Tie2 Phosphorylation Research Articles

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.

Ligand-independent Tie2 Dimers Mediate Kinase Activity Stimulated by High Dose Angiopoietin-1

Tie2 is a receptor tyrosine kinase expressed on vascular endothelial cells (ECs). It has dual roles in promoting angiogenesis and stabilizing blood vessels, and it has been suggested that Tie2 forms dimers and/or oligomers in the absence of angiopoietin-1 (Ang1); however, the mechanism of ligand-independent dimerization of Tie2 and its biological significance have not been clarified. Using a bimolecular fluorescence complementation assay and a kinase-inactive Tie2 mutant, we show here that ligand-independent Tie2 dimerization is induced without Tie2 phosphorylation. Moreover, based on the fact that Tie1 never forms heterodimers with Tie2 in the absence of Ang1 despite having high amino acid sequence homology with Tie2, we searched for ligand-independent dimerization domains of Tie2 by reference to the difference with Tie1. We found that the YIA sequence of the intracellular domain of Tie2 corresponding to the LAS sequence in Tie1 is essential for this dimerization. When the YIA sequence was replaced by LAS in Tie2 (Tie2YIA/LAS), ligand-independent dimer was not formed in the absence of Ang1. When activation of Tie2YIA/LAS was induced by a high dose of Ang1, phosphorylation of Tie2 was limited compared with wild-type Tie2, resulting in retardation of activation of Erk downstream of Tie2. Therefore, these data suggest that ligand-independent dimerization of Tie2 is essential for a strong response upon stimulation with high dose Ang1.

A peptide from the extracellular region of the synaptic protein α Neurexin stimulates angiogenesis and the vascular specific tyrosine kinase Tie2

Neurexin (NRXN) and Neuroligin (NLGN) are trans-synaptic proteins involved in vascular biology. NRXN is encoded in long (α) and short (β) isoforms. We have shown that βNRXN modulates blood vessel development in synergy with VEGFA and associates with NLGN. On the other hand αNRXN is also expressed in blood vessels but does not interact with NLGN or act in synergy with VEGFA, thus demonstrating a differential role.To find clues into the vascular functions of αNRXN, we chose a 7 aa motif that is part of its extracellular region and was formerly selected through a proteomic search for interactors of the vascular receptor Tie2. Next we (a) synthetized and modeled such peptide in order to determine its biological activity towards Tie2 in in vitro and in vivo angiogenesis assays and (b) evaluated if αNRXN and Tie2 physically associate in situ during vascular development.We used biochemical and cellular assays to prove that the synthetic αNRXN peptide (a) modulates the angiogenic phenotype of cultured endothelial cells and angiogenesis in vivo and (b) efficiently stimulates Tie2 phosphorylation and downstream mediators in endothelial cells. Moreover, we show that αNRXN and Tie2 can be reciprocally immunoprecipitated from chicken blood vessels at late stages of vascular development.These data have a double significance, i.e. provide a novel tool to modulate Tie2 and further suggest the involvement of the NRXN family of synaptic protein in the vascular system through their interaction with a fundamental vascular player.

The effect of chinese medicine pu-ren-dan on pancreatic angiogenesis in high fat diet/streptozotocin-induced diabetic rats

Objectives:The islet vascular system is critical for β-cell function. This study investigated the antidiabetic effect of the Chinese Pu-Ren-Dan (PRD) recipe by regulating the pancreatic angiogenic factors in T2DM rats.Materials Methods:High fat diet/streptozotocin-induced obese type-2 diabetes mellitus rats were developed and treated with PRD for 4 weeks. Then glucolipid metabolism, insulin secretion, pancreatic blood flow, ultrastructure of islet β-cell, histological changes of islet and protein expressions of pancreatic angiogenic factors were investigated.Results:PRD-reduced T2DM rats’ body weight and blood glucose level resisted the lipid metabolism disturbance, and ameliorated the insulin resistance and β-cell function. In addition, the histological and morphological studies proved that PRD could maintain the normal distribution of endocrine cell in islet and normal ultrastructure of β cell. An increased pancreatic blood flow was observed after the PRD treatment. In the investigation of pancreatic angiogenic factors, PRD inhibited the decreased expression of VEGF and Ang-1, and reversed the reduction of VEGFR2 and Tie2 phosphorylation in T2DM rats; the Ang-2 and TGFβ expression were up-regulated by PRD while PKC was activated; endostatin and angiostatin were down-regulated by PRD.Conclusions:The results suggest that increasing VEGF expression, regulating VEGF/VEGFR2 signaling, stimulating Ang-1/Tie-2 signaling pathway, and inhibiting PKC-TGFβ signaling and antiangiogenic factors might be the underlying mechanism of PRD's antidiabetic effect.

