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.     

Molecular control of angiopoietin signalling

The angiopoietins act through the endothelial receptor tyrosine kinase Tie2 to regulate vessel maturation in angiogenesis and control quiescence and stability of established vessels. The activating ligand, Ang1 (angiopoietin-1), is constitutively expressed by perivascular cells, and the ability of endothelial cells to respond to the ligand is controlled at the level of the Ang1 receptor. This receptor interacts with the related protein Tie1 on the cell surface, and Tie1 inhibits Ang1 signalling through Tie2. The responsiveness of endothelium to Ang1 is determined by the relative levels of Tie2 and the inhibitory co-receptor Tie1 in the cells. Tie1 undergoes regulated ectodomain cleavage which is stimulated by a range of factors including VEGF (vascular endothelial growth factor), inflammatory cytokines and changes in shear stress. Ectodomain cleavage of Tie1 relieves inhibition of Tie2 and enhances Ang1 signalling. This mechanism regulates Ang1 signalling without requiring changes in the level of the ligand and allows Ang1 signalling to be co-ordinated with other signals in the cellular environment. Regulation of signalling at the level of receptor responsiveness may be an important adaptation in systems in which an activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.     

血管生成系及其相关蛋白的研究进展

对血管生成素及其相关蛋白的研究进展和分子生物学特性进行了综述,迄今,已经发现了4种血管生成素及某些血管生成素的同工蛋白或变异体。这些蛋白质具有共同的结构,即氨基端与分泌相关的信号肽,介导同源寡聚体形成的螺旋样结构域,和羧基末端介导配体活性的纤维蛋白原样结构域。血管生成素均可与Tie-2受体结合,但只有血管生成素1和4可激活Tie-2受体。血管生成素1和4为效应剂而血管生成素2和3为拮抗剂。对于血管生成素是否可与Tie-1结合尚不清楚。血管生成素相关蛋白具有公认的血管生成素的特征性结构,对其功能及其作用途径还不十分了解,有待于进一步研究。Tie-1和Tie-2的其他配体以及血管生成素相关蛋白类似物的特异受体有可能在今后得到阐明。     

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.     

Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell-matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell-cell contacts and the formation of homotypic Tie2-Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell-matrix and cell-cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization.     

Angiopoietin-1 and -2 coiled coil domains mediate distinct homo-oligomerization patterns, but fibrinogen-like domains mediate ligand activity.

Activity of endothelial Tie2 receptor tyrosine kinase is modulated by two naturally occurring, secreted ligands, angiopoietin-1 and -2, which have opposing effects on its phosphorylation. Receptor tyrosine kinase activation requires receptor dimerization/multimerization, which, for many receptors, is mediated by homo-oligomeric ligands binding to and bridging receptor molecules. We show here that angiopoietin-1 and -2 form distinct arrays of disulfide-linked homo-oligomeric complexes. Their mobilities on nonreducing gels suggest that angiopoietin-2 exists predominantly as a homodimer but also forms higher order multimers. In contrast, angiopoietin-1 forms some homotrimers, but predominantly exists in higher order multimers. These two structurally related, 60% homologous ligands are predominantly composed of an amino-terminal coiled coil domain and a carboxyl-terminal fibrinogen-like domain. We show that their distinct oligomerization patterns are determined by their coiled coil domains and, furthermore, that their coiled coil domains, but not their fibrinogen-like domains, are sufficient to mediate formation of disulfide-linked homo-oligomers. In contrast, the differential effects of these ligands on endothelial Tie2 phosphorylation is mediated by their fibrinogen-like domains. We conclude from these studies that the coiled coil and fibrinogen-like domains of the angiopoietins have distinct functions with the coiled coil domain mediating ligand homo-oligomerization and the fibrinogen-like domain mediating ligand activity.     

Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex

The Tie receptor tyrosine kinases and their angiopoietin (Ang) ligands play central roles in developmental and tumor-induced angiogenesis. Here we present the crystal structures of the Tie2 ligand-binding region alone and in complex with Ang2. In contrast to prediction, Tie2 contains not two but three immunoglobulin (Ig) domains, which fold together with the three epidermal growth factor domains into a compact, arrowhead-shaped structure. Ang2 binds at the tip of the arrowhead utilizing a lock-and-key mode of ligand recognition-unique for a receptor kinase-where two complementary surfaces interact with each other with no domain rearrangements and little conformational change in either molecule. Ang2-Tie2 recognition is similar to antibody-protein antigen recognition, including the location of the ligand-binding site within the Ig fold. Analysis of the structures and structure-based mutagenesis provide insight into the mechanism of receptor activation and support the hypothesis that all angiopoietins interact with Tie2 in a structurally similar manner.     

Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling

Angiopoietins are ligands of the Tie2 receptor that control angiogenic remodeling in a context-dependent manner. Tie signaling is involved in multiple steps of the angiogenic remodeling process during development, including destabilization of existing vessels, endothelial cell migration, tube formation and the subsequent stabilization of newly formed tubes by mesenchymal cells. Beyond this critical role in blood vessel development, recent studies suggest a wider role for Tie2 and angiopoietins in lymphangiogenesis and the development of the hematopoietic system, as well as a possible role in the regulation of certain non-endothelial cells. The outcome of Tie signaling depends on which vascular bed is involved, and crosstalk between different VEGFs has an important modulating effect on the properties of the angiopoietins. Signaling through the Tie1 receptor is not well understood, but Tie1 may have both angiopoietin-dependent and ligand-independent functions. Changes in the expression of Tie receptors and angiopoietins occur in many pathological conditions, and mutations in the Tie2 gene are found in familial cases of vascular disease.     

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.     

The angiopoietin:Tie 2 interaction: A potential target for future therapies in human vascular disease

Angiopoietin-1 and -2 are endogenous ligands for the vascular endothelial receptor tyrosine kinase Tie2. Signalling by angiopoietin-1 promotes vascular endothelial cell survival and the sprouting and reorganisation of blood vessels, as well as inhibiting activation of the vascular endothelial barrier to reduce leakage and leucocyte migration into tissues. Angiopoietin-2 generally has an opposing action, and is released naturally at times of vascular growth and inflammation. There is a significant body of emerging evidence that promoting the actions of angiopoietin-1 through Tie2 is of benefit in pathologies of vascular activation, such as sepsis, stroke, diabetic retinopathy and asthma. Similarly, methods to inhibit the actions of angiopoietin-2 are emerging and have been demonstrated to be of preclinical and clinical benefit in reducing tumour angiogenesis. Here the author reviews the evidence for potential benefits of modulation of the interaction of angiopoietins with Tie2, and the potential applications. Additionally, methods for delivery of the complex protein angiopoietin-1 are discussed, as well as potentially deleterious consequences of administering angiopoietin-1.     

Angiopoietins-1 and -2 are both capable of mediating endothelial PAF synthesis: intracellular signalling pathways

Vascular endothelial growth factor (VEGF) is the only angiogenic growth factor capable of inducing an inflammatory response and we have recently demonstrated that its inflammatory effect is mediated by the endothelial synthesis of platelet-activating factor (PAF). Recently discovered, Ang1 and Ang2, upon binding to Tie2 receptor, modulate vascular permeability and integrity, contributing to angiogenesis. Ang1 was initially identified as a Tie2 agonist whereas Ang2 can behave as a context-dependent Tie2 agonist or antagonist. We sought to determine if Ang1 and/or Ang2 could modulate PAF synthesis in bovine aortic endothelial cells (BAEC) and if so, through which intracellular signalling pathways. Herein, we report that Ang1 and Ang2 (1 nM) are both capable of mediating a rapid Tie2 phosphorylation and a rapid, progressive and sustained endothelial PAF synthesis maximal within 4 h (1695% and 851% increase, respectively). Angiopoietin-mediated endothelial PAF synthesis requires the activation of the p38 and p42/44 MAPKs, PI3K intracellular signalling pathways, and a secreted phospholipase A(2) (sPLA(2)-V). Furthermore, angiopoietin-mediated PAF synthesis is partly driven by a relocalization of endogenous VEGF to the cell surface membrane. Our results demonstrate that the angiopoietins constitute another class of angiogenic factors capable of mediating PAF synthesis which may contribute to proinflammatory activities.     

