Tie2 Phosphorylation Research Articles

VEPTP inhibition with an extracellular domain targeting antibody did not restore albuminuria in a mouse model of diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease. DKD is a heterogeneous disease with complex pathophysiology where early endothelial dysfunction is associated with disease progression. The Tie2 receptor and Angiopoietin 1 and 2 ligands are critical for maintaining endothelial cell permeability and integrity. Tie2 signaling is negatively regulated by the endothelial specific transmembrane receptor Vascular Endothelial Protein Tyrosine Phosphatase (VEPTP). Genetic deletion of VEPTP protects from hypertension and diabetes induced renal injury in a mouse model of DKD. Here, we show that VEPTP inhibition with an extracellular domain targeting VEPTP antibody induced Tie2 phosphorylation and improved VEGF-A induced vascular permeability both invitro and invivo. Treatment with the VEPTP blocking antibody decreased the renal expression of endothelial activation markers (Angpt2, Edn1, and Icam1) but failed to improve kidney function in db/db uninephrectomized ReninAAV DKD mice.

Flavan-3-ols, flavonoids, anthocyanidins and triterpenoids induces TIE2 phosphorylation -a candidate target for the vascular protective effects

Vascular system is essential for the body to maintain health. Dysregulated vascular system leads to cardiovascular diseases and are observed in ischaemic stroke, Alzheimer’s disease, amyotrophic lateral sclerosis, and diabetes. TIE2 is a tyrosine kinase receptor expressed on vascular endothelial cells and contributes to the maintenance of a vascular system. In this paper, we screened for natural products with an activity to induce phosphorylation of TIE2, which will be beneficial for protection of a vascular system. Employing HeLa cells expressing TIE2, flavan-3-ols, flavonoids, anthocyanidins and triterpenoids were identified as active compounds that induce TIE2 phosphorylation. Several of the identified compounds are previously reported to protect endothelial cells from inflammation. Thus, the result provided TIE2 as the candidate receptor protein of those compounds for the protective effect of endothelial cells and the identified compounds will be a good candidate for maintenance of a vascular system.

Heterogeneity of endothelial VE-PTP downstream polarization, Tie2 activation, junctional claudin-5, and permeability in the aorta and vena cava

Endothelial cells of mammalian blood vessels have multiple levels of heterogeneity along the vascular tree and among different organs. Further heterogeneity results from blood flow turbulence and variations in shear stress. In the aorta, vascular endothelial protein tyrosine phosphatase (VE-PTP), which dephosphorylates tyrosine kinase receptor Tie2 in the plasma membrane, undergoes downstream polarization and endocytosis in endothelial cells exposed to laminar flow and high shear stress. VE-PTP sequestration promotes Tie2 phosphorylation at tyrosine992 and endothelial barrier tightening. The present study characterized the heterogeneity of VE-PTP polarization, Tie2-pY992 and total Tie2, and claudin-5 in anatomically defined regions of endothelial cells in the mouse descending thoracic aorta, where laminar flow is variable and IgG extravasation is patchy. We discovered that VE-PTP and Tie2-pY992 had mosaic patterns, unlike the uniform distribution of total Tie2. Claudin-5 at tight junctions also had a mosaic pattern, whereas VE-cadherin at adherens junctions bordered all endothelial cells. Importantly, the amounts of Tie2-pY992 and claudin-5 in aortic endothelial cells correlated with downstream polarization of VE-PTP. VE-PTP and Tie2-pY992 also had mosaic patterns in the vena cava, but claudin-5 was nearly absent and extravasated IgG was ubiquitous. Correlation of Tie2-pY992 and claudin-5 with VE-PTP polarization supports their collective interaction in the regulation of endothelial barrier function in the aorta, yet differences between the aorta and vena cava indicate additional flow-related determinants of permeability. Together, the results highlight new levels of endothelial cell functional mosaicism in the aorta and vena cava, where blood flow dynamics are well known to be heterogeneous.