Tie2 Receptor Dimerization Mediated by its Extracellular FNIII Domains

The Tie family of receptor tyrosine kinases (RTKs) regulate a number of angiogenic processes that are critical in vascular development, as well as vascularization of tumor masses. The current signaling paradigm is that angiopoietin (Ang)1 binds to Tie2, promoting Tie2 homodimerization and autophosphorylation. This in turn results in cell proliferation, vessel branching, and sprouting. The extracellular regions of the Tie receptors contain three each of immunoglobulin-like (Ig) domains, EGF-like domains, and fibronectin type III (FNIII) domains. The structure of the Ig/EGF domain region of Tie2 in complex with an Ang protein has been described. However, this structure does not fully explain receptor activation or dimerization. Focusing on the three membrane-proximal FNIII domains - missing from the previously reported structure - we have found that this region can independently drive Tie2 dimerization, indicating that the FNIII domains play an important role in defining the activated dimeric configuration of this receptor. To determine the molecular basis for this observation we have solved a 2.5 A resolution crystal structure of the Tie2 FNIII domains, which reveals a domain architecture with intermolecular interactions between the second and third FNIII domains that are highly reminiscent of those seen in ligand-induced dimers of the hGHR receptor. Guided by this crystal structure we have generated mutations in the region that defines receptor dimerization. These mutations appear to reduce dimer formation in solution, and to reduce Ang1 stimulated phosphorylation of Tie2 in a cellular context.

Angiopoietins regulate vascular reactivity after haemorrhagic shock in rats through the Tie2-nitric oxide pathway

Vascular reactivity shows biphasic changes after severe trauma or shock. Our aim was to elucidate the mechanisms of biphasic-changed vascular reactivity after haemorrhagic shock by observing the regulation of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) on it. Haemorrhagic-shock Sprague-Dawley rats, hypoxia-treated superior mesenteric arteries (SMAs) with intact endothelia, and a cell mixture of vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) were adopted to evaluate the regulatory effects of Ang-1 and Ang-2 on vascular reactivity and their relationship to Tie2 (receptor tyrosine kinase)-Akt-endothelial nitric oxide synthase (eNOS) and Tie2-extracellular signal-regulated kinase (Erk)-inducible nitric oxide synthase (iNOS) signal pathways. Ang-1 expression, Tie2 phosphorylation, and nitric oxide (NO) release were increased at early shock. Exogenous Ang-1 maintained the vascular reactivity of SMAs after early hypoxia. Tie2-blocking antibody and the antagonists of Akt and eNOS antagonized Ang-1-induced maintenance in vascular reactivity and a slight release in NO at the early stage of shock. Ang-2 expression, Tie2 phosphorylation, and NO release were greatly increased at late shock, but exogenous Ang-2 further decreased the vascular reactivity of SMAs after late hypoxia. Tie2-blocking antibody and the antagonists of Erk and iNOS andtagonized the Ang-2-induced decrease in vascular reactivity and a large release of NO at the late stage of shock. Ang-1 and Ang-2 participated in the regulation of vascular reactivity after haemorrhagic shock. Ang-1 was mainly responsible for the hyperreactivity at early shock through the Tie2-Akt-eNOS pathway and an appropriate amount of NO release. Ang-2 was mainly responsible for the hyporeactivity at late shock through the Tie2-Erk-iNOS pathway and the release of a large amount of NO.

