Angiogenesis

, 12:125

The role of the Angiopoietins in vascular morphogenesis

Authors

  • Markus Thomas
    • Joint Research Division Vascular Biology, Medical Faculty Mannheim (CBTM)University of Heidelberg
    • Roche Diagnostics GmbHPharma Research Penzberg
    • German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance)
    • Joint Research Division Vascular Biology, Medical Faculty Mannheim (CBTM)University of Heidelberg
    • German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance)
Original paper

DOI: 10.1007/s10456-009-9147-3

Cite this article as:
Thomas, M. & Augustin, H.G. Angiogenesis (2009) 12: 125. doi:10.1007/s10456-009-9147-3

Abstracts

The Angiopoietin/Tie system acts as a vascular specific ligand/receptor system to control endothelial cell survival and vascular maturation. The Angiopoietin family includes four ligands (Angiopoietin-1, Angiopoietin-2 and Angiopoietin-3/4) and two corresponding tyrosine kinase receptors (Tie1 and Tie2). Ang-1 and Ang-2 are specific ligands of Tie2 binding the receptor with similar affinity. Tie2 activation promotes vessel assembly and maturation by mediating survival signals for endothelial cells and regulating the recruitment of mural cells. Ang-1 acts in a paracrine agonistic manner inducing Tie2 phosphorylation and subsequent vessel stabilization. In contrast, Ang-2 is produced by endothelial cells and acts as an autocrine antagonist of Ang-1-mediated Tie2 activation. Ang-2 thereby primes the vascular endothelium to exogenous cytokines and induces vascular destabilization at higher concentrations. Ang-2 is strongly expressed in the vasculature of many tumors and it has been suggested that Ang-2 may act synergistically with other cytokines such as vascular endothelial growth factor to promote tumor-associated angiogenesis and tumor progression. The better mechanistic understanding of the Ang/Tie system is gradually paving the way toward the rationale exploitation of this vascular signaling system as a therapeutic target for neoplastic and non-neoplastic diseases.

Keywords

AngiogenesisEndothelial cellAngiopoietinTie

Structure and expression of Angiopoietin ligands and Tie receptors

Structure and expression of Angiopoietin-1 and Angiopoietin-2

The Angiopoietins have been identified in the mid 1990s as a family of growth factors that are essential for blood vessel formation (Fig. 1). There are four Angiopoietins known, Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Angiopoietin-3 (Ang-3), and Angiopoietin-4 (Ang-4). The best characterized Angiopoietins are Ang-1 and Ang-2. Ang-3 and Ang-4 are orthologs found in mouse and human, respectively. The Angiopoietins are all ligands for the Tie2 receptor [15]. Structurally, the Angiopoietins are composed of two domains. There is a N-terminal coiled-coil domain which is responsible for ligand homo-oligomerization of the ligands. Electron microscopy experiments have demonstrated that Ang-1 and Ang-2 can form heterogeneous multimers with trimers, tetramers and pentamers [6]. Furthermore, oligomerization is necessary for receptor activation but not for receptor binding. This is mediated by the fibrinogen-like domain which is located in the C-terminus [1, 7].
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Fig. 1

A possible mechanism for the initiation of angiogenesis receptors in the early 1990s, the angiopoietin ligands were identified few years later. Major milestones include the genetic manipulation of receptors and ligands in loss-of-function and gain-of-function experiments as well as the unraveling of relevant signaling pathways, cellular readouts of receptor activation, and adult manipulatory experiments in neoplastic and non-neoplastic settings

The Angiopoietins are secreted glycoproteins with a dimeric molecular weight of approximately 75 kDa. Ang-1 has 498 aa and is located on chromosome 8q22. Ang-2 has 496 aa and is located on chromosome 8q23. Both molecules show sequence homology of about 60% [1, 2]. Ang-1 is expressed by smooth muscle cells and other perivascular cells. Like Ang-2, it binds Tie2 with an affinity of about 3 nM [2] at the IgG-like domain and the EGF-like domain of Tie2 [8]. Ang-1 is produced as four different splice variants. The splice variants with 1.5 kb (full length Ang-1) and 1.3 kb bind the receptor and induce its autophosphorylation. The proteins coded by the 0.9 kb and 0.7 kb also bind Tie2, but do not induce autophosphorylation [9]. A novel Ang-2 splice variant, Ang-2B, with a truncated amino-terminal domain has been detected in chicken [10]. An additional splice variant (Ang-2(443)) has been identified which lacks parts of the coiled-coil domain and cannot stimulate Tie2 phosphorylation [11]. Ang-1 acts as an agonist of the Tie2 receptor, whereas Ang-2 is the antagonist [2]. However, Ang-2 has also been reported to context-dependently induce receptor phosphorylation. The molecular basis for agonistic versus antagonistic functions of Ang-2 have not been unraveled. Cell type specific effects, the degree of endothelial confluence, the duration of Ang-2 stimulation, concentration-dependent effects, as well as the presence of co-receptors such as Tie1 have all been implicated in controlling agonistic versus antagonistic functions of Ang-2 [1214].

