ORIGINAL PAPER

The role of the Angiopoietins in vascular morphogenesis

Markus Thomas Æ Hellmut G. Augustin

Received: 3 February 2009 / Accepted: 24 April 2009 / Published online: 16 May 2009

� Springer Science+Business Media B.V. 2009

Abstracts The Angiopoietin/Tie system acts as a vascu-

lar specific ligand/receptor system to control endothelial

cell survival and vascular maturation. The Angiopoietin

family includes four ligands (Angiopoietin-1, Angiopoie-

tin-2 and Angiopoietin-3/4) and two corresponding tyro-

sine 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 phos-

phorylation 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 exoge-

nous 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 grad-

ually paving the way toward the rationale exploitation of

this vascular signaling system as a therapeutic target for

neoplastic and non-neoplastic diseases.

Keywords Angiogenesis � Endothelial cell �Angiopoietin � Tie

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 [1–5]. Structurally, the Angiopoietins are com-

posed of two domains. There is a N-terminal coiled-coil

domain which is responsible for ligand homo-oligomeri-

zation of the ligands. Electron microscopy experiments

have demonstrated that Ang-1 and Ang-2 can form heter-

ogeneous 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-ter-

minus [1, 7].

The Angiopoietins are secreted glycoproteins with a

dimeric molecular weight of approximately 75 kDa. Ang-1

M. Thomas � H. G. Augustin (&)

Joint Research Division Vascular Biology, Medical Faculty

Mannheim (CBTM), University of Heidelberg, Im Neuenheimer

Feld 280, 69120 Heidelberg, Germany

e-mail: augustin@angiogenese.de

Present Address:M. Thomas

Roche Diagnostics GmbH, Pharma Research Penzberg,

Penzberg, Germany

M. Thomas � H. G. Augustin

German Cancer Research Center Heidelberg (DKFZ-ZMBH

Alliance), Im Neuenheimer Feld 280, 69120 Heidelberg,

Germany

123

Angiogenesis (2009) 12:125–137

DOI 10.1007/s10456-009-9147-3

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 var-

iant (Ang-2(443)) has been identified which lacks parts of the

coiled-coil domain and cannot stimulate Tie2 phosphoryla-

tion [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 conflu-

ence, 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 [12–14].

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 con-

cept 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 [17–20]. 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 [22–24] and in the tumor cells themselves [25–27].

Moreover, retinal neurons [28] and Muller 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 myocar-