Recovery From Radiation-induced Bone Marrow Damage by HSP25 Through Tie2 Signaling

Whole-body radiation therapy can cause severe injury to the hematopoietic system, and therefore it is necessary to identify a novel strategy for overcoming this injury. Mice were irradiated with 4.5 Gy after heat shock protein 25 (HSP25) gene transfer using an adenoviral vector. Then, peripheral blood cell counts, histopathological analysis, and Western blotting on bone marrow (BM) cells were performed. The interaction of HSP25 with Tie2 was investigated with mouse OP9 and human BM-derived mesenchymal stem cells to determine the mechanism of HSP25 in the hematopoietic system. HSP25 transfer increased BM regeneration and reduced apoptosis following whole-body exposure to ionizing radiation (IR). The decrease in Tie2 protein expression that followed irradiation of the BM was blocked by HSP25 transfer, and Tie2-positive cells were more abundant among the BM cells of HSP25-transferred mice, even after IR exposure. Following systemic RNA interference of Tie2 before IR, HSP25-mediated radioprotective effects were partially blocked in both mice and cell line systems. Stability of Tie2 was increased by HSP25, a response mediated by the interaction of HSP25 with Tie2. IR-induced tyrosine phosphorylation of Tie2 was augmented by HSP25 overexpression; downstream events in the Tie2 signaling pathway, including phosphorylation of AKT and EKR1/2, were also activated. HSP25 protects against radiation-induced BM damage by interacting with and stabilizing Tie2. This may be a novel strategy for HSP25-mediated radioprotection in BM.

Abstract 5280: Activation of sphingosine kinase 1 is necessary for angiopoietin 2-induced hemangiogenesis and lymphangiogenesis

Abstract INTRODUCTION: Sphingosine kinase 1 (SphK1) is one of the two isoenzymes that generate the potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P). S1P promotes cancer cell survival, proliferation and hemangiogenesis. The angiopoietin2 (Ang2)-Tie2 axis also plays a significant role in hemangiogenesis and lymphangiogenesis. The aim of this study was to investigate the involvement of SphK1 in Ang2-induced hem- and lymphangiogenesis. METHODS: Human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were treated without or with an isozyme-specific inhibitor of SphK1 (SK1-I) and qPCR, western blot, ELISA, and tube formation assays carried out. We also used a new method we developed to quantify both hem- and lymphangiogenesis in the same sample in vivo by combining Directed In Vivo Angiogenesis Assays (DIVAA) with Fluorescence Activated Cell Sorting (DIVAA/FACS). RESULTS: qPCR revealed that human HLECs like HUVECs express high levels of SphK1 and the S1PR1 receptor. S1P rapidly stimulated xocytosis of Ang2 from HLEC storage granules determined by ELISA. Ang2 activated Tie2 in HLECs as demonstrated by its rapid phosphorylation on Tyr992, a major site responsible for its activation. Similar to Ang2, S1P also stimulated phosphorylation of Tie2, which may be due to secretion of Ang2. S1P is a potent hem- and lymphangiogenic factor for HUVECs and HLECs, respectively, as demonstrated by enhanced tube formation. Ang2-induced hem- and lymphangiogenesis was mediated at least in part by SphK1 as shown by the inhibitory effect of SK1-I. Furthermore, both S1P and Ang2 significantly induced hem- and lymphangiogenesis in vivo determined by DIVAA/FACS. Similar to its effect on in vitro hem- and lymphangiogenesis, inclusion of SK1-I in the angioreactors together with Ang2 completely abolished its ability to stimulate both of these processes. CONCLUSION: Activation of SphK1 is necessary for Ang2-induced hemangiogenesis and lymphangiogenesis through the SphK1/S1P and Ang2/Tie2 axes. This work was supported by NIH (K12HD055881) and Susan G. Komen for the Cure (KG090510) to KT, NCI (R01CA61774) to SS, and Sumitomo Life Social Welfare Services Foundation grant to MN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5280. doi:1538-7445.AM2012-5280

Angiopoietin-1 mediates inhibition of hypertension-induced release of angiopoietin-2 from endothelial cells

