Nicholas P.J. Brindle

Angiogenesis and the atherosclerotic carotid plaque: An association between symptomatology and plaque morphology

Purpose: Symptomatic carotid disease resulting from generation of thromboemboli has been associated with plaque instability and intraplaque hemorrhage. These features of the lesion could be influenced by the fragility and position of neovessels within the plaque. The purpose of this study was to determine whether any association exists between neovessel density, position, morphology, and thromboembolic sequelae. Methods: Carotid endarterectomy samples were collected from 15 asymptomatic patients with greater than 80% stenoses and from 13 highly symptomatic patients who had suffered ipsilateral carotid stenotic events within 1 month of surgery. Both groups were matched for gender, age, risk factors, degree of carotid artery stenosis, and plaque size. Samples were stained with hematoxylin/eosin and van Geison. Immunohistochemistry was performed by using an endothelial specific antibody to CD31. Plaques were assessed for histologic characteristics, and neovessels were counted and characterized by size, site, and shape. Results: There were significantly more neovessels in plaques (P < .00001) and fibrous caps (P < .0001) in symptomatic compared with asymptomatic plaques. Neovessels in symptomatic plaques were larger (P < .004) and more irregular. There was a significant increase in plaque necrosis and rupture in symptomatic plaques. Plaque hemorrhage and rupture were associated with more neovessels within the plaque (P < .017, P < .001) and within the fibrous cap (P < .046, P < .004). Patients with preoperative and intraoperative embolization had significantly more plaque and fibrous cap neovessels (P < .025, P < .001). Conclusion: Symptomatic carotid disease is associated with increased neovascularization within the atherosclerotic plaque and fibrous cap. These vessels are larger and more irregular and may contribute to plaque instability and the onset of thromboembolic sequelae. (J Vasc Surg 1999;30:261-8.)

Signaling and Functions of Angiopoietin-1 in Vascular Protection

Angiopoietin-1 (Ang1) has powerful vascular protective effects: suppressing plasma leakage, inhibiting vascular inflammation, and preventing endothelial death. Preclinical studies indicate that Ang1 may be therapeutically useful in a number of situations, including treatment of edema, endotoxemia, and transplant arteriosclerosis. However, the ligand has also been implicated in vessel remodeling, induction of angiogenesis and pulmonary hypertension, indicating that strategies to minimize any deleterious effects while optimizing vessel protection are likely to be needed. This review surveys the published data on vascular protective effects of Ang1 and highlights the therapeutic potential of this ligand, as well as possible limitations to its use. We also consider the data on Ang1 receptors and speculate on how to maximize therapeutic benefit by targeting the Tie receptors.

Multiple angiopoietin recombinant proteins activate the Tie1 receptor tyrosine kinase and promote its interaction with Tie2

The Tie1 receptor tyrosine kinase was isolated over a decade ago, but so far no ligand has been found to activate this receptor. Here, we have examined the potential of angiopoietins, ligands for the related Tie2 receptor, to mediate Tie1 activation. We show that a soluble Ang1 chimeric protein, COMP-Ang1, stimulates Tie1 phosphorylation in endothelial cells with similar kinetics and angiopoietin dose dependence when compared with Tie2. The phosphorylation of overexpressed Tie1 was weakly induced by COMP-Ang1 also in transfected cells that do not express Tie2. When cotransfected, Tie2 formed heteromeric complexes with Tie1, enhanced Tie1 activation, and induced phosphorylation of a kinase-inactive Tie1 in a ligand-dependent manner. Tie1 phosphorylation was also induced by native Ang1 and Ang4, although less efficiently than with COMP-Ang1. In conclusion, we show that Tie1 phosphorylation is induced by multiple angiopoietin proteins and that the activation is amplified via Tie2. These results should be important in dissecting the signal transduction pathways and biological functions of Tie1.

The Antiinflammatory Endothelial Tyrosine Kinase Tie2 Interacts With a Novel Nuclear Factor-κB Inhibitor ABIN-2