Ang-2 is almost exclusively expressed by endothelial cells where it is stored in Weibel-Palade bodies (WPB) [15]. Following cytokine activation of the endothelium (e.g., by Histamine or Thrombin), Ang-2 is rapidly released from WPB [15]. It acts in an autocrine manner on the Tie2 receptor by binding as homodimers or multimers [7]. Recent studies have shown that endogenous Ang-2 may act through an internal autocrine loop mechanism. This concept is based on cellular experiments showing that endogenously released Ang-2 cannot be inhibited by exogenous soluble Tie2 receptor [16].

Ang-2 levels are upregulated by hypoxia [1720]. Under physiological conditions, Ang-2 is expressed in regions of vascular remodeling, for example during vascularization of the retina or during vessel regression in the cyclic ovarian corpus luteum [2, 21]. Ang-2 expression is also upregulated under pathological condition, e.g., in the endothelium of tumors [2224] and in the tumor cells themselves [2527]. Moreover, retinal neurons [28] and Müller cells [29] are a source of Ang-2.

In contrast to Ang-2, Ang-1 is primarily expressed by mesenchymal cells and acts in a paracrine manner on the endothelium. It is abundantly expressed by the myocardium during early development and by perivascular cells later during development and in adult tissues [2, 30, 31]. Ang-1 is also expressed by tumor cells [22, 32] and neuronal cells of the brain [32].

Expression and structure of Tie1 and Tie2

Tie1 and Tie2 are endothelial cell-specific receptors with similar molecular weight of approximately 135 and 150 kDa, respectively. Originally identified as orphan receptors in the early nineteen nineties (Fig. 1), they are expressed by vascular and lymphatic endothelial cells. Both receptors are structurally similar in the cytoplasmic region (76% sequence identity), but show only 33% similarity in the extracellular part [33]. Tie1 and Tie2 are tyrosine kinases with Ig-like and EGF-like homology domains. The extracellular domain consists of three immunoglobulin (Ig)-like domains that are flanked by three epidermal growth factor (EGF)-like cysteine repeats followed by three fibronectin type III domains (Fig. 2a). The smaller intracellular domains of both receptors consist of a split kinase domain which can bind different molecules after autophosphorylation. Crystal structures analyses showed that the ligands Ang-1 and Ang-2 bind with almost similar affinity to the same site of the Tie2 receptor [8]. They bind to the second Ig-like loop flanked by the first Ig-like loop and the EGF-like repeats [8, 34].
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Fig. 2

Schematic overview of the Tie receptors. a The extracellular domain of Tie1 and Tie2 consists of three immunoglobulin (Ig)-like domains, one EGF-like domain and three fibrinogen-like domains. The intracellular part contains the split kinase domain. b Tie2 has three phosphotyrosine residues (1101, 1107, 1112), whereas, Tie1 has only two (1113 and 1124). The equivalent to Tie2 pTyr1107 is missing. Grb2 and p85 both bind to pTyr1101, Dok-R to pTyr1107 and SHP2 to pTyr1112 of Tie2

During early development, Tie1 can be detected from E8.5 in differentiating angioblasts of the head mesenchyme, in the splanchnopleura and in the dorsal aorta but also in migrating endothelial cells of the developing heart [35]. Tie1 is almost exclusively expressed by endothelial cells. Full length Tie1 is thought to heterodimerize with Tie2 [36]. Other studies have shown that the ectodomain of Tie1 is proteolytically cleaved following VEGF stimulation. The membrane-anchored cleaved form, containing the cytoplasmic domain, may interact with Tie2 and is supposed to be involved in Tie2 signaling [3638]. Receptor shedding also occurs after stimulation with the phorbol ester PMA, stimulation with tumor necrosis factor alpha (TNF-α), and by shear stress [3941]. Tie1 is still largely considered as an orphan receptor. Yet, recent work suggests that COMP-Ang-1, a designed pentameric form of Ang-1, can bind to Tie1 under certain conditions [42].