Glycation of fibronectin impairs angiopoietin-1/Tie-2 signaling through uncoupling Tie-2-α5β1 integrin crosstalk

The dysfunction of angiopoietin-1 (Ang-1)/Tie-2 signaling pathways has been implicated in diabetic complications. However, the underlying molecular mechanisms remain unclear. Fibronectin (FN) is thought to have an important role in regulating Ang-1/Tie-2 signaling activation. But no previous study has investigated the effects of FN glycation on Ang-1/Tie-2 signaling. In the present study, FN was glycated by methylglyoxal (MGO) to investigate whether the glycation of FN contributes to diabetes-induced Ang-1/Tie-2 signaling impairment and to understand the molecular mechanisms involved. The results demonstrated that MGO-glycated FN significantly impaired Ang-1-evoked phosphorylation of Tie-2 and Akt, Ang-1-induced endothelial cell migration and tube formation and Ang-1-mediated cell survival. The glycation of FN also inhibited the binding of α5β1 integrin to Tie-2. Moreover, FN was remarkably modified by AGEs in aortae derived from db/db mice, indicating the glycation of FN in vivo. Ang-1-induced aortic ring vessel outgrowth and Ang-1-mediated cell survival were also both significantly inhibited in aortae from db/db mice compared to that from the wild type littermates. Moreover, FN, rather than glycated FN partly restored aortic ring angiogenesis in db/db mice, indicating that the angiogenesis defect in the db/db mice are due to FN glycation. Collectively, the results in the present study suggest that the glycation of FN impairs Ang-1/Tie-2 signaling pathway by uncoupling Tie-2-α5β1 integrin crosstalk. This may provide a mechanism for Ang-1/Tie-2 signaling dysfunction and angiogenesis failure in diabetic ischaemic diseases.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

Cooperation of Angiopoietin-2 and Angiopoietin-4 in Schlemm's Canal Maintenance.

PurposeDefects in the iridocorneal angle tissues, including the trabecular meshwork (TM) and Schlemm's canal (SC), impair aqueous humor flow and increase the intraocular pressure (IOP), eventually resulting in glaucoma. Activation of endothelial tyrosine kinase receptor Tie2 by angiopoietin-1 (Angpt1) has been demonstrated to be essential for SC formation, but roles of the other two Tie2 ligands, Angpt2 and Angpt4, have been controversial or not yet characterized, respectively.MethodsAngpt4 expression was investigated using genetic cell fate mapping and reporter mice. Congenital deletion of Angpt2 and Angpt4 and tamoxifen-inducible deletion of Angpt1 in mice were used to study the effects of Angpt4 deletion alone and in combination with the other angiopoietins. SC morphology was examined with immunofluorescent staining. IOP measurements, electron microscopy, and histologic evaluation were used to study glaucomatous changes.Results Angpt4 was postnatally expressed in the TM. While Angpt4 deletion alone did not affect SC and Angpt4 deletion did not aggravate Angpt1 deletion phenotype, absence of Angpt4 combined with Angpt2 deletion had detrimental effects on SC morphology in adult mice. Consequently, Angpt2−/−;Angpt4−/− mice displayed glaucomatous changes in the eye. Mice with Angpt2 deletion alone showed only moderate SC defects, but Angpt2 was necessary for proper limbal vasculature development. Mechanistically, analysis of Tie2 phosphorylation suggested that Angpt2 and Angpt4 cooperate as agonistic Tie2 ligands in maintaining SC integrity.ConclusionsOur results indicated an additive effect of Angpt4 in SC maintenance and Tie2 activation and a spatiotemporally regulated interplay between the angiopoietins in the mouse iridocorneal angle.

Adjunctive therapy with the Tie2 agonist Vasculotide reduces pulmonary permeability in Streptococcus pneumoniae infected and mechanically ventilated mice

Community acquired pneumonia, mainly caused by Streptococcus pneumoniae (S.pn.), is a common cause of death worldwide. Despite adequate antibiotic therapy, pneumococcal pneumonia can induce pulmonary endothelial hyperpermeability leading to acute lung injury, which often requires mechanical ventilation (MV) causing ventilator-induced lung injury (VILI). Endothelial stabilization is mediated by angiopoietin-1 induced Tie2 activation. PEGylated (polyethylene glycol) Tie2-agonist Vasculotide (VT) mimics Angiopietin-1 effects. Recently, VT has been shown to reduce pulmonary hyperpermeability in murine pneumococcal pneumonia. The aim of this study was to determine whether VT reduces lung damage in S.pn. infected and mechanically ventilated mice. Pulmonary hyperpermeability, immune response and bacterial load were quantified in S.pn. infected mice treated with Ampicillin + /−VT and undergoing six hours of MV 24 h post infection. Histopathological lung changes, Tie2-expression and -phosphorylation were evaluated. VT did not alter immune response or bacterial burden, but interestingly combination treatment with ampicillin significantly reduced pulmonary hyperpermeability, histological lung damage and edema formation. Tie2-mRNA expression was reduced by S.pn. infection and/or MV but not restored by VT. Moreover, Tie2 phosphorylation was not affected by VT. These findings indicate that VT may be a promising adjunctive treatment option for prevention of VILI in severe pneumococcal pneumonia.

