changes of lung tissues were observed under light microscope and the ultrastructural changes of alveolar epithelial barrier under transmission electron microscope.Results Compared with group C,the lung water content and protein concentrations in BALF were significantly increased,and the expression of Ang-1 and phosphorylated Tie-2 was down-regulated in the other three groups (P ＜ 0.05).Compared with group ALI,the lung water content and protein concentrations in BALF were significantly decreased,and the expression of Ang-1 and phosphorylated Tie-2 was up-regulated in H-PHC group (P ＜ 0.05),and no significant changes were found in the parameters mentioned above in group L-PHC (P ＞0.05).The damage to lung tissues was significantly reduced in group H-PHC as compared with group ALI.Conclusion PHC can improve the permeability of pulmonary microvascular and reduce injury to alveolar epithelial barrier,thus ameliorating endotoxin-induced ALI in rats,and the effect is dose-related and up-regulation of Ang-1 expression and inhancement of Tie-2 activity are involved in the mechanism. Key words: Cholinergic antagonist ; Respiratory distress syndrome, adult ; Endotoxemia; Angiopoietin-1 ; Receptor, protein-tyrosine kinases

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.

TIE-2 and VEGFR Kinase Activities Drive Immunosuppressive Function of TIE-2–Expressing Monocytes in Human Breast Tumors

Tumor-associated TIE-2-expressing monocytes (TEM) are highly proangiogenic cells critical for tumor vascularization. We previously showed that, in human breast cancer, TIE-2 and VEGFR pathways control proangiogenic activity of TEMs. Here, we examine the contribution of these pathways to immunosuppressive activity of TEMs. We investigated the changes in immunosuppressive activity of TEMs and gene expression in response to specific kinase inhibitors of TIE-2 and VEGFR. The ability of tumor TEMs to suppress tumor-specific T-cell response mediated by tumor dendritic cells (DC) was measured in vitro. Characterization of TEM and DC phenotype in addition to their interaction with T cells was done using confocal microscopic images analysis of breast carcinomas. TEMs from breast tumors are able to suppress tumor-specific immune responses. Importantly, proangiogenic and suppressive functions of TEMs are similarly driven by TIE-2 and VEGFR kinase activity. Furthermore, we show that tumor TEMs can function as antigen-presenting cells and elicit a weak proliferation of T cells. Blocking TIE-2 and VEGFR kinase activity induced TEMs to change their phenotype into cells with features of myeloid dendritic cells. We show that immunosuppressive activity of TEMs is associated with high CD86 surface expression and extensive engagement of T regulatory cells in breast tumors. TIE-2 and VEGFR kinase activity was also necessary to maintain high CD86 surface expression levels and to convert T cells into regulatory cells. These results suggest that TEMs are plastic cells that can be reverted from suppressive, proangiogenic cells into cells that are able to mediate an antitumoral immune response.

Hypoxia reduces endothelial Ang1-induced Tie2 activity in a Tie1-dependent manner

Despite the altered expression of Tie receptors and angiopoietin ligands during hypoxic conditions, the effect of hypoxia on Tie-mediated endothelial responses has not been elucidated. In this study, we found that hypoxia increased Tie receptor expression but attenuated angiopoietin-1 (Ang1)-induced Tie2 activity, including Tie2 phosphorylation, Tie2 downstream signaling activation, and endothelial cell tube formation. However, Ang1 binding to endothelial cells was increased during hypoxic conditions. We demonstrated that Tie1 suppression restored the Tie2 activity and that Tie1-mediated Tie2 suppression was independent of tyrosine phosphatase activity. These results suggest that under hypoxic conditions, Tie1 is critical for reducing Ang1-induced Tie2 activity and angiogenesis.

AB0069 TIE2 signalling induces a pro-inflammatory and pro-angiogenic phenotype in differentiated macrophages, independently of macrophage polarization conditions

BackgroundAngiogenesis, the induction and growth of new blood vessels, has a pivotal role in mammalian embryogenesis and homeostatic processes, as well as in chronic inflammatory diseases like rheumatoid arthritis (RA)....

Angiopoietin-2 Functions as a Tie2 Agonist in Tumor Models, Where It Limits the Effects of VEGF Inhibition

The angiopoietins Ang1 (ANGPT1) and Ang2 (ANGPT2) are secreted factors that bind to the endothelial cell-specific receptor tyrosine kinase Tie2 (TEK) and regulate angiogenesis. Ang1 activates Tie2 to promote blood vessel maturation and stabilization. In contrast, Ang2, which is highly expressed by tumor endothelial cells, is thought to inhibit Tie2 activity and destabilize blood vessels, thereby facilitating VEGF-dependent vessel growth. Here, we show that the inhibition of tumor xenograft growth caused by an Ang2-specific antibody (REGN910) is reversed by systemic administration of the Tie2 agonist Ang1. These results indicate that Ang2 blockade inhibits tumor growth by decreasing Tie2 activity, showing that Ang2 is a Tie2 activator. REGN910 treatment of tumors resulted in increased expression of genes that are repressed by Tie2 activation, providing further evidence that REGN910 inhibits Tie2 signaling. Combination treatment with REGN910 plus the VEGF blocker aflibercept reduced tumor vascularity and tumor perfusion more dramatically than either single agent, resulting in more extensive tumor cell death and more potent inhibition of tumor growth. Challenging the prevailing model of Ang2 as a destabilizing factor, our findings indicate that Ang2 plays a protective role in tumor endothelial cells by activating Tie2, thereby limiting the antivascular effects of VEGF inhibition. Thus, blockade of Ang2 might enhance the clinical benefits currently provided by anti-VEGF agents. .

