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.

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

Purpose: To 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). Methods: Subjects 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. Results: A 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. Conclusions: Topical ocular razuprotafib as an adjunct to latanoprost therapy was well tolerated and significantly reduced IOP in patients with OAG/OHT. Translational Relevance: These data support the IOP lowering efficacy of targeting Tie2 activation in Schlemm's canal in the relevant patient population.

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.

VE-PTP inhibition elicits eNOS phosphorylation to blunt endothelial dysfunction and hypertension in diabetes.

AIMS: Receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) dephosphorylates Tie-2 as well as CD31, VE-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2). The latter form a signal transduction complex that mediates the endothelial cell response to shear stress, including the activation of the endothelial nitric oxide (NO) synthase (eNOS). As VE-PTP expression is increased in diabetes, we investigated the consequences of VE-PTP inhibition (using AKB-9778) on blood pressure in diabetic patients and the role of VE-PTP in the regulation of eNOS activity and vascular reactivity. METHODS AND RESULTS: In diabetic patients AKB-9778 significantly lowered systolic and diastolic blood pressure. This could be linked to elevated NO production, as AKB increased NO generation by cultured endothelial cells and elicited the NOS inhibitor-sensitive relaxation of endothelium-intact rings of mouse aorta. At the molecular level, VE-PTP inhibition increased the phosphorylation of eNOS on Tyr81 and Ser1177 (human sequence). The PIEZO1 activator Yoda1, which was used to mimic the response to shear stress, also increased eNOS Tyr81 phosphorylation, an effect that was enhanced by VE-PTP inhibition. Two kinases, i.e. abelson-tyrosine protein kinase (ABL)1 and Src were identified as eNOS Tyr81 kinases as their inhibition and down-regulation significantly reduced the basal and Yoda1-induced tyrosine phosphorylation and activity of eNOS. VE-PTP, on the other hand, formed a complex with eNOS in endothelial cells and directly dephosphorylated eNOS Tyr81 in vitro. Finally, phosphorylation of eNOS on Tyr80 (murine sequence) was found to be reduced in diabetic mice and diabetes-induced endothelial dysfunction (isolated aortic rings) was blunted by VE-PTP inhibition. CONCLUSIONS: VE-PTP inhibition enhances eNOS activity to improve endothelial function and decrease blood pressure indirectly, through the activation of Tie-2 and the CD31/VE-cadherin/VEGFR2 complex, and directly by dephosphorylating eNOS Tyr81. VE-PTP inhibition, therefore, represents an attractive novel therapeutic option for diabetes-induced endothelial dysfunction and hypertension.

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

Purpose: Tyrosine 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. Methods: AKB-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. Results: AKB-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. Conclusions: This 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.

Controversial roles for dexamethasone in glioblastoma - Opportunities for novel vascular targeting therapies.

Glioblastoma is a highly aggressive and treatment resistant primary brain tumor. Features of glioblastoma include peritumoral cerebral edema, the major contributor to neurological impairment. Although the current clinical approach to edema management is administration of the synthetic corticoid dexamethasone, increasing evidence indicates numerous adverse effects of dexamethasone on glioblastoma burden at the molecular, cellular and clinical level. The contradictions of dexamethasone for glioblastoma and brain metastasis therapy are discussed in this article. Finally, alternative strategies for cerebrovascular edema therapy with vascular stabilizing, anti-permeability agents that are either approved or in clinical trials for diabetic retinopathy and macula edema, are addressed.

Context-dependent functions of angiopoietin 2 are determined by the endothelial phosphatase VEPTP.

The angiopoietin (ANGPT)-TIE2/TEK signaling pathway is essential for blood and lymphatic vascular homeostasis. ANGPT1 is a potent TIE2 activator, whereas ANGPT2 functions as a context-dependent agonist/antagonist. In disease, ANGPT2-mediated inhibition of TIE2 in blood vessels is linked to vascular leak, inflammation, and metastasis. Using conditional knockout studies in mice, we show TIE2 is predominantly activated by ANGPT1 in the cardiovascular system and by ANGPT2 in the lymphatic vasculature. Mechanisms underlying opposing actions of ANGPT2 in blood vs. lymphatic endothelium are poorly understood. Here we show the endothelial-specific phosphatase VEPTP (vascular endothelial protein tyrosine phosphatase) determines TIE2 response to ANGPT2. VEPTP is absent from lymphatic endothelium in mouse in vivo, permitting ANGPT2/TIE2-mediated lymphangiogenesis. Inhibition of VEPTP converts ANGPT2 into a potent TIE2 activator in blood endothelium. Our data support a model whereby VEPTP functions as a rheostat to modulate ANGPT2 ligand effect on TIE2.

