Tie2 Activation Research Articles

Targeting the Tie-2 Receptor With Faricimab in Central Serous Chorioretinopathy: A Case Series Motivated by a Genetic Finding

PurposeTo investigate the effects of faricimab, a bispecific antibody targeting VEGF and Ang-2 (thus increasing Tie-2 activity), in patients with CSC based on a recent genetic study that implicated Tie-2 signaling in CSC pathophysiology. DesignA retrospective interventional multicenter case series. MethodsWe included patients with chronic CSC (persistent or recurrent SRF for ≥6 months) who received at least one faricimab 6mg injection between January 1 2022 and April 1 2024. Study sites included Massachusetts Eye and Ear and University of California San Francisco. Patients with evidence of a choroidal neovascular membrane on color photos, optical coherence tomography (OCT) and/or fluorescein angiography were excluded. 16 eyes (15 patients) met the inclusion criteria. The median central macular thickness at each visit from 52 weeks before to 52 weeks after the first faricimab injection was calculated using automated Heidelberg Spectralis ETDRS subfield measurements. ResultsPrior to treatment with faricimab, CSC had been diagnosed a median of 4.1 years (range 0.9–8) earlier and SRF (and intraretinal fluid [IRF] in a subset) had been continuously present for a median of 30 weeks (range 9–257). Decreases in macular thickness were observed in 14/16 eyes after the first faricimab injection and in 14/16 eyes in the full follow-up period compared with prior, 10 of which experienced complete resolution of SRF following the start of the first series of injections at a median of 4 weeks (range 2—25). One eye worsened after the second injection. The median improvement in macular thickness was 40μm [range -3 to 89.5] (P=0.0007). Upon review of OCT images, reductions in macular thickness were consistent with reductions in SRF and/or IRF. Visual acuity improved by 2 lines or more in 6/16 eyes. ConclusionsIn a retrospective case series of patients with chronic CSC and longstanding SRF, we observed improvement in macular thickness after intravitreal faricimab. While the small number of patients and variable natural history of CSC preclude definitive conclusions, a randomized controlled trial seems warranted.

#2114 TIE2 activation promotes vascular protection of tubules to reduce PDGFB driven tubulointerstitial fibrosis in experimental chronic kidney disease

Abstract Background and Aims The relevance of endothelial dysfunction for tubulointerstitial fibrosis in chronic kidney disease (CKD) is poorly understood. Here, we utilize gain- and loss-of-function experiments to investigate the role of endothelial tyrosine kinase TIE2 for vascular-mediated protection in experimental CKD. Method TIE2 activation was achieved using a human TIE2-activating antibody (ABX), or conditional endothelial knockout of vascular endothelial protein tyrosine phosphatase (Veptp). Conditional endothelial knockout of Tie2 served as a TIE2-incompetent signalling model. CKD was induced by unilateral ureter obstruction (UUO). Results ABX treated and Veptp knockout mice were significantly protected from UUO-induced endothelial dysfunction, tubular injury, and tubulointerstitial fibrosis. Capillary density was preserved as well as fenestrations of peritubular capillaries. In contrast, Tie2 knockout mice had aggravated disease. Mechanistically, the link between endothelial dysfunction and tubulointerstitial fibrosis was tubular expression of PDGFB, which was reduced by ABX treatment and Veptp knockout. A role for endothelial-to-mesenchymal transition was ruled out by careful lineage tracing of endothelial cells and reporter for mesenchymal cells. UUO-induced tubulointerstitial fibrosis was reduced in Pdgfb knockout mice, supporting causality of PDGFB in mesenchymal activation and fibrosis. ABX treated and Veptp knockout mice had significantly lower UUO-induced PDGFB levels. Conclusion Our data supports a key role of the endothelium and TIE2 signalling in CKD. We introduce ABX as a new therapeutic approach in CKD and demonstrate the sequence of events from endothelial dysfunction to tubulointerstitial fibrosis.

TIE1 promotes cervical cancer progression via Basigin-matrix metalloproteinase axis.