Abstract— Tie2 is a receptor tyrosine kinase expressed predominantly in endothelial cells and is essential for blood vessel formation and maintenance. The receptor has potent antiinflammatory effects on endothelial cells, suppressing vascular endothelial growth factor– and tumor necrosis factor–induced expression of leukocyte adhesion molecules and procoagulant tissue factor and inhibiting vascular leakage. To delineate the signaling pathways utilized by Tie2, we performed yeast two-hybrid screening of a human endothelial cell cDNA library and identified a novel protein interacting with the intracellular domain of the receptor. This protein was found to be human A20 binding inhibitor of NF-&kgr;B activation-2, ABIN-2, an inhibitor of NF-&kgr;B–mediated inflammatory gene expression. Coexpression of Tie2 and ABIN-2 in CHO cells confirmed the interaction occurs in mammalian cells. In contrast, Tie1 did not interact with ABIN-2 in the yeast two-hybrid system or mammalian cells. Deletion analysis identified the Tie2 binding motif to be encompassed between residues 171 and 272 in ABIN-2. Interaction was dependent on Tie2 autophosphorylation but ABIN-2 was not tyrosine phosphorylated by Tie2. Furthermore, in endothelial cells the interaction was stimulated by the Tie2 ligand angiopoietin-1. Expression of ABIN-2 deletion mutants in endothelial cells suppressed the ability of angiopoietin-1 to inhibit phorbol ester–stimulated NF-&kgr;B–dependent reporter gene activity. These findings provide the first direct link between Tie2 and a key regulator of inflammatory responses in endothelial cells. Interaction between Tie2 and ABIN-2 may be important in the vascular protective antiinflammatory actions of Tie2.

Regulated Proteolytic Processing of Tie1 Modulates Ligand Responsiveness of the Receptor-tyrosine Kinase Tie2

Regulated ectodomain shedding followed by intramembrane proteolysis has recently been recognized as important in cell signaling and for degradation of several type I transmembrane proteins. The receptor-tyrosine kinase Tie1 is known to undergo ectodomain cleavage generating a membrane-tethered endodomain. Here we show Tie1 is a substrate for regulated intramembrane proteolysis. After Tie1 ectodomain cleavage the newly formed 45-kDa endodomain undergoes additional proteolytic processing mediated by γ-secretase to generate an amino-terminal-truncated 42-kDa fragment that is subsequently degraded by proteasomal activity. This sequential processing occurs constitutively and is stimulated by phorbol ester and vascular endothelial growth factor. To assess the biological significance of regulated Tie1 processing, we analyzed its effects on angiopoietin signaling. Activation of ectodomain cleavage causes loss of phosphorylated Tie1 holoreceptor and generation of phosphorylated receptor fragments in the presence of cartilage oligomeric protein angiopoietin 1. A key function of γ-secretase is in preventing accumulation of these phosphorylated fragments. We also find that regulated Tie1 processing modulates ligand responsiveness of the Tie-1-associated receptor Tie2. Activation of Tie1 ectodomain cleavage increases cartilage oligomeric protein angiopoietin 1 activation of Tie2. This correlates with increased ability of Tie2 to bind ligand after shedding of the Tie1 extracellular domain. A similar enhancement of ligand activation of Tie2 is seen when Tie1 expression is suppressed by RNA interference. Together these data indicate that Tie1, via its extracellular domain, limits the ability of ligand to bind and activate Tie2. Furthermore the data suggest that regulated processing of Tie1 may be an important mechanism for controlling signaling by Tie2.

Vascular endothelial growth factor and angiopoietins in neurovascular regeneration and protection following stroke.

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) were originally identified as endothelial-specific ligands regulating key functions of the vasculature important in stroke. There is increasing evidence that these ligands also exert effects on neurons. Here we review the neuronal effects of VEGF and Ang1 and highlight their potential for therapeutic manipulation in stroke. VEGF stimulates angiogenesis whereas Ang1 suppresses leakage, inflammation and regression of microvessels. Expression of both ligands change dramatically in the brain in experimental stroke, correlating with increased vascular leakage and inflammation. In addition to vascular effects, VEGF can stimulate survival, migration and proliferation of neurons suggesting roles in neural protection and possible therapeutic applications, an idea supported by preclinical studies. Recent reports now demonstrate that Ang1 can also act directly on neurons and enhance neural repair. The realization that VEGF and Ang1 have effects on both neural and vascular compartments impacted by stroke provides new opportunities for therapeutic manipulation to promote neuroprotection and extend the thrombolytic window, as well as stimulating neurogenesis and revascularization.

Evidence for Heterotypic Interaction between the Receptor Tyrosine Kinases TIE-1 and TIE-2

The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membrane-localized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.

Effects of angiopoietins-1 and -2 on the receptor tyrosine kinase Tie2 are differentially regulated at the endothelial cell surface.