Targeting Tie2 for Treatment of Diabetic Retinopathy and Diabetic Macular Edema.

Tie2 is a tyrosine kinase receptor located predominantly on vascular endothelial cells that plays a central role in vascular stability. Angiopoietin-1 (Angpt1), produced by perivascular cells, binds, clusters, and activates Tie2, leading to Tie2 autophosphorylation and downstream signaling. Activated Tie2 increases endothelial cell survival, adhesion, and cell junction integrity, thereby stabilizing the vasculature. Angiopoietin-2 (Angpt2) and vascular endothelial-protein tyrosine phosphatase (VE-PTP) are negative regulators increased by hypoxia; they inactivate Tie2, destabilizing the vasculature and increasing responsiveness to vascular endothelial growth factor (VEGF) and other inflammatory cytokines that stimulate vascular leakage and neovascularization. AKB-9778 is a small-molecule antagonist of VE-PTP which increases phosphorylation of Tie2 even in the presence of high Angpt2 levels. In preclinical studies, AKB-9778 reduced VEGF-induced leakage and ocular neovascularization (NV) and showed additive benefit when combined with VEGF suppression. In two clinical trials in diabetic macular edema (DME) patients, subcutaneous injections of AKB-9778 were safe and provided added benefit to VEGF suppression. Preliminary data suggest that AKB-9778 monotherapy improves diabetic retinopathy. These data suggest that Tie2 activation may be a valuable strategy to treat or prevent diabetic retinopathy.

Angiopoietin-2-induced blood-brain barrier compromise and increased stroke size are rescued by VE-PTP-dependent restoration of Tie2 signaling.

The homeostasis of the central nervous system is maintained by the blood-brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.

Targeting VE-PTP activates TIE2 and stabilizes the ocular vasculature.

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

Targeting protein tyrosine phosphatase to enhance insulin action for the potential treatment of diabetes.

The increasing prevalence of type 2 diabetes has sparked interest in the development of agents that treat and prevent the disease. Mounting evidence indicates that protein tyrosine phosphatase (PTP)1B negatively regulates insulin and leptin signaling making it a prime target for enhancing insulin sensitivity and controlling body mass. Despite intense efforts, development of orally bioavailable small-molecule PTP1B inhibitors has been a challenge. This review focuses on recent advances in the validation of PTP1B and in the development of approaches to modulate its activity.

Engineering the catalytic domain of human protein tyrosine phosphatase beta for structure-based drug discovery.

Protein tyrosine phosphatases (PTPs) play roles in many biological processes and are considered to be important targets for drug discovery. As inhibitor development has proven challenging, crystal structure-based design will be very helpful to advance inhibitor potency and selectivity. Successful application of protein crystallography to drug discovery heavily relies on high-quality crystal structures of the protein of interest complexed with pharmaceutically interesting ligands. It is very important to be able to produce protein-ligand crystals rapidly and reproducibly for as many ligands as necessary. This study details our efforts to engineer the catalytic domain of human protein tyrosine phosphatase beta (HPTPbeta-CD) with properties suitable for rapid-turnaround crystallography. Structures of apo HPTPbeta-CD and its complexes with several novel small-molecule inhibitors are presented here for the first time.

Design and synthesis of potent, non-peptidic inhibitors of HPTPbeta.

The sulfamic acid phosphotyrosine mimetic was coupled with a previously known malonate template to obtain highly selective and potent inhibitors of HPTPbeta. Potentially hydrolyzable malonate ester functionalities were replaced with 1,2,4-oxadiazoles without a significant effect on HPTPbeta potency.

1,2,3,4-Tetrahydroisoquinolinyl sulfamic acids as phosphatase PTP1B inhibitors.

High-throughput screening of the P&GP corporate repository against several protein tyrosine phosphatases identified the sulfamic acid moiety as potential phosphotyrosine mimetic. Incorporation of the sulfamic acid onto a 1,2,3,4-tetrahydroisoquinoline scaffold provided a promising starting point for PTP1B inhibitor design.