Background: Tyrosine kinase with immunoglobulin and EGF-like domains 1 (TIE1) is known as an orphan receptor prominently expressed in endothelial cells and participates in angiogenesis by regulating TIE2 activity. Our previous study demonstrated elevated TIE1 expression in cervical cancer cells. However, the role of TIE1 in cervical cancer progression, metastasis and treatment remains elusive. Methods: Immunohistochemistry staining for TIE1 and Basigin was performed in 135 human cervical cancer tissues. Overexpressing vectors and siRNAs were used to manipulate gene expression in tumor cells. Colony formation, wound healing, and transwell assays were used to assess cervical cancer cell proliferation and migration in vitro. Subcutaneous xenograft tumor and lung metastasis mouse models were established to examine tumor growth and metastasis. Co-Immunoprecipitation and Mass Spectrometry were applied to explore the proteins binding to TIE1. Immunoprecipitation and immunofluorescence staining were used to verify the interaction between TIE1 and Basigin. Cycloheximide chase assay and MG132 treatment were conducted to analyze protein stability. Results: High TIE1 expression was associated with poor survival in cervical cancer patients. TIE1 overexpression promoted the proliferation, migration and invasion of cervical cancer cells in vitro, as well as tumor growth and metastasis in vivo. In addition, Basigin, a transmembrane glycoprotein, was identified as a TIE1 binding protein, suggesting a pivotal role in matrix metalloproteinase regulation, angiogenesis, cell adhesion, and immune responses. Knockdown of Basigin or treatment with the Basigin inhibitor AC-73 reversed the tumor-promoting effect of TIE1 in vitro and in vivo. Furthermore, we found that TIE1 was able to interact with and stabilize the Basigin protein and stimulate the Basigin-matrix metalloproteinase axis. Conclusion: TIE1 expression in cervical cells exerts a tumor-promoting effect, which is at least in part dependent on its interaction with Basigin. These findings have revealed a TIE2-independent mechanism of TIE1, which may provide a new biomarker for cervical cancer progression, and a potential therapeutic target for the treatment of cervical cancer patients.

Type I Interferons induce endothelial destabilization in Systemic Lupus Erythematosus in a Tie2-dependent manner.

Endothelial cell (EC) dysfunction is a hallmark of Systemic Lupus Erythematosus (SLE) and Tie2 is a receptor essential for vascular stability. Inflammatory processes promote inhibition of Tie2 homeostatic activation, driving vascular dysfunction. In this work we determined whether type I Interferons (IFN) induce Tie2 signalling-mediated endothelial dysfunction in patients with SLE. Serum levels of Angiopoietin (Ang)-1, Ang-2 and soluble (s)Tie1 in patients with SLE and healthy controls were measured by ELISA. Monocytes from patients with SLE and Human Umbilical Vein EC (HUVEC) were stimulated with IFN-α, IFN-β (1000 I.U.) or SLE serum (20%). mRNA and protein expression, phosphorylation and translocation were determined by quantitative PCR, ELISA, Western Blot, flow cytometry and confocal microscopy. Viability and angiogenic capacity were determined by calcein and tube formation assays. We found that sTie1 and Ang-2 serum levels were increased and Ang-1 decreased in patients with SLE and were associated with clinical characteristics. Type I IFN significantly decreased Ang-1 and increased Ang-2 in monocytes from patients with SLE. Type I IFN increased sTie1 and Ang-2 secretion and reduced Tie2 activation in HUVEC. Functionally, type I IFN significantly reduced EC viability and impaired angiogenesis in a Tie2 signalling-dependent manner. Finally, SLE serum increased Ang-2 and sTie1 secretion and significantly decreased tube formation. Importantly, Tie1 and IFNAR1 knockdown reversed these effects in tube formation. Overall, type I IFN play an important role in the stability of EC by inhibiting Tie2 signalling, suggesting that these processes may be implicated in the cardiovascular events observed in patients with SLE.

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.

Activation of Angiopoietin-Tie2 Signaling Protects the Kidney from Ischemic Injury by Modulation of Endothelial-Specific Pathways.