Isolation and characterization of endothelial cells from intramuscular hemangioma

BackgroundIntramuscular hemangiomas (IMHs) are benign vascular tumors of deep soft tissue characterized by endothelial cell (EC) proliferation. The purpose of this study was to isolate ECs from IMH, characterize their angiogenic phenotype and functional characteristics, and search for a possible signaling pathway related to IMH development. MethodsEC Isolation from IMH was performed by digestion, filtration, washing, incubation, and purification in sequence. Tie2 expression was compared between ECs from IMH and controls using reverse transcriptase polymerase chain reaction (RT-PCR). Cell invasion and proliferation assays were used to analyze functional responses of ECs to angiopoietin 1 (Ang1) and vascular endothelial growth factor (VEGF). Expression of downstream targets was analyzed using Western blot analysis. ResultsIsolated ECs showed typical cobblestone appearance under light microscopy and formed capillary-like tubular structures using Matrigel tube-forming assay. RT-PCR of isolated ECs from six patients showed increased expression of Tie2 and VEGF receptor 1 (VEGFR1) compared with control ECs. Tie2 activation by Ang1 compared with VEGFR1 by VEGF resulted in increased EC migration and proliferation. Western blot analysis showed increased Tie2 expression in hemangioma samples compared with normal ECs. Phosphorylated Akt and phosphorylated forkhead box O1 (FOXO1) expression was observed in hemangioma samples only. ConclusionEC isolation from IMH could be a useful tool for further research. These results suggest that increased Tie2 expression, via Akt-FOXO1 pathway activation, may play an important role in IMH pathogenesis.

The STAT3-Ser/Hes3 signaling axis: an emerging regulator of endogenous regeneration and cancer growth

Stem cells, by definition, are able to both self-renew (give rise to more cells of their own kind) and demonstrate multipotential (the ability to differentiate into multiple cell types). To accommodate this unique dual ability, stem cells interpret signal transduction pathways in specialized ways. Notable examples include canonical and non-canonical branches of the Notch signaling pathway, with each controlling different downstream targets (e.g., Hes1 vs. Hes3) and promoting either differentiation or self-renewal. Similarly, stem cells utilize STAT3 signaling uniquely. Most mature cells studied thus far rely on tyrosine phosphorylation (STAT3-Tyr) to promote survival and growth; in contrast, STAT3-Tyr induces the differentiation of neural stem cells (NSCs). NSCs use an alternative phosphorylation site, STAT3-Ser, to regulate survival and growth, a site that is largely redundant for this function in most other cell types. STAT3-Ser regulates Hes3, and together they form a convergence point for several signals, including Notch, Tie2, and insulin receptor activation. Disregulation and manipulation of the STAT3-Ser/Hes3 signaling pathway is important in both tumorigenesis and regenerative medicine, and worthy of extensive study.

Angiopoietin-2 promotes inflammatory activation of human macrophages and is essential for murine experimental arthritis

BackgroundAngiopoietin (Ang)-1 and Ang-2, and their shared receptor Tie2, are expressed in rheumatoid arthritis (RA) synovial tissue, but the cellular targets of Ang signalling and the relative contributions of Ang-1...

Impaired function of the Tie-2 receptor contributes to vascular leakage and lethality in anthrax

The anthrax lethal toxin (LT) enters host cells and enzymatically cleaves MAPKKs or MEKs. How these molecular events lead to death from anthrax remains poorly understood, but published reports suggest a direct effect of LT on vascular permeability. We have found that LT challenge in mice disrupts signaling through Tie-2, a tonically activated receptor tyrosine kinase in the endothelium. Genetic manipulations favoring Tie-2 activation enhanced interendothelial junctional contacts, prevented vascular leakage, and promoted survival following a lethal dose of LT. Cleavage of MEK1/2 was necessary for LT to induce endothelial barrier dysfunction, and activated Tie-2 signaled through the uncleaved fraction of MEKs to prevent LT's effects on the endothelium. Finally, primates infected with toxin-secreting Bacillus anthracis bacilli developed a rapid and marked imbalance in the endogenous ligands that signal Tie-2, similar to that seen in LT-challenged mice. Our results show that B. anthracis LT blunts signaling through Tie-2, thereby weakening the vascular barrier and contributing to lethality of the disease. Measurement of circulating Tie-2 ligands and manipulation of Tie-2 activity may represent future prognostic and therapeutic avenues for humans exposed to B. anthracis.

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.