Efficacy of systemic administration of SDF-1 in a model of vascular insufficiency: support for an endothelium-dependent mechanism.

OBJECTIVE: Studies have reported that administration of stromal cell-derived factor-1 (SDF-1), the ligand for the G-protein coupled receptor CXCR4, increased collateral blood flow in a mouse model of vascular insufficiency via recruitment of endothelial precursor cells (EPC). The present study investigated the contribution of mature endothelial cells in the actions of SDF-1. METHODS: The regulation of SDF-1 and CXCR4 was examined in the rat cornea cauterization (CC) and aortic ring (AR) model. The functional significance of the SDF-1/CXCR4 pathway was explored in cultured endothelial cells, the AR model, and on collateral blood flow in a rat model of vascular insufficiency. RESULTS: In the present study, the CXCR4 transcript was dramatically upregulated in the rat CC and AR explants, systems containing and lacking bone marrow-derived EPCs, respectively. Addition of AMD3100, a selective CXCR4 antagonist, had no effect on vessel growth in the AR alone, but completely inhibited SDF-1 mediated increases in vascular sprouting. In cultured endothelial cells, SDF-1 alone or in combination with vascular endothelial growth factor (VEGF) significantly enhanced cell survival and migration. Finally, systemic administration of SDF-1 in a rat model of arterial insufficiency enhanced collateral blood flow above vehicle control and equal to that of VEGF after 2 weeks of treatment. CONCLUSION: These studies support activation of the SDF-1/CXCR4 axis as a means to promote blood vessel growth and enhance collateral blood flow, at least in part, via direct effects on vascular endothelial cells.

A nonspecific phosphotyrosine phosphatase inhibitor, bis(maltolato)oxovanadium(IV), improves glucose tolerance and prevents diabetes in Zucker diabetic fatty rats.

The molecular basis of insulin resistance, a major risk factor for development of Type II diabetes, involves defective insulin signaling. Insulin-mediated signal transduction is negatively regulated by the phosphotyrosine phosphatase, PTP1B, and numerous studies have demonstrated that organo-vanadium compounds, which are nonselective phosphotyrosine phosphatase inhibitors, have insulin-mimetic properties. However, whether or not vanadium compounds can prevent the transition from insulin resistance to overt diabetes is unknown. We compared the ability of bis(maltolato)oxovanadium(IV) (BMOV), an orally bioavailable organo-vanadium compound, and rosiglitazone maleate (RSG), a known insulin sensitizer, to prevent development of diabetes in Zucker diabetic fatty (ZDF) rats. Treatment began at 6 weeks of age when animals are insulin resistant and hyperinsulinemic, but not yet hyperglycemic, and ended at 12 weeks of age, which is 4 weeks after ZDF rats typically develop overt diabetes. BMOV-treated ZDF rats did not develop hyperglycemia, showed significant improvement in insulin sensitivity, and retained normal pancreatic islet morphology and endocrine cell distribution, similar to RSG-treated animals. BMOV and RSG treatment also prevented the hyper-phagia and polydipsia present in untreated ZDF rats; however, BMOV-treated ZDF rats gained much less weight than did RSG-treated animals. Circulating levels of adiponectin decreased in untreated ZDF rats compared to lean controls, but these levels remained normal in BMOV-treated ZDF rats. In contrast, in RSG-treated ZDF rats, plasma adiponectin levels were nearly 4-fold higher than in lean control rats, primarily as a result of a large increase in the amount of low-molecular weight forms of adiponectin in circulation. These data demonstrate that phosphatase inhibition offers a new approach to diabetes prevention, one that may have advantages over current approaches.

Tyrosine phosphatases in vessel wall signaling.

Protein tyrosine phosphatases (PTPs) are critical regulators of cellular processes like migration, proliferation and differentiation that are involved in physiological and pathological vessel wall function. In this review, we summarize the biochemistry of this enzyme family, discuss the present knowledge concerning the identity and involvement of PTPs in vascular cells and in pathways of relevance to cardiovascular diseases. We also briefly introduce ongoing efforts to develop inhibitors of PTPs, and finally point to some opportunities for use of such agents in novel treatment strategies.

Preclinical models of human peripheral arterial occlusive disease: implications for investigation of therapeutic agents.