Ischemia-reperfusion AKI (IR-AKI) is common and causes significant morbidity. Effective treatments are lacking. However, preclinical studies suggest that inhibition of angiopoietin-Tie2 vascular signaling promotes injury, whereas activation of Tie2 is protective. We show that kidney ischemia leads to increased levels of the endothelial-specific phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP; PTPRB), which inactivates Tie2. Activation of Tie2 through VE-PTP deletion, or delivery of a novel angiopoietin mimetic (Hepta-ANG1), abrogated IR-AKI in mice. Single-cell RNAseq analysis showed Tie2 activation promotes increased Entpd1 expression, downregulation of FOXO1 target genes in the kidney vasculature, and emergence of a new subpopulation of glomerular endothelial cells. Our data provide a molecular basis and identify a candidate therapeutic to improve endothelial integrity and kidney function after IR-AKI. Ischemia-reperfusion AKI (IR-AKI) is estimated to affect 2%-7% of all hospitalized patients. The significant morbidity and mortality associated with AKI indicates urgent need for effective treatments. Previous studies have shown activation of the vascular angiopoietin-Tie2 tyrosine kinase signaling pathway abrogates ischemia-reperfusion injury (IRI). We extended previous studies to (1) determine the molecular mechanism(s) underlying kidney injury and protection related to decreased or increased activation of Tie2, respectively, and (2) to test the hypothesis that deletion of the Tie2 inhibitory phosphatase vascular endothelial protein tyrosine phosphatase (VE-PTP) or injection of a new angiopoietin mimetic protects the kidney from IRI by common molecular mechanism(s). Bilateral IR-AKI was performed in VE-PTP wild-type or knockout mice and in C57BL/6J mice treated with Hepta-ANG1 or vehicle. Histologic, immunostaining, and single-cell RNA sequencing analyses were performed. The phosphatase VE-PTP, which negatively regulates the angiopoietin-Tie2 pathway, was upregulated in kidney endothelial cells after IRI, and genetic deletion of VE-PTP in mice protected the kidney from IR-AKI. Injection of Hepta-ANG1 potently activated Tie2 and protected the mouse kidney from IRI. Single-cell RNAseq analysis of kidneys from Hepta-ANG1-treated and vehicle-treated mice identified endothelial-specific gene signatures and emergence of a new glomerular endothelial subpopulation associated with improved kidney function. Overlap was found between endothelial-specific genes upregulated by Hepta-ANG1 treatment and those downregulated in HUVECs with constitutive FOXO1 activation, including Entpd1 / ENTPD1 that modulates purinergic receptor signaling. Our data support a key role of the endothelium in the development of IR-AKI, introduce Hepta-ANG1 as a putative new therapeutic biologic, and report a model to explain how IRI reduces Tie2 signaling and how Tie2 activation protects the kidney. This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_05_23_JSN_Ang_EP23_052323.mp3.

Shear stress control of vascular leaks and atheromas through Tie2 activation by VE‐PTP sequestration

Vascular endothelial protein tyrosine phosphatase (VE‐PTP) influences endothelial barrier function by regulating the activation of tyrosine kinase receptor Tie2. We determined whether this action is linked to the development of atherosclerosis by examining the influence of arterial shear stress on VE‐PTP, Tie2 activation, plasma leakage, and atherogenesis. We found that exposure to high average shear stress led to downstream polarization and endocytosis of VE‐PTP accompanied by Tie2 activation at cell junctions. In aortic regions with disturbed flow, VE‐PTP was not redistributed away from Tie2. Endothelial cells exposed to high shear stress had greater Tie2 activation and less macromolecular permeability than regions with disturbed flow. Deleting endothelial VE‐PTP in VE‐PTPiECKO mice increased Tie2 activation and reduced plasma leakage in atheroprone regions. ApoE−/− mice bred with VE‐PTPiECKO mice had less plasma leakage and fewer atheromas on a high‐fat diet. Pharmacologic inhibition of VE‐PTP by AKB‐9785 had similar anti‐atherogenic effects. Together, the findings identify VE‐PTP as a novel target for suppression of atherosclerosis.

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.