dium 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 neu-

ronal 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% simi-

larity in the extracellular part [33]. Tie1 and Tie2 are

tyrosine kinases with Ig-like and EGF-like homology

1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

Ang-1 actsthrough Akt

Ang-2 hasagonistic function

Generation ofTie2-Cre mice

Role of Ang-2 as acontextual remodelling

molecule

Role of Ang-2 role indiabetic retinopathy

Ang-1 restores vesselarchitecture in absence

of mural cells

Ang-2 nullmice

Ang-2 stored inWP bodies

Tie2-positivemonocytes

Low affinity Ang-1 /Tie1 binding

Ang-1 stimulateslymphangiogenesis

Destabilizing role ofautocrine Ang-2

Tie1 cleavage byshear stress

Ang-2 survival factorin stressed EC

Ang-2 role in hyperoxia-induced lung injury

Ang-1 interactionwith mDia

Ang-2 in macrophagecell death switch

Contextual Tie2signalling

Discoveryof Tie1

Constitutive pTie2in quiescent EC

Role of Tie2 inhematopoiesis

Tie2 associateswith Dok-R

Discoveryof Tie2

Knockout ofTie1 & Tie2

No role of Tie1 inhematopoiesis

Identificationof Ang-2

Identificationof Ang-1

Contextuality modelof Ang-2 function

Ang-1-inducedvascularization

Cooperativity betweenAngs and VEGF

Ang-1 vesselsealing function

Role of Ang-2 invessel cooption

Role of Tie-2 in AVmalformations

Ang-1 as an anti-apotopic molecule

Ang-2 primarilyproduced by EC

Ang-1 and Ang-2molecular structure

Ang-1-inducedEC sprouting

Cytokine regulation ofAng-2 expression

Identification ofAng-3/4

Rec

epto

rsLi

gand

s

Ang-1 reducesinflammatory markers

Ang-1 protects againstplasma leakage

Ang-1 controls thestem cell niche

Ang1-FOXO1 axisestablished

COMP-Ang-1developed

Ang-2 facilitates ECresponsiveness to

inflammatory stimuli

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

126 Angiogenesis (2009) 12:125–137

123

domains. The extracellular domain consists of three

immunoglobulin (Ig)-like domains that are flanked by three

epidermal growth factor (EGF)-like cysteine repeats fol-

lowed 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].

During early development, Tie1 can be detected from

E8.5 in differentiating angioblasts of the head mesen-

chyme, 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 stimula-

tion. The membrane-anchored cleaved form, containing the

cytoplasmic domain, may interact with Tie2 and is sup-

posed to be involved in Tie2 signaling [36–38]. Receptor

shedding also occurs after stimulation with the phorbol

ester PMA, stimulation with tumor necrosis factor alpha

(TNF-a), and by shear stress [39–41]. Tie1 is still largely

considered as an orphan receptor. Yet, recent work sug-

gests 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, endo-

thelial 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 macro-

phages [47]. Endothelial cells in larger vessels express Tie2

more abundantly when compared with smaller vessels [33,

43]. Tie2 expression is upregulated during tumor angio-

genesis [48–50]. 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).

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. Ves-

sels 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

Ig-like

EGF-like

Ig-like

FN III

cell membrane

TK I

TK II

extracellular

intracellular

Tie1/Tie2

A B

Tie1 Tie2

RKTY - 1101VNTTLY - 1107EKFTY - 1112AGI

RKA

1113 - YVNMSL

1119 - FENFT

1124 - YAGI

kinase domain

Grb2

p85

Dok-R

Shp-2

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

Angiogenesis (2009) 12:125–137 127

123

occur after PMA stimulation [56]. Circulating concentra-

tions of soluble Tie2 are increased in several vasculopa-

thies, 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 differ-

entiation 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 com-

plex ventricular endocardium. The endocardium is col-

lapsed and appears retracted from the myocardial wall. The

endothelial lining in the atria is collapsed and the trabec-

ulae are absent. Ang-1 deficiency also causes severe vas-

cular defects [30]. The mice show a much simpler and

immature primary capillary plexus. The distinction

between larger and smaller vessels is much less pro-

nounced. 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 dra-

matically 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 devel-

opment of the mice. Yet, overexpression in the adult has little

effect on vessel structure and heart development.

Transgenic mice overexpressing Ang-1 under the con-

trol of the keratin 14 (K14) promoter are viable and gen-

erally 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 nor-

mal cell–cell contacts between endothelial cells and

between endothelial and perivascular cells. The inter-

Fig. 3 Schematic representation of Angiopoietin signaling in regu-

lating the quiescent and the activated phenotype of the endothelium. aAng-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 sub-

sequent 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, inflam-

mation 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 autophos-

phorylation 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 NFjB

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 30-kinase;

Akt, protein kinase B; FAK, focal adhesion kinase; eNOS, endothelial

nitric oxide synthase; FKHR, forkhead transcription factor; VE-PTP,

vascular endothelial tyrosine phosphatase

c

128 Angiogenesis (2009) 12:125–137

123

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

P

P

p110p85

Grb2 Akt

FAKCas

Paxillinp85

p110

α vβ3

αvβ3

nucleus

Survivin

Caspase-9

eNOS

Bad

FOXO-1

Rho

Src mDiaSrcP

P

VE-PTP

Tie2

VE-cadherin

Ang-1

Ang-2

extracellular matrix

SerotoninHGF

HB-EGFPDGF-B

TGFβBMP pathway

Dok-R

Ras MAPKcascade

Cellproliferation

Vesselstabilization

Pericyte/SMCrecruitment

nucleus

EC

pericyte

P

P

EC

A

B

VEGFR2VEGF

Abin-2

IKK complex

NFκB NF B

inactive active

inflammation, thrombosis

Rho

extracellular matrix

mDia Src-P VEGFR2-P

VE-cadherin-P

Vascular leakage

Dok-RP

PNck Pak

RasGAP

Shp-2

p85

p110 Grb7P

P

P

P

P

FAK

Cell migrationEC

Tie2

Ang-1

Ang-2

κ

Angiogenesis (2009) 12:125–137 129

123

acts in a stimulating, agonistic manner on Tie2, whereas,

Ang-2 exerts antagonistic functions on Ang-1/Tie2 sig-

naling. 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 experi-

ments 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, 65–67].