Peripheral arterial occlusive disease (PAOD) is now recognized as a combination of clinical syndromes that are associated with significant morbidity and mortality. The primary pathophysiology of PAOD is impaired perfusion to the lower extremity. Effective pharmacotherapy designed to increase perfusion in PAOD is lacking, and revascularization options are suboptimal. New and more efficacious therapies that improve blood flow are definitely needed, and thus designing, describing, and validating these new therapies in preclinical PAOD models will be essential. This study describes the various preclinical PAOD models presently in use, correlates the models to human PAOD, and reviews the available end points that can be used to detect a response to therapy.

Tyrosine phosphatase inhibition augments collateral blood flow in a rat model of peripheral vascular disease.

During embryonic development, the growth of blood vessels requires the coordinated activation of endothelial receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptor-2 (VEGFR-2) and Tie-2. Similarly, in adulthood, activation of endothelial RTKs has been shown to enhance development of the collateral circulation and improve blood flow to ischemic tissues. Recent evidence suggests that RTK activation is negatively regulated by protein tyrosine phosphatases (PTPs). In this study, we used the nonselective PTP inhibitor bis(maltolato)oxovanadium IV (BMOV) to test the potential efficacy of PTP inhibition as a means to enhance endothelial RTK activation and improve collateral blood flow. In cultured endothelial cells, pretreatment with BMOV augmented VEGFR-2 and Tie-2 tyrosine phosphorylation and enhanced VEGF- and angiopoietin-1-mediated cell survival. In rat aortic ring explants, BMOV enhanced vessel sprouting, a process that can be influenced by both VEGFR-2 and Tie-2 activation. Moreover, 2 wk of BMOV treatment in a rat model of peripheral vascular disease enhanced collateral blood flow similarly to VEGF, and after 4 wk, BMOV was superior to VEGF. Taken together, these studies provide evidence that PTPs are important regulators of endothelial RTK activation and for the first time demonstrate the potential utility of phosphatase inhibition as a means to promote collateral development and enhance collateral blood flow to ischemic tissue.

Functional significance of Tie2 signaling in the adult vasculature.

Abundant data now demonstrate that the growth of new blood vessels, termed angiogenesis, plays both pathological and beneficial roles in human disease. Based on these data, a tremendous effort has been undertaken to understand the molecular mechanisms that drive blood vessel growth in adult tissues. Tie2 recently was identified as a receptor tyrosine kinase expressed principally on vascular endothelium. Disrupting Tie2 function in mice resulted in embryonic lethality with defects in embryonic vasculature, suggesting a role in blood vessel maturation and maintenance. Based on these studies, we undertook a series of studies to probe the function of Tie2 in adult vasculature that will form the focus of this chapter. Consistent with a role in blood vessel growth in adult vasculature, Tie2 was upregulated and activated in the endothelium of rat ovary and in healing rat skin wounds, both areas of active angiogenesis. Moreover, Tie2 was upregulated in the endothelium of vascular "hot spots" in human breast cancer specimens. Surprisingly, Tie2 also was expressed and activated in the endothelium of all normal rat tissues examined, suggesting a role in maintenance of adult vasculature. To determine the functional role of Tie2 in tumor vasculature, a soluble Tie2 extracellular domain (ExTek) was designed that blocked the activation of Tie2 by its activating ligand, angiopoietin 1 (Ang1). Administration of recombinant ExTek protein or an ExTek adenovirus inhibited tumor growth and metastasis in rodent tumor models, demonstrating a functional role for Tie2 in pathological angiogenesis in adult tissues. To begin to understand the endothelial signaling pathways and cellular responses that mediate Tie2 function, we identified signaling molecules that are recruited to the activated, autophosphorylated Tie2 kinase domain. Two of these molecules, SHP2 and GRB2, are part of the pathway upstream of mitogen-activated protein kinase (MAPK) activation, a pathway that may be responsible for morphogenetic effects of Tie2 on endothelial cells. Another signaling molecule, p85, is responsible for recruitment of phosphatidylinositol 3 kinase (PI3-K) and activation of the Akt/PI3-K pathway. Akt/PI3-K has emerged as a critical pathway downstream of Tie2 that is necessary for cell survival effects as well as for chemotaxis, activation of endothelial nitric oxide synthase, and perhaps for anti-inflammatory effects of Tie2 activation. Taken together, these studies and many others demonstrate that the Tie2 pathway has important functions in adult tissues, in both quiescent vasculature and during angiogenesis, and help to validate the Tie2 pathway as a therapeutic target.