Anti-angiogenic collagen IV-derived peptide target engagement with αvβ3 and α5β1 in ocular neovascularization models

AXT107, a collagen-derived peptide that binds integrins αvβ3 and α5β1 with high affinity, suppresses vascular endothelial growth factor (VEGF) signaling, promotes angiopoietin 2-induced Tie2 activation, and suppresses neovascularization (NV) and vascular leakage. Immunohistochemical staining for αvβ3 and α5β1 was markedly increased in NV compared with normal retinal vessels. After intravitreous injection of AXT107, there was no staining with an anti-AXT107 antibody on normal vessels but robust staining of NV that co-localized with αvβ3 and α5β1. Likewise, after intravitreous injection, fluorescein amidite-labeled AXT107 co-localized with αvβ3 and α5β1 on NV but not normal vessels. AXT107 also co-localized with αv and α5 at cell-cell junctions of human umbilical vein endothelial cells (HUVECs). AXT107-integrin binding was demonstrated by exvivo cross-linking/pull-down experiments. These data support the hypothesis that AXT107 therapeutic activity is mediated through binding αvβ3 and α5β1 which are markedly upregulated on endothelial cells in NV providing selective targeting of diseased vessels which has therapeutic and safety benefits.

Prothrombotic Transformation of the Endothelium Is Markedly Augmented By Cytokine Priming and Inhibited By Tie2 Activation

The endothelium is an intrinsically anticoagulant surface that upon viral or bacterial infection promotes thrombosis via the upregulation of pro-coagulatory mechanisms and loss of anticoagulant properties. The COVID-19 pandemic has underscored the association between respiratory infection and thrombosis by highlighting the importance of host cytokine responses in mediating systemic vasculopathy. Immune activation within the alveolar-capillary unit precedes systemic pathogen spread and induces marked increases in plasma cytokine levels prior to pathogen circulation. We therefore tested the hypothesis that cytokine exposure primes ECs toward a pro-coagulatory state that is subsequently activated by pathogen-associated molecular patterns (PAMPs). In initial studies, human ECs were exposed to cytokines elevated in the setting of pneumonia including TNFα, IFNα, IFNβ, IFNγ, IL-1β, IL-6, IL-10, and MCP for 24 hours prior to induction with poly(I:C), a viral PAMP mimetic and TLR3 agonist, and subsequently evaluated for thrombin generation in a plasma-based assay. Pre-incubation of ECs with several candidate cytokines enhanced responses to poly(I:C) by 10-20-fold. Focusing on the antiviral type-II interferon cytokine IFNγ, we found that its pre-incubation with ECs enhanced thrombin generation induced by poly(I:C) in a dose-dependent manner. IFNγ followed by poly(I:C) also enhanced factor VIIa-dependent factor Xa (FXa) generation, implicating upregulation of TF activity. SARS-CoV-2 nucleocapsid and spike proteins showed little prothrombotic effects in ECs by themselves. However, following incubation of ECs with IFNγ, both proteins stimulated FXa generation. These observations were not limited to viral PAMPs, as pre-incubation with IFNγ enhanced thrombin and FXa generation in response to lipopolysaccharide (LPS), a bacterial PAMP. Exposure of ECs to IFNγ also resulted in a reduction in total thrombomodulin, even prior to PAMP exposure. To better replicate the alveolar-capillary functional unit, we used an alveolus-on-chip model. The chip includes primary pulmonary microvascular ECs cultured under flow conditions in a vascular channel and small airway epithelial cells in an alveolus channel. The two channels are separated by a collagen matrix and the chip is subject to cyclic stretch to mimic breathing. Exposure of pneumocytes in the pulmonary channel to IFNγ and either poly(I:C) or LPS resulted in decreased production of thrombomodulin in the vascular channel, validating the monoculture results. One strategy for preventing the prothrombotic transformation of ECs is to stimulate cytoprotective endothelial pathways. Tie2 is a cytoprotective receptor found primarily on ECs. Incubation of ECs with the Tie2 ligand angiopoietin-1 (Angpt-1) significantly reversed the marked thrombin generation observed with IFNγ-priming followed by poly(I:C) exposure. Angpt-1 did not directly inhibit thrombin or FXa generation, as measured in standard thrombin time and aPTT assays. However, exposure to Angpt-1 did reverse depletion of protein C generation induced by poly(I:C) or LPS. Angpt-1 also reversed thrombomodulin reduction, providing an explanation for the ability of Angpt-1 to suppress cytokine/PAMP-induced prothrombotic changes on the ECs. To test the effect of Tie2 activation on thrombus formation in vivo, we used an LPS model since LPS was the most thrombotic of the PAMPs that we tested in mice. We used the small molecule [4-[(2S)-2-[[(2S)-2-(methoxycarbonylamino)-3-phenylpropanoyl]amino]-2-(2-thiophen-2-yl-1,3-thiazol-4-yl)ethyl]phenyl]sulfamic acid to promote Tie2 activation. Infusion of this Tie2 activating compound reduced LPS-induced accumulation of fibrin in both lung and liver. Taken together, these studies show that cytokines prime ECs for prothrombotic changes in response to PAMP exposure. Decreased thrombomodulin expression upon cytokine priming with resultant reduction in activated protein C production is essential to these prothrombotic changes. Tie2 activation inhibits the prothrombotic transformation and represents a potential strategy for reversing thrombosis associated with respiratory infection.

