Signaling Mechanisms of the EndothelialAngiopoietin-Tie Pathway.

The regulation and effects of proteolytic processingof Tie1 receptor and autocrine Ang2 secretion inendothelial cells.

Prson Gautam

Degree project in applied biotechnology, Master of Science (2 years), 2011Examensarbete i tillämpad bioteknik 45 hp till masterexamen, 2011Biology Education Centre, Uppsala University, and Molecular Cancer Biology Program, ResearchPrograms Unit, Faculty of Medicine, Biomedicum Helsinki, University of HelsinkiSupervisor: Docent Pipsa Saharinen

Abstract

The vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-Tie signaling pathways

have pivotal roles in regulating endothelial cell functions during angiogenesis and

lymphangiogenesis. The angiopoietin-Tie pathway comprises Tie1 and Tie2 receptors and the

angiopoietin growth factors (Ang1, Ang2, Ang4, and its mouse ortholog Ang3). The Ang-Tie

pathway is important for the remodeling and maturation of the developing vasculature, and it also

contributes to pathological angiogenesis, such as in the tumors. Interestingly, blocking the

endothelial cell secreted growth factor, Ang2, has shown promising results in tumor growth

inhibition. Tie1 is regarded as an orphan receptor as the ligand for it has not been discovered,

while Tie2 is the receptor for the angiopoietins. However, it has been shown that Tie1 is

activated by angiopoietins, most likely via Tie2. Metalloproteases can cleave the ectodomain of

Tie1, and it has been suggested to regulate angiopoietin signaling. We investigated the signaling

mechanisms of Ang2 and Tie1, whose molecular functions are not well understood. Specifically,

we analyzed the regulation and effects of the proteolytic cleavage of the Tie1 ectodomain as well

as of autocrine Ang2 secretion in endothelial cells. The results suggest that the Tie1 ectodomain

cleavage might be differentially regulated in different endothelial cell types, and enlighten the

consequences of autocrine Ang2 secretion on Tie2 activation.

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Table of content

Abbreviations................................................................................................................................. iii

1. Introduction .................................................................................................................................1

1.1. Regulation of Angiogenesis and Lymphangiogenesis ..........................................................2

1.2. Angiopoietin-Tie Signaling pathway....................................................................................2

1.3. Tie1 .......................................................................................................................................5

1.3.1. Tie1 cleavage by Metalloproteases ................................................................................6

1.3.1.1. PMA ............................................................................................................................7

1.3.1.2. VEGF ..........................................................................................................................7

1.4. Tie2/TEK ..............................................................................................................................7

1.5. Angiopoietins ........................................................................................................................8

2. Aims of the research ....................................................................................................................9

3. Materials and Methods ..............................................................................................................10

3.1. Reagents ..............................................................................................................................10

3.2. Cell Culture .........................................................................................................................11

3.3. Retroviruses ........................................................................................................................12

3.4. Lentiviruses.........................................................................................................................13

3.5. Immunofluorescence staining .............................................................................................13

3.6. Cell lysis and immunoprecipitation ....................................................................................14

3.7. SDS-PAGE and Western blot .............................................................................................14

3.8. Hypoxia treatment...............................................................................................................15

4. Results .......................................................................................................................................16

4.1. PMA induced Tie1 cleavage varies between different endothelial cells/cell lines.............16

4.2. VEGF differentially stimulates Tie1 cleavage in BECs and LECs ....................................18

4.3. Cleavage-resistant Tie1mutant............................................................................................22

4.4. PMA induces the secretion of Ang2 in endothelial cells ....................................................23

4.5. Hypoxia-induced autocrine Ang2 secretion results in Tie2 translocation to cell-cell

junctions .....................................................................................................................................27

5. Discussion..................................................................................................................................29

6. Acknowledgements ...................................................................................................................32

7. References .................................................................................................................................33