The second Angiopoietin receptor, Tie2, is expressed by endothelial cells as well as by hematopoietic cells, endothelial precursor cells [43, 44] and tumor cells (e.g., Kaposi sarcoma cells [45] and melanoma cells [46]). A Tie2-positive subpopulation of monocytes is associated with the angiogenic activity of recruited tumor-associated macrophages [47]. Endothelial cells in larger vessels express Tie2 more abundantly when compared with smaller vessels [33, 43]. Tie2 expression is upregulated during tumor angiogenesis [4850]. The receptor dimerizes by ligand binding. Following binding of the activating ligand Ang-1, Tie2 is autophosphorylated and intracellular signaling pathways are activated (Fig. 3a).
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Fig. 3

Schematic representation of Angiopoietin signaling in regulating the quiescent and the activated phenotype of the endothelium. a Ang-1 is produced in non-endothelial cells and binds to Tie2 inducing Tie2 autophosphorylation. In a next step, PI3-K and Akt are activated which in turn promotes survival or anti-apoptotic signals through proteins like, Survivin, Caspase-9, eNOS and Bad. Inactivated FAK in the cell further supports survival of endothelial cells through Akt. On the other hand, Rho GTPases are activated by Ang-1 which reduces endothelial cell permeability by sequestering Src through mDia. Thereby, VEGF-R2-mediated Src phosphorylation and subsequent VE-cadherin internalization is inhibited. VE-PTP interacts with Tie2 in the presence, but not in the absence of cell–cell contacts. VE-PTP inhibition in endothelial cells is associated with increased permeability. Furthermore, several proteins like Dok-R or Grb14 associate with phosphorylated Tie2 and thereby inhibit endothelial cell proliferation. Ang-1/Tie2 signaling is required for vessel stabilization. Ang-2 acts as an antagonistic regulator on endothelial cells and thereby leads to vessel destabilization and pericyte dropout. The exact molecular mechanisms of how this process is regulated are not known. Potential molecules that are involved in this process are mentioned in the scheme. FOXO transcription factors are also involved in Ang/Tie signaling by regulating protein synthesis. Their phosphorylation leads to an inactive form which promotes endothelial cell survival, quiescence and vascular stabilization, whereas, the activated form supports vascular destabilization and apoptosis. b Tie2 activation under certain conditions results in cell migration, inflammation and vascular leakage. Cell migration is mediated by the activation of FAK by PI3-K, adaptor proteins of Dok-R, e.g., Nck and PAK and by SHP-2, which is thought to dephosphorylate autophosphorylation sites of Tie2. Translocation of Tie2 to cell-matrix attachment sites in subconfluent cells promotes endothelial cell migration through the activation of Dok-R and its adaptor proteins. The interaction of ABIN-2 with Tie2 is thought to inactivate NFκB via the IKK complex and thereby induces destabilization and inflammation. Rho activation is blocked during Ang/Tie-mediated vascular leakage, which liberates Src from mDia. VEGF promotes VEGF-R2 activation which in turn activates Src and induces VE-cadherin internalization. Abbreviations: Ang, Angiopoietin; SMC, smooth muscle cell; HB-EGF, heparin-binding epidermal growth factor-like growth factor; PDGF, platelet-derived growth factor; TGF, transforming growth factor; BMP, bone morphogenetic protein; Dok-R, docking protein R; MAPK, mitogen-activated protein kinase; PAK, p21-activated kinase; PI3-K, phosphatidylinositol 3′-kinase; Akt, protein kinase B; FAK, focal adhesion kinase; eNOS, endothelial nitric oxide synthase; FKHR, forkhead transcription factor; VE-PTP, vascular endothelial tyrosine phosphatase

Physiological roles of Tie receptors and Angiopoietin ligands during development and in the adult

Tie receptors

Tie2-deficient mouse embryos die at E10.5 due to vessel remodeling defects in the plexus of the yolk sac, of the brain and severe heart defects. The mice show 30 and 75% less endothelial cells at E8.5 and E9.5, respectively. Vessels are only poorly organized, have fewer branches and have reduced pericyte coverage [30, 44, 51, 52]. Tie2 also exerts critical roles during hematopoiesis [53]. Loss of Tie2 function leads to endothelial cell apoptosis which in turn results in hemorrhage [54]. These results suggest that the Ang-Tie system plays a key role during vessel remodeling, maturation and stabilization of the cardiovascular system.