Genetic and Pharmacological Activation of Endothelial Tie2 in Experimental CKD Alleviate Endothelial Injury and Reduces Tubulointerstitial Fibrosis by Decreasing Tubular PDGFB

Genetic and Pharmacological Activation of Endothelial Tie2 in Experimental CKD Alleviate Endothelial Injury and Reduces Tubulointerstitial Fibrosis by Decreasing Tubular PDGFB

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.

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.

Tie2 Activation via VE-PTP Inhibition With Razuprotafib as an Adjunct to Latanoprost in Patients With Open Angle Glaucoma or Ocular Hypertension.

PurposeTo evaluate the ocular hypotensive efficacy and safety of razuprotafib, a novel Tie2 activator, when used as an adjunct to latanoprost in patients with open-angle glaucoma (OAG) or ocular hypertension (OHT).MethodsSubjects with OAG or OHT and an unmedicated IOP from ≥22 mm Hg to <36 mm Hg were randomized to one of three treatment arms: razuprotafib every day (QD) + latanoprost; razuprotafib twice daily (BID) + latanoprost; or latanoprost monotherapy. The primary endpoint was change in mean diurnal IOP from baseline at day 28.ResultsA total of 194 subjects were randomized, and 193 (99.5%) completed the study. Razuprotafib BID + latanoprost resulted in a significantly larger reduction in diurnal IOP than latanoprost alone (7.95 ± 0.26 mmHg vs. 7.04 ± 0.26 mm Hg, P < 0.05). A smaller improvement was observed after 14 days of treatment (7.62 ± 0.26 mm Hg vs. 7.03 ± 0.26 mm Hg, P = 0.11). Razuprotafib QD dosing did not demonstrate additional IOP lowering compared to latanoprost alone. Conjunctival hyperemia on Day 28 increased by 1.1 units on the four-point Efron scale two hours post dose from a baseline value of 0.6 units, and decreased thereafter.ConclusionsTopical ocular razuprotafib as an adjunct to latanoprost therapy was well tolerated and significantly reduced IOP in patients with OAG/OHT.Translational RelevanceThese data support the IOP lowering efficacy of targeting Tie2 activation in Schlemm's canal in the relevant patient population.

Patho-Mechanisms for Hemorrhage/Sepsis-Induced Indirect Acute Respiratory Distress Syndrome: A Role for Lung TIE1 and Its Regulation by Neutrophils.

Severe hemorrhage (Hem) has been shown to be causal for the development of extra-pulmonary/indirect acute respiratory distress syndrome (iARDS) and is associated with severe endothelial cell (EC) injury. EC growth factors, Angiopoietin (Ang)-1 and -2, maintain vascular homeostasis via tightly regulated competitive interaction with the tyrosine kinase receptor, Tie2, expressed on ECs. Since it has been reported that the orphan receptor, Tie1, may be able to play a role in Ang:Tie2 signaling; we chose to examine Tie1's capacity to alter the lung Ang:Tie2 interaction in response to the sequential insults of shock/sepsis (cecal ligation and puncture [CLP]), culminating in iARDS. Male mice were subjected to Hem alone or sequential Hem followed 24 hours later by CLP that induces iARDS. Changes in lung and/or plasma levels of Tie1, Tie2, Ang-1, Ang-2, various systemic cytokine/chemokines and indices of lung injury/inflammation were then determined. The role of Tie1 was established by intravenous administration of Tie1 specific or control siRNA at 1 h post-Hem. Alternatively, the contribution of neutrophils was assessed by pre-treating mice with anti-neutrophil antibody depletion 48 h prior to Hem. Lung tissue levels of Tie1 expression elevated over the first 6 to 24 h post-Hem alone. Subsequently, we found that treatment of Hem/CLP mice with Tie1-specific siRNA not only decreased Tie1 expression in lung tissue compared to control siRNA, but, suppressed the rise in lung inflammatory cytokines, lung MPO and the rise in lung protein leak. Finally, much as we have previously shown that neutrophil interaction with resident pulmonary vascular ECs contribute significantly to Ang-2 release and EC dysfunction, central to the development of iARDS. Here, we report that depletion of neutrophils also decreased lung tissue Tie1 expression and increased Tie2 activation in Hem/CLP mice. Together, these data imply that shock-induced increased expression of Tie1 can contribute to EC activation by inhibiting Ang:Tie2 interaction, culminating in EC dysfunction and the development of iARDS.

