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.

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.

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.

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.

Mechanism of insulin sensitization by BMOV (bis maltolato oxo vanadium); unliganded vanadium (VO4) as the active component.

Organovanadium compounds have been shown to be insulin sensitizers in vitro and in vivo. One potential biochemical mechanism for insulin sensitization by these compounds is that they inhibit protein tyrosine phosphatases (PTPs) that negatively regulate insulin receptor activation and signaling. In this study, bismaltolato oxovanadium (BMOV), a potent insulin sensitizer, was shown to be a reversible, competitive phosphatase inhibitor that inhibited phosphatase activity in cultured cells and enhanced insulin receptor activation in vivo. NMR and X-ray crystallographic studies of the interaction of BMOV with two different phosphatases, HCPTPA (human low molecular weight cytoplasmic protein tyrosine phosphatase) and PTP1B (protein tyrosine phosphatase 1B), demonstrated uncomplexed vanadium (VO(4)) in the active site. Taken together, these findings support phosphatase inhibition as a mechanism for insulin sensitization by BMOV and other organovanadium compounds and strongly suggest that uncomplexed vanadium is the active component of these compounds.

Determination of endogenous extracellular signal-regulated protein kinase by microchip capillary electrophoresis.

The application of microchip capillary electrophoresis (CE) to the assay of extracellular signal-regulated protein kinase (ERK) is presented. In this assay, ERK catalyzes the transfer of gamma-phosphate from adenosine 5(')-triphosphate to the threonine residue of a fluorescently labeled nonapeptide (APRTPGGRR), and the phosphorylated and nonphosphorylated peptides were detected by fluorescence. The phosphorylated and nonphosphorylated peptides and the internal standard were separated within 20s, and the increase in magnitude of the phosphorylated peptide peak was monitored to assess ERK activity. ERK reactions were prepared off-chip and analyzed on a single-lane glass microchip fabricated by standard methods. It was demonstrated that microchip CE could be used to measure endogenous amounts of ERK by spiking known concentrations of recombinant ERK2 into the lysates of serum-starved human umbilical vein endothelial cells (HUVEC) and recovering between 90 and 100% for all samples. Endogenous ERK activity was determined by microchip where HUVEC were stimulated with 500pM vascular endothelial growth factor (VEGF) at different times before cell lysis. The results showed a transient VEGF-mediated ERK activation that peaked at 10min, which was consistent with previous reports using conventional techniques. The microchip assay provided a rapid, accurate, and precise alternative to conventional methods of determining endogenous ERK activity.

Neovascularization in intimal hyperplasia is associated with vein graft failure after coronary artery bypass surgery.

Vein graft atherosclerosis is the major limitation of arterial bypass surgery. This study was carried out to determine if the number of microvessels per area of intimal hyperplasia correlated with vein graft disease. Vein grafts (n=24, graft age range 2-19 years) were taken from 22 patients undergoing redo-coronary artery bypass surgery. Mean age of the patients was 68 +/- 9 years; 92% were males. Samples were divided into three groups (n=8 per group): in group I segments were from grafts without angiographic or histologic disease, in groups II and III segments were from grafts with significant angiographic stenosis, without (group II) and with (group III) atheroma. Intimal hyperplasia was identified by Masson staining, morphometric analysis was performed with NIH image analysis software. Microvessels in the intimal hyperplasia were identified using immunohistochemical techniques. Significance was determined by single-factor ANOVA p < 0.05. The mean area of intimal hyperplasia was similar in groups I and II at 1.06 +/- 0.25 mm2 and 0.97 +/- 0.37 mm2, respectively. The extent of intimal hyperplasia was significantly greater in group III, 1.70 +/- 0.62 mm2 (p < 0.01). In group I, the microvessel count in the intimal hyperplasia was 5.62 +/- 3.89 vessels/mm2, while in group III it was 15.26 +/- 3.66/mm2 (p < 0.01 versus group I). Interestingly the number of microvessels per area of intimal hyperplasia in group II was similar to that in group III). In this study, the extent of neovascularization in intimal hyperplasia correlated with stenoses in human vein grafts. Strategies designed to limit neovascularization in intimal hyperplasia may lead to novel therapies to prevent vein graft failure.

Deletion of the carboxyl terminus of Tie2 enhances kinase activity, signaling, and function. Evidence for an autoinhibitory mechanism.

Tie2 is an endothelial receptor tyrosine kinase that is required for both embryonic vascular development and tumor angiogenesis. There is considerable interest in understanding the mechanisms of Tie2 activation for therapeutic purposes. The recent solution of the Tie2 crystal structure suggests that Tie2 activity is autoinhibited by its carboxyl terminus. Here we investigated the role of the C tail in Tie2 activation, signaling, and function both in vitro and in vivo by deleting the C terminus of Tie2 (Delta CT). Compared to wild type Tie2, in vitro autophosphorylation and kinase activity were significantly enhanced by the Delta CT mutation. In NIH 3T3 cells expressing chimeric Tie2 receptors, both basal and ligand-induced tyrosine phosphorylation were markedly enhanced compared to wild type in several independent clones of Tie2-Delta CT. Moreover, the Delta CT mutation enhanced basal and ligand-dependent activation of Akt and extracellular signal-regulated kinase. Enhanced Akt activation correlated with significant inhibition of staurosporine-induced apoptosis. These findings demonstrate that the Tie2 C tail performs a novel negative regulatory role in Tie2 signaling and function, and they provide important insights into the mechanisms by which the Tie2 kinase is activated.

The endothelial receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt to inhibit apoptosis.

Tie1 is an orphan receptor tyrosine kinase that is expressed almost exclusively in endothelial cells and that is required for normal embryonic vascular development. Genetic studies suggest that Tie1 promotes endothelial cell survival, but other studies have suggested that the Tie1 kinase has little to no activity, and Tie1-mediated signaling pathways are unknown. To begin to study Tie1 signaling, a recombinant glutathione S-transferase (GST)-Tie1 kinase fusion protein was produced in insect cells and found to be autophosphorylated in vitro. GST-Tie1 but not a kinase-inactive mutant associated with a recombinant p85 SH2 domain protein in vitro, suggesting that Tie1 might signal through phosphatidylinositol (PI) 3-kinase. To study Tie1 signaling in a cellular context, a c-fms-Tie1 chimeric receptor (fTie1) was expressed in NIH 3T3 cells. Ligand stimulation of fTie1 resulted in Tie1 autophosphorylation and downstream activation of PI 3-kinase and Akt. Stimulation of fTie1-expressing cells potently inhibited UV irradiation-induced apoptosis in a PI 3-kinase-dependent manner. Moreover, both Akt phosphorylation and inhibition of apoptosis were abrogated by mutation of tyrosine 1113 to phenylalanine, suggesting that this residue is an important PI 3-kinase binding site. These findings are the first biochemical demonstration of a signal transduction pathway and corresponding cellular function for Tie1, and the antiapoptotic effect of Tie1 is consistent with the results of previous genetic studies.