Injection of soluble Tie2 (sTie2-Fc) was shown to inhibit ischemia-induced retinal neovascularization in a mouse model [55]. This soluble form is also present under physiological conditions in the serum resulting from Tie2 cleavage which has been shown in cellular experiments to occur after PMA stimulation [56]. Circulating concentrations of soluble Tie2 are increased in several vasculopathies, including coronary artery disease [57].

Abnormal vessel structures are not only caused by Tie2-deficiency. A constitutively active Tie2 mutant has been identified in patients with venous malformations [58]. This leads to enlarged veins with pronounced proliferation of endothelial cells. The endothelial cells are surrounded by several layers of smooth muscle cells. The range is between areas with normal coverage and areas completely devoid of SMC.

Mice lacking the Tie1 gene die between E13.5 and P1 due to a loss of structural integrity of vascular endothelial cells, resulting in severe edema and hemorrhage [44]. Developmental angiogenesis is not perturbed. Unlike Tie2-deficient mice, hematopoiesis occurs normally in Tie1-deficient mice [59]. The genetic experiments suggest that Tie1 plays important roles during endothelial cell differentiation and in the regulation of vessel integrity.

Double-knockout mice for Tie1 and Tie2 have been created to shed further light into the signaling pathways of both receptors during vascular development. These mice die like Tie2-deficient mice around E10.5 not only due to cardiovascular defects but also as a consequence of severe defects in the vascular system. Vasculogenesis proceeds normally in these mice. The authors concluded from their results that Tie1 and Tie2 are essential for maintaining the integrity of mature vessels but that they are dispensable for early angiogenic sprouting [60].

Angiopoietin ligands

Angiopoietin-1 deficiency results in lethality at E11–E12.5 [30]. The phenotype of Ang-1-deficient mice is similar to the phenotype of Tie2-deficient mice but not as severe. These mice have growth-retarded hearts with a less complex ventricular endocardium. The endocardium is collapsed and appears retracted from the myocardial wall. The endothelial lining in the atria is collapsed and the trabeculae are absent. Ang-1 deficiency also causes severe vascular defects [30]. The mice show a much simpler and immature primary capillary plexus. The distinction between larger and smaller vessels is much less pronounced. Periendothelial cells are scarce in Ang-1-deficient embryos and not associated with endothelial cells but appear separated from rounded endothelial cells.

Myocardial overexpression of Ang-1 under the control of the tetracycline promoter shed further light in the importance of Ang-1 during heart development. Most of these mice (90%) die between E12.5 and E15.5 as a result of cardiac hemorrhage. The myocardial walls of both atria and the ventricles are thinned and the density of trabeculae is dramatically reduced. The mice show hemorrhages around the heart and the atria are enlarged. The outflow tract is collapsed and mice lack an intact endocardium and coronary arteries. Ten percent of the mice survive with cardiac hypertrophy and a dilation of the right atrium [61]. These studies showed that Ang-1 overexpression dramatically affects early development of the mice. Yet, overexpression in the adult has little effect on vessel structure and heart development.

Transgenic mice overexpressing Ang-1 under the control of the keratin 14 (K14) promoter are viable and generally healthy [62]. Newborn mice show larger vessels in the skin. Additionally, the skin of older mice is more reddish than those of normal mice. Transmission electron microscopic analysis confirmed that these mice have normal cell–cell contacts between endothelial cells and between endothelial and perivascular cells. The inter-endothelial distance is slightly increased but mice show no plasma leakage or edema. The experiments demonstrated that the vasculature is largely intact and functional.

Angiopoietin-2 transgenic mice show severe vascular defects including disruption of vessel integrity [2]. The endocardial lining is collapsed and detached from the underlying myocardium. Trabecular folds are completely absent. The systemic Ang-2 overexpression phenotype is highly reminiscent of the phenotype of Ang-1- and Tie2-deficient mice which supported the hypothesis that Ang-1 acts in a stimulating, agonistic manner on Tie2, whereas, Ang-2 exerts antagonistic functions on Ang-1/Tie2 signaling. Endothelial-specific overexpression of Ang-2 in adult mice confirmed this hypothesis exhibiting a complete suppression of Ang-1-mediated Tie2 phosphorylation in addition to arteriogenesis defects [63]. It has also been reported that injection of Ang-2 has an effect on pericyte coverage in the mouse retina. Ang-2 has in these experiments been injected intravitreally which resulted in the dropout of pericytes [64]. Ang-2 has also been reported to have different effects dependent on the cytokine milieu. Ang-2 and VEGF act together to induce angiogenesis. However, Ang-2 induces vessel regression in the absence/inhibition of VEGF [2, 6567].