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 postna-

tally 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 analy-

ses 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 lym-

phatic 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 [71–75]. 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]. How-

ever, 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 endo-

thelial 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 anti-

body 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

PDGFRb which is expressed by pericytes. PDGF-B

thereby acts as chemoattractant which promotes the pro-

liferation of SMCs and pericytes during their recruitment to

the endothelium [82]. PDGFRb 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 PDGFRb 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 PDGFRb-defi-

cient mice indicating additional mechanisms of pericyte

recruitment. Another regulator of SMC differentiation is

TGF-b which is upregulated by Ang-1 following PDGF-B

stimulation. In turn, Ang-1 is downregulated by TGF-b.

130 Angiogenesis (2009) 12:125–137

123

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-b is responsible for SMC differentiation and to render

the vasculature in its quiescent state [84–86]. 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 acti-

vated 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 ras-

GAP, 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 para-

cellular permeability. Additionally, Akt is activated to

induce endothelial cell survival and stability of the endo-

thelium through the phosphorylation of eNOS (Fig. 3a)

[88, 89]. Src is activated during VEGF-mediated angio-

genesis. 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 recrui-

ted 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 phenyl-

alanine markedly decreased the interaction of Grb2 with

Tie2. SHP2 association with phosphorylated Tie2 remained

unaffected. Conversely, mutation of pTyr-1112 to phenyl-

alanine reduced the association of SHP2 with the phos-

phorylated kinase domain (Fig. 2b) [96, 98]. These

findings indicate that Grb2 and SHP2 associate with

phosphorylated Tie2, thereby supporting intracellular sig-

naling processes, like activation of MAPK. Indeed, Ang-1

can activate MAPK in endothelial cells and in the aortic

ring assay [99–101]. 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 autophosphoryla-

tion 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 stim-

ulation [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 pre-

vents 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

Angiogenesis (2009) 12:125–137 131

123

adhesion to endothelial cells [106]. Cardiac allograft ath-

erosclerosis [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-jB

signaling [109–111]. 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-a [112, 113]. TNF-a but also IL-1b 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 tran-

scription 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 aur-

eus-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 [116–119]. 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 metalloproteas-

es, 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-a.

Thrombin, an angiogenesis promoting molecule, but also

hypoxia, is able to induce Ang-2 expression [17, 19, 20,

124–128]. Hegen and co-workers could show that the

activity of the Ang-2 promoter is regulated by the tran-

scription 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 sup-

presses 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 endo-

thelium [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 cover-

age [134]. Ang-2 is also detectable in significant con-

centrations 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 glio-

blastomas, 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 dem-

onstrated 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

132 Angiogenesis (2009) 12:125–137

123

can also stimulate breast cancer metastasis in a Tie2-

independent pathway by binding directly to integrin a5b1

[143]. However, Ang-2 overexpression in Lewis lung car-

cinoma 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 [145–147].

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 Angio-

poietins can activate endothelial or tumor cells in a Tie2-

independent manner. It was shown that endothelial cells

can adhere to immobilized Angiopoietins via avb3 and

a5b1 integrin [151]. The direct binding of Ang-2 to a5b1

stimulates breast cancer metastasis through an a5b1

integrin-mediated pathway via Akt [pS473] [143] and

induces glioma cell invasion by stimulating matrix me-

talloprotease-2 expression through avb1 integrin and FAK

[152]. However, Ang-1 further triggers signaling path-

ways of Tie2 and a5b1 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 hypothe-

sized that Angiopoietins can interact with integrins

expressed by neuronal cells [156]. However, Ang-1 pro-

motes 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 hemato-

poietic 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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