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Abbreviations

Ab antibody

ABIN2 A20-binding inhibitor of nuclear factor-!B (NF- !B) activation2

Ang angiopoietin (ANGPT)

BEC blood microvascular endothelial cell

BSA bovine serum albumin

COMP-Ang1 cartilage oligomeric protein angiopoietin 1

DAPI 4’,6-diamidino-2-phenylindole

DMEM Dulbecco’s modified eagle medium

Dok-R Dok-related docking protein (DOK2)

DPBS Dulbecco’s phosphate buffer saline

ECBM endothelial cell basal medium

EGTA ethylene glycol tetraacetic acid

FBS fetal bovine serum

GRB2 growth factor receptor-bound protein2

HMEC-1 immortalized human microvascular endothelial cell

HUVEC human umbilical vein endothelial cells

HDMEC human dermal microvascular endothelial cells

Ig immunoglobulin

IP immunoprecipitation

kDa kilodalton

LEC lymphatic endothelial cell

NF- !B nuclear factor-!B

NRP neuropilin

PAGE polyacrylamide gel electrophoresis

PBS phosphate buffered saline

PFA paraformaldehyde

PI3K phophoinositide 3-kinase

PMA phorbol myristate acetate (12-O-tetradecanoylphorbol-13-acetate)

PMSF phenylmethylsulfonyl fluoride

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SDS sodium dodecyl sulphate

SHP2 SH2 domain-containing phosphatase

TBS tris buffered saline

VEGF vascular endothelial growth factor

VEGFR vascular endothelial growth factor receptor

VE-PTP vascular endothelial phosphotyrosine phosphatase (also known as PTPRB, protein

tyrosine phosphatase receptor type B)

Tie tyrosine kinase with immunoglobulin and EGF homology domains

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1. Introduction

In a vertebrate embryo, the first functional organ system to develop is the cardiovascular system,

which is required for the transport of oxygen and nutrients (and waste) to support the growth and

development of the embryo. The development of the vascular system occurs via two different

processes: vasculogenesis followed by angiogenesis. The formation of blood vessels by the de

novo formation of vascular endothelium is known as vasculogenesis, where the endothelial cells

are formed from mesoderm derived precursors known as angioblasts (Lancrin et al., 2009). After

the formation of a primitive vascular plexus through vasculogenesis further formation of new

blood vessels occurs by sprouting or splitting of the pre-existing blood vessels in the process of

angiogenesis (Risau, 1997).

Another vascular system, namely the lymphatic vascular system helps to return the fluid, solutes

and macromolecules that have extravasated from blood capillaries to the interstitial space, back

to the bloodstream (Witte et al., 2001). In contrast to blood circulation, the lymphatic system is

unidirectional carrying the fluid from tissues back to the bloodstream. Lymphatic system also

plays a vital role in eliciting the acquired immune response of the body as it carries the

lymphocytes and antigen presenting cells (APCs) to the lymph nodes (Olszewski, 2003).

The formation of the new lymphatic vasculature is termed as lymphangiogenesis. The lymphatic

vasculature develops after the formation of a functional blood vasculature. After the venous and

arterial specification of endothelial cells, a subpopulation of cardinal vein endothelial cells starts

to express lymphatic markers, such as the homeobox transcription factor Prox1, which regulates

the expression of lymphatic endothelial specific genes (Banerji et al., 1999; Wigle and Oliver,

1999). The specialized lymphatic precursor cells then migrate along the VEGF-C (vascular

endothelial growth factor C) growth factor gradient to form the first lymphatic structures, termed

the jugular lymphatic sacs.