Adequate endothelial cell stimulation is a prerequisite for the adaptive remodelling of macro- and microvessels. A pivotal autocrine mechanism following endothelial cell activation is the release of angiopoietin-2 (Ang-2), which subsequently antagonizes the binding of Ang-1 to the Tie-2 receptor, thus sensitizing the endothelial cells to pro-angiogenic and/or pro-inflammatory stimuli. Based on the observation that hypertension in mice reduces the abundance of Ang-2 stored in arterial endothelial cells, this study was aimed at testing the hypothesis that an increase in wall stress (WS) or stretch-a hallmark of hypertension-is sufficient to release Ang-2 from endothelial cells. In fact, stretching of isolated perfused mouse arteries or human cultured endothelial cells rapidly elicited an increased release of Ang-2. In the cultured endothelial cells, this was preceded by a transient rise in intracellular free calcium, abrogated through calcium chelation and accompanied by a decrease in Tie-2 phosphorylation. Interestingly, Ang-1 abolished the stretch-induced release of Ang-2 from both cultured and native endothelial cells through inhibiting the stretch-dependent mobilization of intracellular calcium. Collectively, these results indicate that increased WS or stretch facilitates the release of Ang-2 from endothelial cell Weibel-Palade bodies, and that Ang-1 can block this by attenuating the stretch-mediated rise in intracellular calcium.

Shear stress-induced activation of Tie2-dependent signaling pathway enhances reendothelialization capacity of early endothelial progenitor cells

Although endothelial progenitor cells (EPCs) play a pivotal role in the endothelial repair following arterial injury and shear stress has a beneficial effect on EPCs, however, the molecular mechanism underlying the influence of EPCs on the endothelial integrity and the regulation of shear stress on the EPC signaling remained to be studied. Here, we investigated the effects of laminar shear stress on the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)-dependent signaling and its relation to in vivo reendothelialization capacity of human early EPCs. The human early EPCs were treated with shear stress. Shear stress in a dose-dependent manner increased angiopoietin-2 (Ang2)-induced migratory, adhesive and proliferatory activities of EPCs. Transplantation of EPCs treated by shear stress facilitated in vivo reendothelialization in nude mouse model of carotid artery injury. In parallel, the phosphorylation of Tie2 and Akt of EPCs in response to shear stress was significantly enhanced. With treatment of Tie2 knockdown or Akt inhibition, shear stress-induced phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) of EPCs was markedly suppressed. After Tie2/PI3K/Akt/eNOS signaling was blocked, the effects of shear stress on in vitro function and in vivo reendothelialization capacity of EPCs were significantly inhibited. The present findings demonstrate for the first time that Tie2/PI3k/Akt/eNOS signaling pathway is, at least in part, involved in the EPCs-mediated reendothelialization after arterial injury. The upregulation of shear stress-induced Tie2-dependent signaling contributes to enhanced in vivo reendothelialization capacity of human EPCs.

The Molecular Balance between Receptor Tyrosine Kinases Tie1 and Tie2 Is Dynamically Controlled by VEGF and TNFα and Regulates Angiopoietin Signalling

Angiopoietin-1 (Ang1) signals via the receptor tyrosine kinase Tie2 which exists in complex with the related protein Tie1 at the endothelial cell surface. Tie1 undergoes regulated ectodomain cleavage in response to phorbol esters, vascular endothelial growth factor (VEGF) and tumour necrosis factor-α (TNFα). Recently phorbol esters and VEGF were found also to stimulate ectodomain cleavage of Tie2. Here we investigate for the first time the effects of factors activating ectodomain cleavage on both Tie1 and Tie2 within the same population of cells, and their impact on angiopoietin signalling. We find that phorbol ester and VEGF activated Tie1 cleavage within minutes followed by restoration to control levels by 24 h. However, several hours of PMA and VEGF treatment were needed to elicit a detectable decrease in cellular Tie2, with complete loss seen at 24 h of PMA treatment. TNFα stimulated Tie1 cleavage, and induced a sustained decrease in cellular Tie1 over 24 h whilst increasing cellular Tie2. These differential effects of agonists on Tie1 and Tie2 result in dynamic modulation of the cellular Tie2∶Tie1 ratio. To assess the impact of this on Ang1 signalling cells were stimulated with VEGF and TNFα for differing times and Ang1-induced Tie2 phosphorylation examined. Elevated Tie2∶Tie1, in response to acute VEGF treatment or chronic TNFα, was associated with increased Ang1-activated Tie2 in cells. These data demonstrate cellular levels of Tie1 and Tie2 are differentially regulated by pathophysiologically relevant agonists resulting in dynamic control of the cellular Tie2∶Tie1 balance and modulation of Ang1 signalling. These findings highlight the importance of regulation of signalling at the level of the receptor. Such control may be an important adaptation to allow modulation of cellular signalling responses in systems in which the activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.