It has recently been observed that the perinatal lethality of Ang-2-deficient mice is strain-dependent. Essentially all Ang-2-deficient mice in the 129/J background die postnatally within 14 days after birth [31]. Ang-2-deficient mice in the C57/Bl6 background are viable with only 10% postnatal lethality [68]. These mice show no vascular defects but develop a severe chylous ascites after birth indicating defects in the lymphatic system. Further analyses revealed that large vessels are disorganized forming a lacy network with poor smooth muscle cell coverage [69]. Small lymphatic vessels in the intestine are disorganized and irregular [31]. Ang-2-deficient mice show only minor vascular defects. Hyaloid vessels in the eye’s lens regress shortly after birth in wild type, but not in Ang-2-deficient mice. This reflects a role of Ang-2 in vessel remodeling and vascular regression [31, 70].

The genetic knock-in of Ang-1 into the Ang-2 locus completely rescues the lymphatic phenotype of Ang-2-deficient mice, but not the vascular remodeling defects [31] supporting the hypothesis that Ang-2 is agonistic in lymphatic vessels and antagonistic in blood vessels. These experiments further support the concept that Ang-2 is dispensable for early development but necessary for vessel remodeling and during later stages of development.

Signaling through Tie receptors

Ang-1 and Ang-2 both bind Tie2, but only Ang-1 induces its autophosphorylation and thereby the activation of the receptor [1]. As antagonistic ligand, Ang-2 does not induce receptor autophosphorylation but competes with Ang-1 to act as an inhibitor of Ang-1/Tie2 signaling [2]. Yet, some studies also identified Ang-2 as an agonist of Tie2 [12, 14] Ang-1 binding to Tie2 leads to an activation of signaling pathways inside the cell by recruiting different adaptor proteins to the receptor. Signaling is related to several processes including cell survival, migration, inflammation and permeability.

Endothelial cell survival and maintenance

Ligand binding of Tie2 leads to phosphorylation of the p85 subunit of phosphatidylinositol 3- kinase (PI3 K). PI3 K activates Akt which in turn phosphorylates and activates the Forkhead transcription factor FOXO-1 (FKHR-1). FKHR-1 is a strong inducer of Ang-2 expression and inhibits Ang-2 liberation [7175]. Activation of Akt also stimulates the phosphorylation and thereby the inhibition of pro-apoptotic proteins, including BAD and procaspase-9 [72, 76]. Additionally, Akt upregulates survivin, a classical apoptosis inhibitor, and thereby supports cell survival (Fig. 3a) [77, 78].

Ang-1- and Tie2-deficient mice show severe defects in the recruitment of pericytes and in their interaction with endothelial cells [30, 44, 51]. In a rat model of diabetic retinopathy, Ang-2 expression was found to be strongly increased leading to the dropout of pericytes [64]. However, the mechanisms involved in Ang/Tie-mediated SMC recruitment are poorly understood. One possible molecule involved in the recruitment of mural cells is the EC-derived heparin binding EGF-like growth factor (HB-EGF). Its expression in endothelial cells is upregulated by Ang-1 [79], but only when they are in contact with mural cells. HB-EGF-mediated receptor (ErbB1 and ErbB2) activation thereby induces SMC migration. However, there is also evidence that hepatocyte growth factor may be involved in Ang-1-mediated SMC recruitment. Stimulation of endothelial cells with Ang-1 induces SMC migration toward endothelial cells in a co-culture assay. This effect could be reversed by the addition of a neutralizing anti-HGF antibody indicating that Ang-1 is regulating HGF expression [80].

In addition to HB-EGF and HGF, PDGF-B is also expressed by endothelial cells and is involved in pericyte recruitment [81]. PDGF-B signals through its receptor PDGFRβ which is expressed by pericytes. PDGF-B thereby acts as chemoattractant which promotes the proliferation of SMCs and pericytes during their recruitment to the endothelium [82]. PDGFRβ antibodies completely block the recruitment of pericytes to the newly formed vasculature in the retina of newborn mice leading to retinal edema and hemorrhage [83]. These vessels are poorly remodeled and leaky. The injection of recombinant Ang-1 almost completely rescued the phenotype caused by the blocking of PDGFRβ antibodies. This suggests coordinated activities of Ang-1 and PDGF-B. The vascular defects in Ang-1- and Tie2-deficient mice occur earlier during development than those of PDGF-B- and PDGFRβ-deficient mice indicating additional mechanisms of pericyte recruitment. Another regulator of SMC differentiation is TGF-β which is upregulated by Ang-1 following PDGF-B stimulation. In turn, Ang-1 is downregulated by TGF-β. These data suggest that pericytes are recruited by PDGF-B which induces pericyte proliferation. Ang-1, which is upregulated by PDGF-B, promotes pericyte migration. TGF-β is responsible for SMC differentiation and to render the vasculature in its quiescent state [8486]. Ang/Tie signaling has also been implicated in the regulation of vessel diameter sensing. The vessel diameter is controlled in a Tie2-dependent manner by autocrine-acting Apelin and its cognate receptor APJ [87].