Glycation of Tie-2 Inhibits Angiopoietin-1 Signaling Activation and Angiopoietin-1-Induced Angiogenesis.

The impairment of the angiopoietin-1 (Ang-1)/Tie-2 signaling pathway has been thought to play a critical role in diabetic complications. However, the underlying mechanisms remain unclear. The present study aims to investigate the effects of Tie-2 glycation on Ang-1 signaling activation and Ang-1-induced angiogenesis. We identified that Tie-2 was modified by advanced glycation end products (AGEs) in aortae derived from high fat diet (HFD)-fed mice and in methylglyoxal (MGO)-treated human umbilical vein endothelial cells (HUVECs). MGO-induced Tie-2 glycation significantly inhibited Ang-1-evoked Tie-2 and Akt phosphorylation and Ang-1-regulated endothelial cell migration and tube formation, whereas the blockade of AGE formation by aminoguanidine remarkably rescued Ang-1 signaling activation and Ang-1-induced angiogenesis in vitro. Furthermore, MGO treatment markedly increased AGE cross-linking of Tie-2 in cultured aortae ex vivo and MGO-induced Tie-2 glycation also significantly decreased Ang-1-induced vessel outgrow from aortic rings. Collectively, these data suggest that Tie-2 may be modified by AGEs in diabetes mellitus and that Tie-2 glycation inhibits Ang-1 signaling activation and Ang-1-induced angiogenesis. This may provide a novel mechanism for Ang-1/Tie-2 signal dysfunction and angiogenesis failure in diabetic ischaemic diseases.

Abstract 5970: Tumor vessel normalization by a novel anti-TIE2 antibody PMC-403 enhances the effectiveness of radiation therapy on cancer therapy

Abstract Purpose: The delivery of drugs and immune cells into the tumor microenvironment is limited due to abnormal tumor vessel which is characterized as low oxygen concentration, low perfusion rate and leakage. It leads to diminish the efficacy radiation therapy. Traditional anti-angiogenesis strategies attempt to reduce the tumor vascular supply, but their success is restricted by insufficient efficacy or development of resistance. To solve the problem, we try to using PMC-403, which can normalize the abnormal condition of tumor vessel. Experimental procedures: Target selectivity: It was measured by Surface Plasmon Resonance (SPR) assay. Human TIE2 Cho-K1 cells or mouse TIE2 Cho-K1 cells were tested for species cross-reactive assay based on flow cytometry (FACS).In vitro studies: Human umbilical vein endothelial cells (HUVEC) were used for TIE2 signal pathway analysis with western blot assay and efficacy analysis with VEGF-induced vessel permeability assay.In vivo study: Glioblastoma model mice were employed for the assessment of tumor vessel normalizing activity of 1 mg/mouse PMC-403. CT26 colon cancer model mice were used to evaluate the anti-tumor efficacy of PMC403 (10 mg/kg) in combination with radiation therapy. Hypoxyprobe was used to measure the improvement of hypoxic conditions in tumor tissue. Summary of data: PMC-403 has a nanomolar range of affinity for both human or mouse TIE2 expressing cells. This contributed to dose-dependent ANG1-like TIE2 and FOXO1 phosphorylation and inhibited VEGF-induced vascular leakage, thereby contributing to ANG1-like vascular normalization. Moreover, similar to ANG-1, p-VEGFR2 and p-VE-Cadherin levels were significantly reduced by PMC-403, suggesting that PMC-403 can normalize blood vessels. In addition, PMC-403 stabilized abnormal tumor blood vessels in the glioblastoma mouse model and significantly reduced tumor growth and tumor hypoxia in the combination treatment (PMC-403 + radiation therapy) compared to radiation therapy alone, although the antitumor effect of PMC-403 alone was not effective in the same condition model. Conclusions: PMC-403, an anti-TIE2 monoclonal antibody, is a novel tumor vessel stabilizer. PMC-403 improved tumor microenvironment hypoxic condition that synergized with radiotherapy when it combined together for the anticancer therapy. These data strongly support the notion that PMC-403 can effectively treat tumors even with low doses of radiation by stabilizing tumor blood vessels. Citation Format: Eun-Ah Lee, Beom Yong Park, Nuri Kang, Cheon Ho Park, Weon Sup Lee. Tumor vessel normalization by a novel anti-TIE2 antibody PMC-403 enhances the effectiveness of radiation therapy on cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5970.