The blood vessels are lined by the blood vascular endothelial cells (BEC), whereas the lymph

vessels are formed by the lymphatic endothelial cells (LEC), which can be differentiated from the

BECs based on the expression of specific lymphatic markers (Petrova et al., 2002; Saharinen et

al., 2004; Wick et al., 2007)

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1.1. Regulation of Angiogenesis and Lymphangiogenesis

Angiogenesis and lymphangiogenesis are complex processes, which are regulated by different

cell-cell and cell-matrix interactions, cytokines and growth factors. Two families of receptor

tyrosine kinases expressed on endothelial cells, the vascular endothelial growth factor receptors

(VEGFRs) and the Tie receptors and their corresponding ligands, the vascular endothelial growth

factors (VEGFs) and angiopoietins, respectively, play vital roles in the development of both

blood and lymphatic vessels (Augustin et al., 2009; Lohela et al., 2009). The VEGF-VEGFR-2

pathway stimulates the initial assembly of the blood vasculature, while the angiopoietin-Tie

system is required later for vessel maturation, integrity, remodeling and endothelial cell survival

(Augustin et al., 2009; Lohela et al., 2009).

Most solid tumors cannot grow unless they induce neoangiogenesis to support their oxygen and

nutrient needs. Limiting harmful angiogenesis via inhibition of the VEGF receptor signaling

pathway has turned out to be a relevant strategy to reduce tumor growth, and inhibitors blocking

VEGF (e.g. Bevacizumab, Avastin) (Ferrara et al., 2005) have been clinically approved for

cancer therapy and are in use in the clinics. However, as with most cancer therapies, the current

anti-angiogenic treatments induce only transient tumor growth control benefit. Thus, novel

molecular targets for tumor therapy are being searched. Recently, several studies have shown that

targeting the angiopoietin-Tie pathway may be the next potential treatment to block tumor

angiogenesis (Oliner et al., 2004; Hu and Cheng, 2009; Brown et al., 2010; Hashizume et al.,

2010; Mazzieri et al., 2011). Different molecules, including covX bodies, antibodies and

peptibodies that specifically target angiopoietin-2 (Ang2) and in some cases also Ang1have been

shown to inhibit tumor angiogenesis and tumor growth ( Oliner et al., 2004; Brown et al., 2010;

Huang et al., 2011).

1.2. Angiopoietin-Tie Signaling pathway

The angiopoietin-Tie signaling pathway comprises Tie1 and Tie2 (also called as TEK) receptor

tyrosine kinases (RTK) and their angiopoietin ligands (Ang1, Ang2, Ang4 and its mouse

ortholog Ang3) ( Partanen et al., 1992; Schnurch and Risau, 1993; Dumont et al., 1994; Lee et

al., 2004; Peters et al., 2004). The Tie receptors have a similar domain structure with three

epidermal growth factor (EGF) like domains embedded between three immunoglobulin (Ig) like

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domains, and followed by three fibronectin type III like domains in their extracellular domains

and a split tyrosine kinase domain in the intracellular domain (Figure1) (Partanen et al., 1992;

Sato et al., 1993). The intracellular domains of Tie1 and Tie2 have 76% sequence homology,

while the extracellular domains are less conserved with approximately 33% sequence homology

(Schnurch and Risau, 1993). Tie1 is regarded as an orphan receptor as the ligand for it has not

been discovered, while angiopoietins are the ligands for Tie2 (Suri et al., 1996). However, it has

been shown that Tie1 is activated by angiopoietins, most likely via Tie2 (Saharinen et al., 2005;

Yuan et al., 2007).

Figure 1. Structure of the Tie1 and Tie2 receptors.

Ang1-activated Tie2 activates severel downstream signaling pathways, including the Dok-related

docking protein (Dok-R; also known as DOK2) and growth factor receptor-bound protein2

(GRB2), which subsequently activate other effector molecules resulting in cell migration (Jones

and Dumont, 1999; Master et al., 2001). Cell survival signals are mediated via the SH2 domain-

containing phosphatase (SHP2) and the phophoinositide 3-kinase (PI3K) pathways (Augustin et

al., 2009; Fukuhara et al., 2008; Saharinen et al., 2008). Ang1-activated Tie2 also leads to the

recruitment of A20- binding inhibitor of nuclear factor-!B (NF- !B) activation2 (ABIN2) to

exert anti-inflammatory response by blocking the NF- !B pathway (Hughes et al., 2003). On the

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contrary, depending on the cellular context, Ang2 may counteract Ang1-Tie2 signaling, and in

vivo Ang2 enhances inflammation and vessel destabilization (Fiedler et al., 2006).