Inhibition of Protein Tyrosine Phosphatase Improves Angiogenesis via Enhancing Ang-1/Tie-2 Signaling in Diabetes

Diabetes is associated with impairment of angiogenesis such as reduction of myocardial capillary formation. Our previous studies demonstrate that disruption of Angiopoietin-1 (Ang-1)/Tie-2 signaling pathway contributes to the diabetes-associated impairment of angiogenesis. Protein tyrosine phosphatase (PTP) has a critical role in the regulation of insulin signal by inhibition of tyrosine kinase phosphorylation. In present study, we examined the role of protein tyrosine phosphatase-1 (SHP-1) in diabetes-associated impairment of Ang-1/Tie-2 angiogenic signaling and angiogenesis. SHP-1 expression was significantly increased in diabetic db/db mouse hearts. Furthermore, SHP-1 bond to Tie-2 receptor and stimulation with Ang-1 led to SHP-1 dissociation from Tie-2 in mouse heart microvascular endothelial cell (MHMEC). Exposure of MHMEC to high glucose (HG, 30 mmol/L) increased SHP-1/Tie-2 association accompanied by a significant reduction of Tie-2 phosphorylation. Exposure of MHMEC to HG also blunted Ang-1-mediated SHP-1/Tie-2 dissociation. Knockdown of SHP-1 significantly attenuated HG-induced caspase-3 activation and apoptosis in MHMEC. Treatment with PTP inhibitors restored Ang-1-induced Akt/eNOS phosphorylation and angiogenesis. Our data implicate a critical role of SHP-1 in diabetes-associated vascular complications, and that upregulation of Ang-1/Tie-2 signaling by targeting SHP-1 should be considered as a new therapeutic strategy for the treatment of diabetes-associated impairment of angiogenesis.

Overexpression of angiopoietin‐2 promotes myocardial fibrosis and rarefaction in diabetic db/db mouse model

Diabetic rarefaction is associated with increased endothelial cell (EC) apoptosis and capillary regression. We investigated the potential roles of diabetic-induced angiopoietin-2 (Ang-2) on capillary regression, EC-mesenchymal transition (Endo-MT) and apoptosis in db/db mouse hearts. The db/db mice were intra-myocardial administration of adenovirus Ang-2 (Ad-CMV-Ang-2). Myocardial Ang-2, Tie-2 phosphorylation, caspase-3, Wnt7b expression were measured. Myocardial apoptosis, capillary density, cardiac hypertrophy and fibrosis were analyzed. Administration of Ad-Ang-2 results in Ang-2 overexpressing in db/db mice hearts. This is accompanied by downregulation of Tie-2 phosphorylation. SMC recruitment and coverage in the neovessels of the hearts are severely impaired in the Ang-2 overexpressing db/db mice. Overexpressing Ang-2 leads to a dramatic reduction of capillary density and significant increases in myocardial caspase-3, Wnt7b expression and EC apoptosis. Intriguingly, overexpressing Ang-2 also leads to a significant increase in cardiac fibrosis via Endo-MT. Our data show that increased Ang-2 in diabetic hearts promotes EC apoptosis and capillary regression as well as cardiac fibrosis, thus lead to myocardial rarefaction. Suppression of Ang-2 may represent a potential novel therapeutic strategy for the treatment of the diabetes-associated rarefaction and cardiac dysfunction.

Tie1 attenuation reduces murine atherosclerosis in a dose-dependent and shear stress–specific manner