In vitro inhibition of cancer angiogenesis and migration by a nanobody that targets the orphan receptor Tie1.

The human signaling molecules Tie1 and Tie2 receptor tyrosine kinases (RTKs) play important pathophysiological roles in many diseases, including different cancers. The activity of Tie1 is mediated mainly through the downstream angiopoietin-1 (Ang1)-dependent activation of Tie2, rendering both Tie 1 and the Tie1/Tie2/Ang1 axis attractive putative targets for therapeutic intervention. However, the development of inhibitors that target Tie1 and an understanding of their effect on Tie2 and on the Tie1/Tie2/Ang1 axis remain unfulfilled tasks, due, largely, to the facts that Tie1 is an orphan receptor and is difficult to produce and use in the quantities required for immune antibody library screens. In a search for a selective inhibitor of this orphan receptor, we sought to exploit the advantages (e.g., small size that allows binding to hidden epitopes) of non-immune nanobodies and to simultaneously overcome their limitations (i.e., low expression and stability). We thus performed expression, stability, and affinity screens of yeast-surface-displayed naïve and predesigned synthetic (non-immune) nanobody libraries against the Tie1 extracellular domain. The screens yielded a nanobody with high expression and good affinity and specificity for Tie1, thereby yielding preferential binding for Tie1 over Tie2. The stability, selectivity, potency, and therapeutic potential of this synthetic nanobody were profiled using in vitro and cell-based assays. The nanobody triggered Tie1-dependent inhibition of RTK (Tie2, Akt, and Fak) phosphorylation and angiogenesis in endothelial cells, as well as suppression of human glioblastoma cell viability and migration. This study opens the way to developing nanobodies as therapeutics for different cancers associated with Tie1 activation.

Pericyte Insulin Receptors Modulate Retinal Vascular Remodeling and Endothelial Angiopoietin Signaling.

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRβ-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

Mucopolysaccharide polysulfate promotes microvascular stabilization and barrier integrity of dermal microvascular endothelial cells via activation of the angiopoietin-1/Tie2 pathway

BackgroundMucopolysaccharide polysulfate (MPS) is a heparinoid and MPS-containing formulations are widely used as moisturizers for dry skin and to treat peripheral vascular insufficiency. Although MPS has therapeutic effects in skin diseases with microvascular abnormalities, the effects of MPS on microvascular function remain incompletely understood. ObjectiveThe aim of this study was to evaluate the functional activities of MPS on human pericytes (HPC) and human dermal microvascular endothelial cells (HDMEC) in vitro, and on microvascular permeability of the skin. MethodsThe protein expression of angiopoietin (Ang)-1 in HPC, and platelet-derived growth factor-BB (PDGF-BB) and phosphorylated tyrosine-protein kinase receptor 2 (Tie2) in HDMEC were measured in the presence or absence of MPS. The vascular barrier was evaluated by the expressions of claudin-5 and vascular endothelial (VE)-cadherin, and transendothelial electrical resistance (TEER). ResultsIn HPC, MPS dose-dependently enhanced Ang-1 secretion, which activated Tie2 in HDMEC. In HDMEC, MPS significantly increased the production of PDGF-BB, which is important for the recruitment of HPC to the vascular endothelium, and significantly increased the phosphorylation of Tie2, which results in the activation of the Ang-1/Tie2 signaling . MPS significantly increased the expression of tight junction protein claudin-5 and TEER in the HDMEC. Moreover, the intradermal injection of MPS prevented vascular endothelial growth factor-induced increase in vascular permeability in mouse skin. ConclusionWe found that MPS promoted microvascular stabilization and barrier integrity in HDMEC via Ang-1/Tie2 activation. These results suggest that MPS might improve microvascular abnormalities in various diseases accompanied by disturbances in Ang-1/Tie2 signaling.

Activated mesangial cells induce glomerular endothelial cells proliferation in rat anti-Thy-1 nephritis through VEGFA/VEGFR2 and Angpt2/Tie2 pathway.