Endothelial cell activation and contextual presentation of Tie receptors

Tie2 activation has been related to endothelial migration (Fig. 3B). This seems to be dependent on the contextual presentation of the receptor on the endothelial cell surface [88, 89]. Tie2 is expressed in a polarized manner in activated endothelial cells and translocated to the extracellular matrix where it binds to matrix immobilized Ang-1. The Akt pathway is blocked, whereas, Dok-R (docking protein R) is phosphorylated. Activated Dok-R interacts with rasGAP, Nck and Crk. All these molecules are involved in cell migration, proliferation, cytoskeletal reorganization and the regulation of the ras signaling cascade [90]. In contrast, Tie2 is translocated to cell–cell junctions in quiescent endothelial cells where it engages in trans complexes with other Tie2 molecules of neighboring cells. In this context, Tie2 interacts with VE-PTP, a molecule which is strongly associated with barrier function, thereby inhibiting paracellular permeability. Additionally, Akt is activated to induce endothelial cell survival and stability of the endothelium through the phosphorylation of eNOS (Fig. 3a) [88, 89]. Src is activated during VEGF-mediated angiogenesis. This leads to the activation of VAV, a guanine-nucleotide-exchange factor (GEF) for Rac. Rac further activates VE-cadherin at Ser665. Beta-arrestin-2 is recruited to VE-cadherin which leads to its internalization in a clathrin-dependent manner [91, 92]. This progress supports vascular permeability and migration. Ang-1-mediated Tie2 signaling inhibits this pathway by activation of mDia through Rho. This leads to an association of Src and mDia. Src is not longer available for VE-cadherin activation and internalization [93]. Ang-1 was further shown to inhibit Thrombin-induced permeability by decreasing PKCzeta activation [94]. The same group could show that Ang-1 also prevents vascular permeability in vitro and in vivo by stimulating sphingosine kinase-1 [95]. Other molecules like Grb2, Grb7, ShcA, the protein tyrosine kinase SHP2 and the previously mentioned p85 subunit interact with Tie2 via SH2 domains. These molecules seem to be involved in cell migration, proliferation, differentiation and apoptosis [96, 97]. Fusion proteins of the recombinant Tie2 kinase domain and glutathione-S-transferase (GST) showed that Grb2 interacts with Tie2 at pTyr-1101 (Fig. 2B). A mutation of this tyrosine residue to phenylalanine markedly decreased the interaction of Grb2 with Tie2. SHP2 association with phosphorylated Tie2 remained unaffected. Conversely, mutation of pTyr-1112 to phenylalanine reduced the association of SHP2 with the phosphorylated kinase domain (Fig. 2b) [96, 98]. These findings indicate that Grb2 and SHP2 associate with phosphorylated Tie2, thereby supporting intracellular signaling processes, like activation of MAPK. Indeed, Ang-1 can activate MAPK in endothelial cells and in the aortic ring assay [99101]. Yet, the inhibition of MAPK had no effect on Ang-1-mediated endothelial cell survival and migration [99].

SHP2 is not only involved in signal transduction. It may also act as a negative regulator of Tie2 phosphorylation. Mutation of Tie2 at pTyr1112 enhanced autophosphorylation and downstream signaling [97, 102]. Yeast-two-hybrid experiments revealed that the p85 subunit of PI3 K also interacts with the phosphorylated Tie2 kinase domain [97]. p85 binds like Grb2 to pTyr1101 (Fig. 2b). A mutation of this phosphorylation site reduced the binding of p85 to Tie2. Dok-R has been shown to bind to activated Tie2 at pTyr1107 (Fig 1b). A mutation of this phosphorylation site reduced the binding of Dok-R to Tie2 [102]. Dok-R is immediately phophorylated at multiple sites after binding to activated Tie2 to recruit other molecules. This leads to the activation of several signaling pathways, including migration. However, this phosphotyrosine is missing in Tie1 protein, as shown in Fig. 2b.