Although the response of endothelial cells to the disturbed blood flow in the vicinity of atherosclerotic lesions is known to be distinct from that elicited by nonatherogenic laminar flow, the mechanisms involved are poorly understood. Our initial studies confirmed that expression of the endothelial receptor tyrosine kinase Tie1 was evident at regions of atherogenic flow in mature animals. We therefore hypothesized that Tie1 plays a role in the endothelial response to atherogenic shear stress. Consistent with this, we found that Tie1+/- mice bred to the apoE-deficient background displayed a 35% reduction in atherosclerosis relative to Tie1+/+;Apoe-/- mice. Since deletion of Tie1 results in embryonic lethality secondary to vascular dysfunction, we used conditional and inducible mutagenesis to study the effect of endothelial-specific Tie1 attenuation on atherogenesis in Apoe-/- mice and found a dose-dependent decrease in atherosclerotic lesions. Analysis of primary aortic endothelial cells indicated that atheroprotective laminar flow decreased Tie1 expression in vitro. Attenuation of Tie1 was associated with an increase in eNOS expression and Tie2 phosphorylation. In addition, Tie1 attenuation increased IkBα expression while decreasing ICAM levels. In summary, we have found that shear stress conditions that modulate atherogenic events also regulate Tie1 expression. Therefore, Tie1 may play a novel proinflammatory role in atherosclerosis.

Effect of 8-O-acetyl Shanzhiside Methylester Increases Angiogenesis and Improves Functional Recovery after Stroke

We investigated whether 8-O-acetyl shanzhiside methylester (ND01) regulates angiogenesis and thereby improves functional outcome after stroke. Adult male rats were subjected to 1 hr of middle cerebral artery occlusion (MCAO) and reperfusion, and treated with or without different doses (5 and 10 mg/kg) of ND01, starting 24 hr after ischaemia and reperfusion (I/R) and by intravenous injection daily for 14 days. Neurological functional tests were performed and cerebral Evans blue extravasation was measured. Angiogenesis and angiogenic factor expression were measured by immunohistochemistry and Western blot, respectively. The results indicated that ND01 significantly promoted angiogenesis in the ischaemic brain and improved functional outcome after stroke. ND01 also significantly increased vascularization compared with vehicle treatment. ND01 increased the expression of VEGF, Ang1, phosphorylation of Tie2 and Akt VEGF. The Ang1/Tie2 axis and Akt pathways appear to mediate ND01-induced angiogenesis.

Angiopoietin-2-Driven Vascular Remodeling in Airway Inflammation

Vascular remodeling is a feature of chronic inflammation during which capillaries transform into venules that expand the region of the vasculature in which leakage and leukocyte emigration both occur. Recently, we found that angiopoietin/Tie2 receptor signaling drives the transformation of capillaries into venules at an early stage of the sustained inflammatory response in the airways of mice infected with Mycoplasma pulmonis. However, the precise contributions of both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are not clear. In this study, we sought to determine the contribution of Ang2 to this vascular remodeling. Ang2 mRNA expression levels increased and phosphorylated Tie2 immunoreactivity in mucosal blood vessels decreased, indicative of diminished receptor signaling after infection. Selective inhibition of Ang2 throughout the infection by administration of either of two distinct function-blocking antibodies reduced the suppression of Tie2 phosphorylation and decreased the remodeling of mucosal capillaries into venules, the amount of leukocyte influx, and disease severity. These findings are consistent with Ang2 acting as an antagonist of Tie2 receptors and the reduction of Tie2 phosphorylation in endothelial cells rendering the vasculature more responsive to cytokines that promote both vascular remodeling and the consequences of inflammation after M. pulmonis infection. By blocking such changes, Ang2 inhibitors may prove beneficial in the treatment of sustained inflammation in which vascular remodeling, leakage, and leukocyte influx contribute to its pathophysiology.

Ang1 Functions as An Autocrine Activating Factor of Tie2 Signaling In Hematopoietic Stem Cells.