ObjectivesWe aimed to investigate the underlying mechanism of endothelial cells (ECs) proliferation in anti‐Thy‐1 nephritis.Materials and methodsWe established anti‐Thy‐1 nephritis and co‐culture system to explore the underlying mechanism of ECs proliferation in vivo and in vitro. EdU assay kit was used for measuring cell proliferation. Immunohistochemical staining and immunofluorescence staining were used to detect protein expression. ELISA was used to measure the concentration of protein in serum and medium. RT‐qPCR and Western blot were used to qualify the mRNA and protein expression. siRNA was used to knock down specific protein expression.ResultsIn anti‐Thy‐1 nephritis, ECs proliferation was associated with mesangial cells (MCs)‐derived vascular endothelial growth factor A (VEGFA) and ECs‐derived angiopoietin2 (Angpt2). In vitro co‐culture system activated MCs‐expressed VEGFA to promote vascular endothelial growth factor receptor2 (VEGFR2) activation, Angpt2 expression and ECs proliferation, but inhibit TEK tyrosine kinase (Tie2) phosphorylation. MCs‐derived VEGFA stimulated Angpt2 expression in ECs, which inhibited Tie2 phosphorylation and promoted ECs proliferation. And decline of Tie2 phosphorylation induced ECs proliferation. In anti‐Thy‐1 nephritis, promoting Tie2 phosphorylation could alleviate ECs proliferation.ConclusionsOur study showed that activated MCs promoted ECs proliferation through VEGFA/VEGFR2 and Angpt2/Tie2 pathway in experimental mesangial proliferative glomerulonephritis (MPGN) and in vitro co‐culture system. And enhancing Tie2 phosphorylation could alleviate ECs proliferation, which will provide a new idea for MPGN treatment.

Role of angiopoietin-2 in venous thrombus resolution and chronic thromboembolic disease.

Defective angiogenesis, incomplete thrombus revascularisation and fibrosis are considered critical pathomechanisms of chronic thromboembolic pulmonary hypertension (CTEPH) after pulmonary embolism. Angiopoietin-2 (ANGPT2) has been shown to regulate angiogenesis, but its importance for thrombus resolution and remodelling is unknown. ANGPT2 plasma concentrations were measured in patients with CTEPH (n=68) and acute pulmonary embolism (n=84). Tissue removed during pulmonary endarterectomy (PEA) for CTEPH was analysed (immuno)histologically. A mouse model of inferior vena cava ligation was used to study the kinetics of venous thrombus resolution in wild-type mice receiving recombinant ANGPT2 via osmotic pumps, and in transgenic mice overexpressing ANGPT2 in endothelial cells. Circulating ANGPT2 levels were higher in CTEPH patients compared to patients with idiopathic pulmonary arterial hypertension and healthy controls, and decreased after PEA. Plasma ANGPT2 levels were elevated in patients with pulmonary embolism and diagnosis of CTEPH during follow-up. Histological analysis of PEA specimens confirmed increased ANGPT2 expression, and low levels of phosphorylated TIE2 were observed in regions with early-organised pulmonary thrombi, myofibroblasts and fibrosis. Microarray and high-resolution microscopy analysis could localise ANGPT2 overexpression to endothelial cells, and hypoxia and transforming growth factor-β1 were identified as potential stimuli. Gain-of-function experiments in mice demonstrated that exogenous ANGPT2 administration and transgenic endothelial ANGPT2 overexpression resulted in delayed venous thrombus resolution, and thrombi were characterised by lower TIE2 phosphorylation and fewer microvessels. Our findings suggest that ANGPT2 delays venous thrombus resolution and that overexpression of ANGPT2 contributes to thrombofibrosis and may thus support the transition from pulmonary embolism to CTEPH.

Angiopoietin 2 stimulates trophoblast invasion via a mechanism associated with JNK signaling.