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Figure 2. Angiopoietin1-Tie2 downstream signaling pathways. (Modified from Huang et al., 2010).

The angiopoietin ligands utilize a unique mechanism for Tie receptor activation by inducing

translocation of the Tie receptors to the cell-cell and cell-matrix contacts (Fukuhara et al., 2008;

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Saharinen et al., 2008). In contacting cells Ang1 induces translocation of Tie2 to cell-cell

junction and the assembly of homotypic Tie2-Tie2 trans-associated complexes, which also

include vascular endothelial phosphotyrosine phosphatase (VE-PTP) and Tie1, leading to

establishment of vascular integrity through the activation of the Akt-eNOS signaling pathway. In

the absence of cell–cell contacts, matrix–bound Ang1 induces Tie2 translocation to cell-matrix

contacts and Tie2 activation that preferentially induces cell migration through activation of Dok-

R and Erk pathways (Fukuhara et al., 2008; Saharinen et al., 2008).

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Figure 3. Angiopoietin induced Tie2 translocation to cell-cell and cell-matrix junction. (Modified from Saharinen et

al., 2008)

1.3. Tie1

Tie1 is expressed as a duplet of 125 kDa and 135 kDa forms in endothelial cells, where the 135

kDa form represents the mature fully glycosylated form, and the 125 kDa form represents an

immature unglycosylated form of Tie1 (Yabkowitz et al., 1997). Tie1 is found associated with

Tie2 in ECs and they interact with each other (Saharinen et al., 2008; Seegar et al., 2010).

Although Tie1 does not bind any ligands it has been found to be activated by angiopoietins in

primary endothelial cells and also by Tie2 when co-expressed in non-endothelial cells (Saharinen

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et al., 2005; Yuan et al., 2007). It has also been shown using chimeric Tie1 receptors that Tie1

activation results in the activation of survival signal through PI3K-AKT signaling pathway

(Kontos et al., 2002).

Tie1 deficient mice have decreased vascular endothelial cell integrity, widespread oedema and

hemorrhage resulting eventually in death by E13.5 (Sato et al., 1995). When investigated in a

permissive background, it has been shown that deficiency of Tie1 causes impaired development

of the lymphatic vasculature (D'Amico et al., 2009; Qu et al., 2010).

1.3.1. Tie1 cleavage by metalloproteases

Tie1 is highly regulated in endothelial cells. Tie1 undergoes regulated proteolytic processing and

it has been shown that metalloproteases activated by PMA (phorbol myristate acetate), VEGF

and inflammatory cytokines cleave the Tie1 ectodomain (Yabkowitz et al., 1999; Marron et al.,

2007; Singh et al., 2009). The metalloprotease-mediated cleavage of the Tie1ectodomain occurs

at peptide bond between glutamine749 and serine750 proximal to the transmembrane domain

(Yabkowitz et al., 1999). The cleavage results in the formation of a 45 kDa endodomain, which

further undergoes "-secretase -mediated proteolytic processing resulting in a 42 kDa fragment

and that is consecutively degraded by proteasomal activity (Marron et al., 2007). The ectodomain

cleavage of Tie1 has been suggested to modulate the ligand responsiveness of Tie2, by increasing

the activation of Tie2 by cartilage oligomeric protein angiopoietin 1 (COMP-Ang1). However, it

remains to be found out, if the regulated proteolytic processing of Tie1 has an important function

in controlling Tie2 signaling in vivo.

Figure 4. Metalloprotease mediated Tie1 ectodomain cleavage.