Angiopoietin-1/Tie2 signaling pathway inhibits lipopolysaccharide-induced activation of RAW264.7 macrophage cells

Angiopoietin-1 (Ang1) is a ligand for the endothelial-specific tyrosine kinase receptor Tie2 and has been shown to play an essential role in embryonic vasculature development. There have been many studies about the anti-inflammatory effects of Ang1, most of which focus on endothelium cells. In the present study, we explore the role of Ang1-Tie2 signaling in the activation of macrophages upon lipopolysaccharide (LPS) stimulation. We found that Tie2 receptor is expressed on macrophages and Ang1 could inhibit LPS-induced activation of macrophages, as evidenced by cell migration and TNF-α production, specifically through Tie2 receptor. We further investigated the mechanism and found that Ang1-Tie2 could block LPS-induced activation of NF-κB which has been shown to be necessary for macrophage activation with LPS treatment. Thus, we described, for the first time, the role of Ang1-Tie2 signaling in macrophage activation and the possible mechanisms in response to immune stimulation.     

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.     

Structure of the angiopoietin-2 receptor binding domain and identification of surfaces involved in Tie2 recognition

The angiopoietins comprise a small class of secreted glycoproteins that play crucial roles in the maturation and maintenance of the mammalian vascular and lymphatic systems. They exert their effects through a member of the tyrosine kinase receptor family, Tie2. Angiopoietin/Tie2 signaling is unique among tyrosine kinase receptor-ligand systems in that distinct angiopoietin ligands, although highly homologous, can function as agonists or antagonists in a context-dependent manner. In an effort to understand this molecular dichotomy, we have crystallized and determined the 2.4 A crystal structure of the Angiopoietin-2 (Ang2) receptor binding region. The structure reveals a fibrinogen fold with a unique C-terminal P domain. Conservation analysis and structure-based mutagenesis identify a groove on the Ang2 molecular surface that mediates receptor recognition.     

Angiopoietin-1 but not angiopoietin-2 promotes neutrophil viability: Role of interleukin-8 and platelet-activating factor

We previously reported the expression of angiopoietin receptor Tie2 on human neutrophils. Both angiopoietins (Ang1 and Ang2) induce platelet activating factor (PAF) synthesis from endothelial cells (ECs) and neutrophils. Both angiopoietins can also modulate EC viability and since PAF can promote pro-survival activity on neutrophils, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Neutrophils were isolated from venous blood of healthy volunteers and neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers (annexin-V and propidium iodide (P.I.), respectively). Basal neutrophil viability from 0 to 24 h post-isolation decreased from 98% to ≈45%. Treatment with anti-apoptotic mediators such as interleukin-8 (IL-8; 25 nM) and PAF (100 nM) increased neutrophil basal viability by 34 and 26% (raising it from 43 to 58 and 55%) respectively. Treatment with Ang1 (0.001-50 nM) increased neutrophil viability by up to 41%, while Ang2 had no significant effect. Combination of IL-8 (25 nM) or PAF (100 nM) with Ang1 (10 nM) further increased neutrophil viability by 56 and 60% respectively. We also observed that Ang1, but not Ang2 can promote IL-8 release and that a pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 81% respectively. Pretreatment with a selective PAF receptor antagonist (BN 52021), did abrogate PAF pro-survival activity, without affecting Ang1-induced neutrophil viability. Our data are the first ones to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.     

Effects of protein and gene transfer of the angiopoietin-1 fibrinogen-like receptor-binding domain on endothelial and vessel organization

The vessel-stabilizing effect of angiopoietin-1 (Ang1)/Tie2 receptor signaling is a potential target for pro-angiogenic therapies as well as anti-angiogenic inhibition of tumor growth. We explored the endothelial and vascular specific activities of the Ang1 monomer, i.e. dissociated from its state as an oligomer. A truncated monomeric Ang1 variant (i.e. DeltaAng1) containing the isolated fibrinogen-like receptor-binding domain of Ang1 was created and recombinantly produced in insect cells. DeltaAng1 ligated the Tie2 receptor without triggering its phosphorylation. Moreover, monomeric DeltaAng1 was observed to bind alpha(5)beta(1) integrin with similar affinity compared with Tie2. Unexpectedly, in vitro treatment of endothelial cells with DeltaAng1 showed some of the known effects of full-length Ang1, including inhibition of basal endothelial cell permeability and stimulation of cell adhesion as well as activation of MAPKs. Local treatment of the microvasculature of the developing chicken chorioallantoic membrane with the DeltaAng1 protein led to profound reduction of the mean vascular length density, thinning of vessels, and reduction of the number of vessel branching points. Similar effects were observed in side-by-side experiments with the recombinant full-length Ang1 protein. These effects of simplification of the vessel branching pattern were confirmed through local gene transfer with lentiviral particles encoding DeltaAng1 or full-length Ang1. Together, our findings suggest a potential use for exogenous Ang1 in reducing rather than increasing vascular density. Furthermore, we show that the isolated receptor-binding domain of Ang1 is capable of mediating some effects of full-length Ang1 independently of Tie2 phosphorylation, possibly through integrin ligation.     