Extravillous trophoblast cell (EVT) invasion is tightly controlled, and its dysregulation can lead to altered spiral artery remodeling and contribute to a number of different pregnancy complications. Angiopoietin-2 (Ang-2) is expressed by trophoblast cells and various cells in the decidua, and trophoblast cells express its receptor, Tie2. Ang-2 has been shown to play roles in tumor progression and metastasis but it is not known if it also regulates EVT invasion. Here, we show that both the HTR-8/SVneo cell line and primary isolates of human EVT expressed various integrins and the Tie2 receptor, and Ang-2 stimulated their migration and/or invasion. Ang-2 increased expression of matrix metalloproteinase (MMP)2 and MMP9, altered the cytoskeleton of HTR-8/SVneo cells and also induced phosphorylation of Tie2, JNK and c-Jun. Inhibition of p-JNK (using SP600125) blocked the Ang-2 induced invasion of HTR-8/SVneo cells. In addition, inhibition of Tie2 (pexmetinib) and integrin signaling (RGDS and ATN-161) also blocked Ang-2-induced invasion. In conclusion, we demonstrate that Ang-2 can stimulate EVT invasion via a mechanism associated with activation of both the Tie2 receptor and integrins, which appear to work through different pathways; Tie2 through the JNK/c-JUN pathway and integrins through an as yet unidentified pathway(s). We therefore propose that any alterations in Ang-2 expression in the decidua would lead to an imbalance in pro- and anti-invasive factors, disrupting regulation of EVT invasion and spiral artery remodeling and thereby contribute to the etiology of several complications of pregnancy.

A Small Molecule Inhibitor of VE-PTP Activates Tie2 in Schlemm's Canal Increasing Outflow Facility and Reducing Intraocular Pressure.

PurposeTyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie2) activation in Schlemm's canal (SC) endothelium is required for the maintenance of IOP, making the angiopoietin/Tie2 pathway a target for new and potentially disease modifying glaucoma therapies. The goal of the present study was to examine the effects of a Tie2 activator, AKB-9778, on IOP and outflow function.MethodsAKB-9778 effects on IOP was evaluated in humans, rabbits, and mice. Localization studies of vascular endothelial protein tyrosine phosphatase (VE-PTP), the target of AKB-9778 and a negative regulator of Tie2, were performed in human and mouse eyes. Mechanistic studies were carried out in mice, monitoring AKB-9778 effects on outflow facility, Tie2 phosphorylation, and filtration area of SC.ResultsAKB-9778 lowered IOP in patients treated subcutaneously for diabetic eye disease. In addition to efficacious, dose-dependent IOP lowering in rabbit eyes, topical ocular AKB-9778 increased Tie2 activation in SC endothelium, reduced IOP, and increased outflow facility in mouse eyes. VE-PTP was localized to SC endothelial cells in human and mouse eyes. Mechanistically, AKB-9778 increased the filtration area of SC for aqueous humor efflux in both wild type and in Tie2+/− mice.ConclusionsThis is the first report of IOP lowering in humans with a Tie2 activator and functional demonstration of its action in remodeling SC to increase outflow facility and lower IOP in fully developed mice. Based on these studies, a phase II clinical trial is in progress to advance topical ocular AKB-9778 as a first in class, Tie2 activator for treatment for ocular hypertension and glaucoma.

New soluble angiopoietin analog of Hepta-ANG1 prevents pathological vascular leakage.

Vascular leak is a key driver of organ injury in diseases, and strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the angiopoietin-1 (ANG1)-Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANG1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANG1 with a heptameric scaffold derived from the C-terminus of serum complement protein C4-binding protein α. We refer to this new fusion protein biologic as Hepta-ANG1, which forms a stable heptamer and induces Tie2 phosphorylation in cultured cells, and in the lung following intravenous injection of mice. Injection of Hepta-ANG1 ameliorates vascular endothelial growth factor- and lipopolysaccharide-induced vascular leakage, in keeping with the known functions of Angpt1-Tie2 in maintaining quiescent vascular stability. The new Hepta-ANG1 fusion is easy to produce and displays remarkable stability with high multimericity that can potently activate Tie2. It could be a new candidate ANG1 mimetic therapy for treatments of inflammatory vascular leak, such as acute respiratory distress syndrome and sepsis.

Stage-dependent angiopoietin-Tie2 and nitric oxide signaling of erythrocytes in response to surgical trauma in head and neck cancer