Angiopoietin-1 (Ang1) and Ang2 are ligands for the receptor tyrosine kinase Tie2. Structural data suggest that the two ligands bind Tie2 similarly. However, in endothelial cells Ang1 activates Tie2 whereas Ang2 can act as an apparent antagonist. In addition, each ligand exhibits distinct kinetics of release following binding. These observations suggest that additional factors influence function and binding of angiopoietins with receptors in the cellular context. Previous work has shown that Ang1 binding and activation of Tie2 are inhibited by Tie1, a related receptor that complexes with Tie2 in cells. In this study we have investigated binding of Ang1 and Ang2 to Tie2 in endothelial cells. In contrast to Ang1, binding of Ang2 to Tie2 was found to be not affected by Tie1. Neither PMA-induced Tie1 ectodomain cleavage nor suppression of Tie1 expression by siRNA affected the ability of Ang2 to bind Tie2. Analysis of the level of Tie1 co-immunoprecipitating with angiopoietin-bound Tie2 demonstrated that Ang2 can bind Tie2 in Tie2:Tie1 complexes whereas Ang1 preferentially binds non-complexed Tie2. Stimulation of Tie1 ectodomain cleavage did not increase the agonist activity of Ang2 for Tie2. Similarly, the Tie2-agonist activity of Ang2 was not affected by siRNA suppression of Tie1 expression. Consistent with previous reports, loss of Tie1 ectodomain enhanced the agonist activity of Ang1 for Tie2. Importantly, Ang2 was still able to antagonize the elevated Ang1-activation of Tie2 that occurs on Tie1 ectodomain loss. Together these data demonstrate that Ang1 and Ang2 bind differently to Tie2 at the cell surface and this is controlled by Tie1. This differential regulation of angiopoietin binding allows control of Tie2 activation response to Ang1 without affecting Ang2 agonist activity and maintains the ability of Ang2 to antagonize even the enhanced Ang1 activation of Tie2 that occurs on loss of Tie1 ectodomain. This provides a mechanism by which signalling through Tie2 can be modified by stimuli in the cellular microenvironment.

The endothelial receptor tyrosine kinase tie-1 is upregulated by hypoxia and vascular endothelial growth factor

The receptor tyrosine kinase tie‐1 is essential for angiogenesis where it appears to have a role in vessel maturation. Here we have examined the effects of hypoxia and vascular endothelial growth factor (VEGF) on the level of tie‐1 protein expressed in bovine aortic endothelial cells. Both hypoxia (2% O2) and VEGF were found to increase tie‐1 in a time‐dependent manner. Hypoxic induction was direct and effects of hypoxia and VEGF were not additive. Experiments with actinomycin D indicate that these activators regulate tie‐1 at the transcriptional level.

Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases

The ability of cells to respond appropriately to changes in their environment requires integration and cross-talk between relevant signalling pathways. The vascular endothelial growth factor (VEGF) and angiopoietin families of ligands are key regulators of blood vessel formation. VEGF binds to receptor tyrosine kinases of the VEGF-receptor family to activate signalling pathways leading to endothelial migration, proliferation and survival whereas the angiopoietins interact with the Tie receptor tyrosine kinases to control vessel stability, survival and maturation. Here we show that VEGF can also activate the angiopoietin receptor Tie2. Activation of human endothelial cells with VEGF caused a four-fold stimulation of tyrosine phosphorylation of Tie2. This stimulation was not due to VEGF-induction of Tie2 ligands as soluble ligand binding domain of Tie2 failed to inhibit VEGF activation of the receptor. Immunoprecipitation analysis demonstrated no physical interaction between VEGF receptors and Tie2. However Tie2 does interact with the related receptor tyrosine kinase Tie1 and this receptor was found to be essential for VEGF activation of Tie2. VEGF stimulated proteolytic cleavage of Tie1 generating a truncated Tie1 intracellular domain. Similarly, phorbol ester also both stimulated Tie1 truncation and activated Tie2 phosphorylation. Inhibition of Tie1 cleavage with the metalloprotease inhibitor TAPI-2 suppressed VEGF- and phorbol ester-induced phosphorylation of Tie2. Truncated Tie1 formed in response to VEGF was also found to be tyrosine phosphorylated and this was independent of Tie2, though Tie2 could enhance Tie1 intracellular domain phosphorylation. Together these data demonstrate that VEGF activates Tie2 via a mechanism involving proteolytic cleavage of the associated tyrosine kinase Tie1 leading to trans-phosphorylation of Tie2. This novel mechanism of receptor tyrosine kinase activation is likely to be important in integrating signalling between two of the key receptor groups regulating angiogenesis.