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1.3.1.1. PMA

Phorbol myristate acetate is a biologically active substance extracted from corton oil, which is

regarded as a potential tumor promoting agent. The cellular receptor for PMA is protein kinase

C, whose activation results in a vast number of effects on cells including increased protease

activity and Tie1 ectodomain cleavage as well as secretion of Ang2 from Weibel-Palade bodies

in endothelial cell (Fiedler et al., 2004). It also modulates the synthesis of fibronectin, a

component of the extracellular matrix (Lee et al., 1996).

1.3.1.2. VEGF

VEGF, also known as VEGF-A (Ferrara, 1993), is the ligand for the vascular endothelial growth

factor receptor 1 (VEGFR-1) and VEGFR-2 (de Vries et al., 1992; Quinn et al., 1993). VEGF

also binds to neuropilin 1 (NRP1) and neuropilin 2 (NRP2) (Soker et al., 1998; Gluzman-

Poltorak et al., 2000). VEGF induces endothelial cell survival, proliferation, migration, sprouting

and tube formation (Ferrara, 2005; Gerber et al., 1998; Olsson et al., 2006). One report also

suggests that VEGF interacts and modulates the Tie1-Tie2 receptor complex (Tsiamis et al.,

2002).

1.4. Tie2/ TEK

Tie2 is constitutively expressed in endothelial cells (Augustin et al., 2009). In contrast to the

orphan receptor Tie1, Tie2 has been found to bind members of the angiopoietin family (Davis et

al., 1996; Maisonpierre et al., 1997; Peters et al., 2004). Angiopoietins bind to the second Ig-like

domain of Tie2 receptor that is flanked by the first Ig-like domain and the EGF-like domain

repeats (Barton et al., 2006; Macdonald et al., 2006) as shown in Figure 1. Tie2 is critical for the

development of the heart endocardium during mouse embryogenesis, and the embryos die

between E10.5 to E12.5 (Dumont et al., 1994; Takakura et al., 1998). In the Tie2 deficient

embryos, the primary capillary plexus is also not remodeled, indicating a critical role in vascular

remodeling and maturation (Dumont et al., 1994; Sato et al., 1995).

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1.5. Angiopoietins

Angiopoietins are soluble secreted proteins, which exist in various multimeric forms.

Angiopoietins have an amino (N) terminal superclustering domain, a coiled-coil domain and a

carboxyl (C) terminal fibrinogen-like domain, through which they bind to the Tie2 receptor

(Barton et al., 2006; Procopio et al., 1999). There are three members of angiopoietins that has

been discovered as ligands for Tie2. These are Ang1, Ang2, Ang3 and Ang4, where Ang3 and

Ang4 represent interspecies orthologs from mouse and human respectively (Valenzuela et al.,

1999). Ang1 is produced by vascular smooth muscle cells or pericytes and specialized pericytes

like podocytes of kidney (Satchell et al., 2002), while Ang2 is secreted by endothelial cells,

where Ang2 is stored in Weibel-Palade bodies (Fiedler et al., 2004). During tumor angiogenesis

endothelial Ang2 secretion is upregulated and increased Ang2 levels are found in the serum of

cancer patients (Szarvas et al., 2008). Ang1 and Ang4 act as agonists of Tie2, whereas Ang2 may

act as an agonist or antagonist of Tie2 in a context dependent manner (Maisonpierre et al., 1997;

Valenzuela et al., 1999; Yuan et al., 2009; Lee et al., 2004).

Ang1-deficient mouse embryos exhibit lethality between E9.5 and E12.5, very similarly to Tie2-

deficient embryos (Suri et al., 1996; Jeansson et al., 2011; Saharinen and Alitalo, 2011).