Angiopoietins have distinct modular domains essential for receptor binding,dimerization and superclustering

Angiopoietins are a recently discovered family of angiogenic factors that interact with the endothelial receptor tyrosine kinase Tie2, either as agonists (angiopoietin-1) or as context-dependent agonists/antagonists (angiopoietin-2). Here we show that angiopoietin-1 has a modular structure unlike any previously characterized growth factor. This modular structure consists of a receptor-binding domain, a dimerization motif and a superclustering motif that forms variable-sized multimers. Genetic engineering of precise multimers of the receptor-binding domain of angiopoietin-1, using surrogate multimerization motifs, reveals that tetramers are the minimal size required for activating endothelial Tie2 receptors. In contrast, engineered dimers can antagonize endothelial Tie2 receptors. Surprisingly, angiopoietin-2 has a modular structure and multimerization state similar to that of angiopoietin-1, and its antagonist activity seems to be a subtle property encoded in its receptor-binding domain.     

Notoginsenoside R1 activates the Ang2/Tie2 pathway to promote angiogenesis

BackgroundTherapeutic angiogenesis is a novel strategy for the treatment of ischemic diseases that involves promotion of angiogenesis in ischemic tissues via the use of proangiogenic agents. However, effective proangiogenic drugs that activate the Ang2/Tie2 signaling pathway remain scarce.PurposeWe aimed to investigate the proangiogenic activity of notoginsenoside R1 (NR1) isolated from total saponins of Panax notoginseng with regard to activation of the Ang2/Tie2 signaling pathway.MethodsWe examined the proangiogenic effects of NR1 by assessing the effects of NR1 on the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells (HUVECs). The aortic ring assay and vascular endothelial growth factor receptor inhibitor (VRI)-induced vascular regression in the zebrafish model were used to confirm the proangiogenic effects of NR1 ex vivo and in vivo. Furthermore, the molecular mechanism was investigated by Western blot analysis.ResultsWe found that NR1 promoted the proliferation, mobility and tube formation of HUVECs in vitro. NR1 also increased the number of sprouting vessels in rat aortic rings and rescued VRI-induced vascular regression in zebrafish. NR1-induced angiogenesis was dependent on Tie2 receptor activation mediated by increased autocrine Ang2 in HUVECs, and inhibition of the Ang2/Tie2 pathway abrogated the proangiogenic effects of NR1.ConclusionsOur results suggest that NR1 promotes angiogenesis by activating the Ang2/Tie2 signaling pathway. Thus, NR1-induced activation of the Ang2/Tie2 pathway is an effective proangiogenic approach. NR1 may be useful agent for the treatment of ischemic diseases.     

Osteoblast-specific Angiopoietin 1 overexpression increases bone mass

Although osteoblasts express the angiogenic protein Angiopoietin 1 (Ang1), the role of Ang1 in bone formation remains largely unknown. Here we report that Ang1 overexpression in osteoblasts driven by the osteoblast-specific 2.3 kb alpha 1 type 1 collagen promoter results in increased bone mass in vivo. In Ang1-transgenic mice (Ang1-Tg), bone volume and bone parameters increased significantly compared with wild-type littermates, although the Ang1 receptor, Tie2 was not expressed in osteoblasts. Tie2 is primarily expressed in vascular endothelial cells, and Ang1-Tie2 signaling is reportedly crucial for angiogenesis. We found that the number of vascular endothelial cells was significantly elevated in Ang1-Tg mice compared with that of wild-type littermates, an increase accompanied by increased alkaline-phosphatase activity, a marker of osteoblast activation. The number of osteoclasts in the bone of Ang1-Tg mice did not differ from wild-type littermates. These results indicate that angiogenesis induced by Ang1 expressed in osteoblasts is coupled with osteogenesis.