BackgroundAngiopoietin-Tie2 and nitric oxide pathway is crucial in tumor angiogenesis and closely correlates with tumor development, growth, and metastasis. This study aimed to investigate the angiopoietin-Tie2 and nitric oxide signaling of the erythrocyte membrane in response to surgical trauma in head and neck cancer.MethodsWe prospectively enrolled the patients with histology-proven head and neck squamous cell carcinoma undergoing surgical resection of primary tumors at the medical center between August and November 2019. We measured the preoperative and postoperative levels of angiopoietin-1, angiopoietin-2 in plasma using enzyme-linked immunosorbent assays, nitric oxide in plasma using nitrate/nitrite colorimetric assays, and Tie2 phosphorylation in erythrocyte membrane using Western blotting.ResultsThe plasma angiopoietin-1 was downregulated from the median 971.3 pg/mL (interquartile range [IQR] 532.1–1569.3) to 417.9 (IQR 270.5–597.3) after tumor resection (p = 0.0020). Conversely, the plasma angiopoietin-2 was enhanced from 1173.6 pg/mL (IQR 977.7–1450.2) to 2353.7 (IQR 1352.4–2954.3) after surgery (p = 0.0021), with a concomitant increase in plasma nitric oxide level from 7.73 μM (IQR 5.39–10.06) to 10.50 (IQR 7.65–14.18) after surgical resection (p = 0.0093). Subgroup analyses further showed the angiopoietin-Tie2 and nitric oxide signaling was significant only in stage III and IV cancer.ConclusionsThe dynamic change of angiopoietin-Tie2 signaling in the erythrocyte membrane along with the enhanced nitric oxide in plasma after tumor resection suggests erythrocytes play a significant role in modulating surgery-induced angiogenesis, which may provide a novel marker for cancer surveillance and control.

Aliskiren Improved the Endothelial Repair Capacity of Endothelial Progenitor Cells from Patients with Hypertension via the Tie2/PI3k/Akt/eNOS Signalling Pathway.

Background Studies show that aliskiren exerts favourable effects not only on endothelial progenitor cells (EPCs) but also on endothelial function. However, the mechanism of the favourable effect of aliskiren on EPCs from patients with hypertension is unclear and remains to be further studied. Methods The object of this study was to investigate and assess the in vitro function of EPCs pretreated with aliskiren. After treated with aliskiren, the human EPCs were transplanted into a nude mouse model of carotid artery injury, and the in vivo reendothelialization of injured artery was estimated by staining denuded areas with Evans blue dye via tail vein injection. Results We found that aliskiren increased the in vitro migration, proliferation, and adhesion of EPCs from patients with hypertension in a dose-dependent manner and improved the reendothelialization capability of these EPCs. Furthermore, aliskiren increased the phosphorylation of Tie2, Akt, and eNOS. After the blockade of the Tie2 signalling pathway, the favourable effects of aliskiren on the in vitro function and in vivo reendothelialization capability of EPCs were suppressed. Conclusions This study demonstrates that aliskiren can improve the in vitro function and in vivo reendothelialization capability of EPCs from patients with hypertension via the activation of the Tie2/PI3k/Akt/eNOS signalling pathway. These findings further indicate that aliskiren is an effective pharmacological treatment for cell-based repair in hypertension-related vascular injury.

Targeting the ER\u03b2/Angiopoietin-2/Tie-2 signaling-mediated angiogenesis with the FDA-approved anti-estrogen Faslodex to increase the Sunitinib sensitivity in RCC

Sunitinib has been used as the main therapy to treat the metastatic clear cell renal cell carcinoma (ccRCC) as it could function via suppressing the tumor growth and angiogenesis. Yet most ccRCC tumors may still regrow due to the development of sunitinib-resistance, and detailed mechanisms remain to be further investigated. The angiopoietin family includes angiopoietin-1 and angiopoietin-2 (ANGPT-1 and -2). It was reported that estradiol regulates expression of ANGPT-1, but not ANGPT-2, through estrogen receptor α (ERα) in an experimental stroke model. To date, there is no finding to link the E2/ER signal on regulating ANGPT-2. Our study is the first to explore (i) how estrogen receptor β (ERβ) can up-regulate ANGPT-2 in RCC cells, and (ii) how ERβ-increased ANGPT-2 can promote the HUVEC tube formation and reduce sunitinib sensitivity. Mechanistic studies revealed that ERβ could function via transcriptional regulation of the cytokine ANGPT-2 in the ccRCC cells. We found the up-regulated ANGPT-2 of RCC cells could then increase the Tie-2 phosphorylation to promote the angiogenesis and increase sunitinib treatment resistance of endothelial cells. In addition to the endothelial cell tube formation and aortic ring assay, preclinical studies with a mouse RCC model also confirmed the finding. Targeting this newly identified ERβ/ANGPT-2/Tie-2 signaling pathway with the FDA-approved anti-estrogen, Faslodex, may help in the development of a novel combined therapy with sunitinib to better suppress the ccRCC progression.