Ang-1 is also able to activate focal adhesion kinase (FAK) through Tie2 [103]. This in turn leads to the phosphorylation of paxillin. The MAP kinase ERK is activated in further steps [104] which supports migration. In turn, when blocking Tie2 activation, Ang-1 induced migration via ERK is inhibited. Endothelial cell sprouting is mediated by the secretion of plasminogen and metalloproteinase following Ang-1 stimulation [103]. All these in vitro activation phenotypes of Ang-1 are supported by in vivo studies in mice which have shown that Ang-1 overexpression promotes vessel formation in the heart of mice [62].

Role of Ang/Tie signaling during pathology

Angiopoietin-1 functions during inflammation

Ang-1 acts as an anti-inflammatory cytokine. It protects against endotoxic shock-induced by LPS and thereby prevents microvascular leakage [105]. It blocks the expression and cell surface activity of tissue factor (TF), an initiator of blood coagulation, which is involved in thrombosis and inflammation. Ang-1 reduces VEGF stimulated leukocyte adhesion to endothelial cells [106]. Cardiac allograft atherosclerosis [107] and radiation induced cell damage [108] are protected by Ang-1 and by a designed pentameric Ang-1, named COMP-Ang-1. Furthermore, Tie2 activation leads to ABIN-2 recruitment which interferes with NF-κB signaling [109111]. This prevents endothelial cells from undergoing apoptosis and the induction of inflammation. Subsequent signaling is likely mediated by the PI3 K/Akt pathway because blocking PI3 K results in suppression of ABIN-2-induced inhibition of cell death [111]. Ang-1 may also be involved in inflammatory diseases, like rheumatoid arthritis (RA). Synovial fibroblasts are a key player during RA and a major source of Ang-1. Ang-1 is also upregulated during this disease by inflammation promoting cytokines, including TNF-α [112, 113]. TNF-α but also IL-1β are capable to induce the expression of the transcription factor epithelium-specific Ets-like factor (ESE-1) which is also detectable in the synovium of RA patients [114]. ESE-1 has been shown to upregulate Ang-1 indicating that this transcription factor regulates the high Ang-1 mRNA levels during RA [115].

Angiopoietin-2 functions during inflammation

Little is known about the mechanisms of Ang-2 function on Tie2. Recent studies have shown that Ang-2 supports RhoA and MLC activation and thereby promotes vascular leakage and endothelial cell migration [116]. Other studies have identified Ang-2 as a pro-inflammatory cytokine. Ang-2-deficient mice cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis [68]. Ang-2 serum levels are increased during sepsis. Normal serum levels are in the range of 1–2 ng/ml. During sepsis, Ang-2 levels may increase up to 20 fold (30 ng/ml). Elevated circulating Ang-2 levels have also been associated with mortality. More than 50% of patients with soluble Ang-2 levels in excess, i.e. about 20 ng/ml die [116119]. Furthermore, Ang-2 expression correlates with neovascularization during physiological and pathological processes, like arthritis [120] or psoriasis [121]. In both diseases, Ang-2 expression is not only associated with vessel remodeling but also with VEGF expression. Ang-2 and VEGF act together to induce angiogenesis and the expression of matrix metalloproteases, proteins that degrade the basement membrane [122]. However, Ang-2 induces vessel regression in the absence/inhibition of VEGF [2, 65, 66]. Ang-2 increases the expression level of the matrix metalloprotease MMP-2 in gliomas which is a sign for active angiogenesis [123]. Moreover, an anti-Ang-2 therapy in the cornea of rats was shown to inhibit VEGF-induced neovascularization [24]. Ang-2 expression is highly upregulated by angiogenesis-inducing molecules like VEGF, bFGF or TNF-α. Thrombin, an angiogenesis promoting molecule, but also hypoxia, is able to induce Ang-2 expression [17, 19, 20, 124128]. Hegen and co-workers could show that the activity of the Ang-2 promoter is regulated by the transcription factor Ets-1 [127]. The implication of Ets-1 in neovascularization has been shown in a mouse model of proliferative retinopathy [129]. Ets-1 dominant negative constructs injected in the eye completely blocked this function. Its expression is further upregulated by VEGF and shear stress which in turn increase Ang-2 expression [130, 131]. Ang-2 expression is also regulated by the transcription factor FOXO1. This family of transcription factors is involved in the upregulation of proteins during destabilization and remodeling. Ang-1 negatively interferes with FKHR-associated gene expression and thereby suppresses the production of Ang-2 [132].