Structural insights into the clustering and activation of Tie2 receptor mediated by Tie2 agonistic antibody

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.

Tie2 activation protects against prothrombotic endothelial dysfunction in COVID-19.

Endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. Constitutively, the endothelial surface is anticoagulant, a property maintained at least in part via signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and alterations in the Tie2/angiopoietin axis. Primary HUVECs treated with plasma from patients with severe COVID-19 upregulated the expression of thromboinflammatory genes, inhibited the expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. Lung autopsies from patients with COVID-19 demonstrated a prothrombotic endothelial signature. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity, and the highest levels were associated with worse survival. These data highlight the disruption of Tie2/angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Our findings provide rationale for current trials of Tie2-activating therapy with AKB-9778 in COVID-19.

Targeting the Angiopoietin/Tie Pathway: Prospects for Treatment of Retinal and Respiratory Disorders.

Anti-angiogenic approaches have significantly advanced the treatment of vascular-related pathologies. The ephemeral outcome and known side effects of the current vascular endothelial growth factor (VEGF)-based anti-angiogenic treatments have intensified research on other growth factors. The angiopoietin/Tie (Ang/Tie) family has an established role in vascular physiology and regulates angiogenesis, vascular permeability, and inflammatory responses. The Ang/Tie family consists of angiopoietins 1–4, their receptors, tie1 and 2 and the vascular endothelial-protein tyrosine phosphatase (VE-PTP). Modulation of Tie2 activation has provided a promising outcome in preclinical models and has led to clinical trials of Ang/Tie-targeting drug candidates for retinal disorders. Although less is known about the role of Ang/Tie in pulmonary disorders, several studies have revealed great potential of the Ang/Tie family members as drug targets for pulmonary vascular disorders as well. In this review, we summarize the functions of the Ang/Tie pathway in retinal and pulmonary vascular physiology and relevant disorders and highlight promising drug candidates targeting this pathway currently being or expected to be under clinical evaluation for retinal and pulmonary vascular disorders.

The in vivo disposition of subcutaneous injected 14C-razuprotafib (14C-AKB-9778), a sulphamic acid phosphatase inhibitor, in nonclinical species and human

The disposition of radioactivity following subcutaneous 14C-razuprotafib, a Tie2 activator, was explored in multiple species. The absorption and clearance of razuprotafib and total radioactivity in human plasma are rapid and pharmacokinetics support razuprotafib as primary circulating component. Radioactivity is distributed greater to human plasma than whole blood (B:P = 0.36). In pigmented rats, radioactivity distributes to whole-body tissues rapidly and, within 24 h, is localised to elimination pathway end organs and injection site. Overall recovery of radioactivity across species is >93%, with the majority recovered within 24–48 h, and >80% in faeces. The CYP2C8 enzyme contributes significantly to razuprotafib metabolism. A hydrolysis product of razuprotafib (m/z− 380) is the main component in rat plasma at 2 h (49% peak area radioactivity), while razuprotafib (m/z− 585) is the main component in plasma for dog (58%), monkey (99.3%), and human (100%). Razuprotafib is present in dog, monkey, and human faeces, with the greatest percentage of radioactivity as metabolites. The major metabolite (>25%) in monkey and human, m/z− 633, is an S-methylated oxidised derivative of razuprotafib and is localised in faeces. Overall disposition of 14C-razuprotafib in human is best modelled by monkey over lower order species.