AbstractAbstract 1548Hematopoietic stem cells (HSCs) are clonogenic cells that possess the self-renewal capacity to produce more HSCs, as well as the multilineage potential that gives rise to a defined set of mature differentiated progeny for maintenance or repair of the whole blood system. HSCs lie in the hematopoietic niches located along the inner surface of the bone or the sinusoidal endothelium, and are in contact with niche cells. The cell-cell interactions with niche cells are believed to be an important prerequisite to trigger signaling events in HSCs, thereby controlling the balance between HSC self-renewal and differentiation. However, the precise molecular mechanisms regulating niche cell-HSC interactions are not well understood. One of the key molecules for those interactions is Angiopoietin-1 (Ang1). Ang1 is expressed by the niche cells and has been identified as an activating ligand for Tie2 (tyrosine kinase with Ig-like loops and epidermal growth factor homology domains 2). The expression of Tie2 is dominant in HSCs, and Tie2 in HSCs is supposed to be stimulated by Ang1 derived from niche cells. However, Ang1 is also expressed in HSCs. Detailed analysis has shown that Ang1 expression was found to be restricted in long-term HSCs (CD34-lineage-Sca-1+c-Kit+), indicating that Ang1 derived from HSCs plays a role in regulating HSCs. We attempted to elucidate a novel regulating system for HSCs through Ang1-Tie2 signaling by utilizing a hematopoietic cell line in which Tie2 was stably expressed (Ba/F3-Tie2). In Ba/F3-Tie2 cells, Tie2 was found to be phosphorylated on tyrosine residues, even without exogenous addition of Ang1. In the same cells, the expression level of endogenous Ang1 was increased four-fold. When Ang1 expression was down-regulated by transduction with a lentiviral vector expressing short hairpin RNA (shRNA) for Ang1 (shAng1), the phosphorylation of Tie2 was suppressed, suggesting that Tie2 expressed in Ba/F3-Tie2 cells could be stimulated by endogenous Ang1. To mimic the physiological circumstances of the bone marrow, Ba/F3-Tie2 cells were cultured on OP9 stromal cells. Under these culture conditions, the effect of endogenous Ang1 was investigated. Down-regulation of Ang1 by shAng1 demonstrated an approximate 50% reduction in the proliferation of Ba/F3-Tie2 cells on the OP9 cell layer. A HSC-rich population of cells prepared from bone marrow (lineage-Sca-1+c-Kit+; LSK) was also analyzed on OP9 cell layers. Similar to the results obtained from the analysis of Ba/F3-Tie2 cells, down-regulation of Ang1 by shAng1 resulted in an approximately 70% decrease in the proliferation of LSK cells cultured on OP9 monolayers. We confirmed that the suppressive effect on HSC proliferation was due to the lack of Ang1 from HSCs by culturing on Ang1-defective OP9 cells. Finally, we performed in vivo analysis to confirm the importance of endogenous Ang1 to HSCs. Ly5.2 LSK cells transduced with the shAng1 expressing vector were transplanted along with Ly5.1xLy5.2 bone marrow cells into lethally irradiated Ly5.1 mice. The Ly5.2 donor-derived cells in the recipient's peripheral blood were monitored every 2 weeks. As expected, shAng1-introduced donor cells were at decreased ratios at week four (mean ratios, 31.5% for control vs. 17.5% for shAng1), and were reduced to an even lower level at week 12 (mean ratios, 27.1% for control vs. 6.79% for shAng1). This phenomenon was also confirmed by histochemical results, where statistically fewer HSCs existed in the bone marrow of recipient mice in which shAng1-introduced HSCs were transplanted, as compared to the control. Altogether, our data suggested that Tie2 in HSCs could be stimulated by the Ang1 produced by the surrounding HSCs, and this possible autocrine regulation might control the functions of HSCs. Disclosures:No relevant conflicts of interest to declare.

Anti-Angiogenic Activity of a Tetravalent Bispecific Antibody (TAvi6) Targeting VEGF and Angiopoietin-2