Angiopoietin expression in tumors and tumor associated angiogenesis

Ang-2 is only weakly expressed in endothelial cells under physiological conditions. However, Ang-2 expression is dramatically increased during vascular remodeling, e.g., during tumor growth [133]. For example, glioblastoma show increased levels of Ang-2 in their associated endothelium [32]. Here, Ang-2 is highly expressed in necrotic and hypoxic regions [26]. Vessels in these areas are not covered by smooth muscle cells. Only small vessels in glioblastomas express high amounts of Ang-2 but not larger ones [32]. Overexpression of Ang-2 in a rat glioma model resulted in aberrant vessels with low SMC coverage [134]. Ang-2 is also detectable in significant concentrations in the circulation of tumor patients, e.g., in esophageal squamous cell cancer [135], hepatocellular carcinoma [136], and lung cancer [137]. The expression of Ang-2 in melanomas correlates with tumor progression [46]. Tumor cells have been shown to express Ang-2, e.g., stomach [122], colon [138], bladder carcinoma [139], melanoma [46], and non small cell lung cancer (NSCLC) [140].

In addition to promoting vessel regression as in glioblastomas, Ang-2 induces tumor neovascularization in combination with angiogenic growth factors such as VEGF or bFGF. Blocking experiments with Ang-2 neutralizing antibodies or fusion proteins massively decreased tumor growth [24]. Antibodies against Ang-2 not only inhibited Ang-2- but also VEGF-induced endothelial cell migration and proliferation during angiogenesis [141], which demonstrated enhancing functions of Ang-2 during VEGF-induced angiogenesis. Moreover, Ang-2 aptamers (RNAs that bind and thereby block proteins) inhibit bFGF-induced angiogenesis in the rat corneal assay [142]. In addition to promoting vessel regression and neovascularization, Ang-2 can also stimulate breast cancer metastasis in a Tie2-independent pathway by binding directly to integrin α5β1 [143]. However, Ang-2 overexpression in Lewis lung carcinoma and TA3 mammary carcinoma cells suppressed tumor growth. Angiogenesis was found to be disrupted and apoptosis was enhanced [144].

The role of Ang-1 in tumor-associated angiogenesis remains controversial. Ang-1 overexpression leads to reduced tumor growth in several tumor models [145147]. Pericyte coverage of the tumor vasculature is massively increased and thereby stabilized [147, 148]. Yet, Ang-1 has also been shown to promote tumor growth in rat gliomas [134] and in plasma cell tumors [149]. The downregulation of Ang-1 in HeLa cells by antisense RNA inhibited tumor growth and angiogenesis [150]. These findings suggest that Angiopoietin-1 promoting or inhibiting functions are dependent on the tumor cell type, the dosage and possibly on the amount of Ang-2 in the tumors.

Tie receptor independent signaling and non-vascular Angiopoietin effects

Several studies support the hypothesis that the Angiopoietins can activate endothelial or tumor cells in a Tie2-independent manner. It was shown that endothelial cells can adhere to immobilized Angiopoietins via αvβ3 and α5β1 integrin [151]. The direct binding of Ang-2 to α5β1 stimulates breast cancer metastasis through an α5β1 integrin-mediated pathway via Akt [pS473] [143] and induces glioma cell invasion by stimulating matrix metalloprotease-2 expression through αvβ1 integrin and FAK [152]. However, Ang-1 further triggers signaling pathways of Tie2 and α5β1 through their interaction in endothelial cell plated on fibronectin, thereby promoting angiogenesis [153]. Other studies showed that Ang-1 monomers (that do not activate Tie2) promote cardiac and skeletal myocyte survival and reduce cardiac hypertrophy through integrins [154, 155]. It has also been hypothesized that Angiopoietins can interact with integrins expressed by neuronal cells [156]. However, Ang-1 promotes neurite outgrowth from Tie2-positive dorsal root ganglion cells and activates PI3 K thereby preventing neuronal apoptosis [157].

Tie2 is also expressed by a subpopulation of hematopoietic stem cells and bone marrow osteoblasts. It has been shown that Ang-1, produced by osteoblasts, mediates the adhesion of hematopoietic stem cells to osteoblasts in an integrin-dependent autocrine manner [158]. Therefore, it has been suggested that constitutive Ang-1/Tie2 signaling controls the maintenance of the bone marrow stem cell niche.

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© Springer Science+Business Media B.V. 2009