AbstractAbstract 4304 Background:VEGF blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis. Several publications described that VEGF and Ang-2 are up-regulated in several hematological tumors like B-CLL, AML or multiple myeloma. In particular, Ang-2 may qualify as a novel prognostic marker in AML and CLL. We have generated TAvi6, a novel bispecific antibody targeting VEGF-A and Ang-2 and tested its anti-tumor and anti-angiogenic activity. TAvi6 is a tetravalent IgG-like bispecific antibody based on bevacizumab and targets Ang-2 with 2 disulfide-stabilized scFvs (LC06) fused to the C-terminus of the heavy chain. Material and Methods:TAvi 6 was profiled in biochemical and cellular (angiogenesis) assays. Antitumoral and anti-angiogenic efficacy was assessed in established s.c. Colo205, s.c. Calu-3 and orthotopic i.m.f.p. KPL-4 xenografts in SCID beige mice. Mice were treated with bevacizumab or <Ang-2> antibody LC06 (10 mg/kg), the respective combination (each 10 mg/kg) and TAvi6 (13.3 mg/kg). In addition, TAvi6 was evaluated for inhibition of metastasis to the lung quantified by Alu-PCR. Tumors were explanted for histological analysis of angiogenesis and inhibition of angiogenesis was assessed in the cornea angiogenesis assay. Results:In biochemical assays (affinity, Tie2-Ang-2 interaction) and cellular assays (Tie2 phosphorylation, HUVEC proliferation, tube formation) TAvi6 shows properties identical to the parental antibodies bevacizumab and LC06. In the orthotopic KPL-4-003 xenograft tumor growth inhibition was 79% for bevacizumab; 39% for LC06; 90% for the combination and 91% for TAvi6. In the s.c. Colo205-009 xenograft TGI was 66% for bevacizumab; 50% for LC06; 78% for the combination and 87% for TAvi6. TAvi6 was able to suppress growth of tumors refractory to 1st-line treatment with bevacizumab (Colo205 xenograft) and suppressed metastasis to the lung significantly (KPL-4 xenograft). In the s.c. Calu-3 xenograft we observed a strong inhibition of angiogenesis by in vivo and ex vivo imaging and an advantage of TAvi6 compared to the antibody combination. Furthermore, TAvi6 potently inhibited VEGF-induced angiogenesis in the mouse corneal angiogenesis assay. Conclusions:We have generated a novel tetravalent IgG-like bispecific antibody targeting VEGF-A and Ang-2 simultaneously. TAvi6 shows identical properties compared to the respective parental antibodies. In particular, TAvi6 blocks angiogenesis in vivo efficacously and mediates strong tumor growth inhibition in various xenograft models with a slight advantage of TAvi6 over the combination of the respective single agents bevacizumab and LC06 in several models. Further studies are foreseen to assess the application of bispecific antibodies targeting Ang-2 and VEGF as novel therapeutic approach for the treatment of hematological cancers. Disclosures:Weidner:Roche: Employment, Equity Ownership. Scheuer:Roche: Employment, Patents & Royalties. Thomas:Roche: Employment, Patents & Royalties. Baehner:Roche: Employment, Patents & Royalties. Seeber:Roche: Employment, Patents & Royalties. Kettenberger:Roche: Employment, Patents & Royalties. Schanzer:Roche: Employment, Patents & Royalties. Brinkmann:Roche: Employment, Patents & Royalties. Regula:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit beta3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, alphavbeta3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser(910) upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from alphavbeta3 integrin. The alphavbeta3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/alphavbeta3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.

Abstract 1560: VE-PTP inhibitor AKB-9778 mitigates IL-2-induced VLS and interacts positively with IL-2 against melanoma and renal cancer tumors in mice

Abstract IL-2 induces complete responses (CR) durable more than 10 years in a subpopulation of patients with advanced melanoma or renal cell carcinoma, establishing the clinical effectiveness of immunotherapy for human malignancies. However, the application and clinical efficacy of IL-2 have been limited by its major dose-limiting toxicity vascular leak syndrome (VLS), which occurs in the majority (∼65%) of the patients and often necessitates cessation of the cytokine treatment. Being a life-threatening toxicity, VLS is managed by supportive care with no specific treatment. Therapeutics for IL-2-induced VLS might improve the efficacy and tolerance of the cytokine. Recent studies implicated heightened Ang2 as a potential mediator of VLS via antagonizing Ang1 and thus blocking Tie2/AKT signaling in endothelial cells. Being a regulator of Tie2 phosphorylation, vascular endothelial protein tyrosine phosphatase (VE-PTP) might be targeted to modulate Tie2/AKT signaling although tthe effects and tolerance of targeting VE-PTP were not well-defined. Our work herein identified compound AKB-9778 as a VE-PTP inhibitor capable of inducing pTie2 and down stream pAKT in endothelial cells, interacting positively with Ang1 and/or Ang2 to increase cellular pTie2 levels, reducing IL-2-induced circulating Ang2 in mice and protecting mice from IL-2-induced VLS. AKB-9778 also interacted positively with IL-2 against B16 melanoma and Renca renal cancer tumors in mice in a tolerated manner. These data provide pre-clinical proof of concept evidence of targeting VE-PTP to mitigate IL-2-induced VLS. AKB-9778 may provide beneficial adjunctive therapy in combination with IL-2 in cancer treatment by reducing VLS and enhancing efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1560.