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2. Aims of the research

The project was part of a larger research project ongoing in the host laboratory and was aimed to

dissect the molecular mechanisms of the angiopoietin-Tie receptor tyrosine kinase signaling

pathway. My research was focused on the proteolytic processing of Tie1 receptor tyrosine kinase

by metalloproteases as that were induced by PMA and VEGF. The second topic was to uncover

the consequences of endothelial cell secreted autocrine Ang2 on Tie signaling. The main

objectives are summarized below:

1. To observe the metalloprotease-mediated proteolysis of Tie1 in different endothelial

cell types.

2. To analyze the effect of Tie1 cleavage on Tie2 phosphorylation.

3. To analyze the secretion of Ang2 on endothelial cells and the effect on Tie2

localization.

4. To examine the function of Tie1 in autocrine Ang2-mediated regulation of Tie2.

5. To analyze the hypoxia regulation of autocrine Ang2 secretion and its effects on Tie2.

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3. Materials and Methods

I performed all the experiments unless otherwise mentioned.

3.1. Reagents

All chemicals were purchased from Sigma-Aldrich unless otherwise mentioned. Gelatin was

purchased from Difco Laboratories, BSA from Bovogen Biologicals and Ang2 from R&D

systems. VEGF-A and VEGF-C were provided as a kind gift by Dr. Michael Jeltsch, University

of Helsinki. VEGF-A and VEGF-C were used at a final concentration of 100ng/ml, PMA at

50ng/ml, COMPAng1 at 200ng/ml unless otherwise stated. COMP-Ang1 was a generous gift

from Dr. Gou Young Koh (Korea Advanced Institute of Science and Technology, Republic of

Korea).

Table 1 Various antibodies used.

Antibody Manufacturer Concentration Dilution for

Coverslip

Staining

Dilution for

Western Blot

Analysis

goat anti-hTie1 R&D Systems 0.2mg/ml 1/100 1/4000

goat anti-hTie2 R&D Systems 0.2mg/ml 1/100 1/4000

rabbit-anti Tie2 sc-324 Santa Cruz 0.2mg/ml 1/200

mouse anti-V5-Cy3 Sigma-Aldrich 1mg/ml 1/500

goat anti-hAng2 R&D Systems 0.2mg/ml 1/200

mouse anti-hPECAM1

CD31

eBioscience 1/200

donkey anti-goat IgG Alexa

Fluor® 488

Invitrogen 2mg/ml 1/300

donkey anti-mouse IgG

Alexa Fluor® 488

Invitrogen 2mg/ml 1/300

donkey anti-rabbit IgG

Alexa Fluor® 488

Invitrogen 2mg/ml 1/300

donkey anti-goat IgG Alexa

Fluor® 594

Invitrogen 2mg/ml 1/300

donkey anti-mouse IgG

Alexa Fluor® 594

Invitrogen 2mg/ml 1/300

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donkey anti-goat IgG Alexa

Fluor® 594

Invitrogen 2mg/ml 1/300

donkey anti-rabbit IgG

Alexa Fluor® 594

Invitrogen 2mg/ml 1/300

donkey anti-rabbit IgG

Alexa Fluor® 594

Invitrogen 2mg/ml

1/300

donkey anti-rabbit IgG

Alexa Fluor® 647

Invitrogen 2mg/ml 1/300

biotinylated rabbit anti-goat

IgG

DakoCytomation 1.6mg/ml 1/4000

biotinylated goat anti-

mouse IgG

DakoCytomation 0.79mg/ml 1/4000

mouse anti-

phosphotyrosine, clone

4G10

Millipore 1mg/ml 1/15000

HSC-70 (B-6) Santa Cruz 0.2mg/ml 1/5000

3.2. Cell Culture

Primary human blood microvascular endothelial cells (BECs), primary human lymphatic

endothelial cells (LECs), human dermal microvascular endothelial cells (HDMECs) and human

umbilical vein endothelial cells (HUVECs) were purchased from PromoCell and cultured on

fibronectin coated plates (Greiner Bio One), except for HUVECs which were cultured on gelatin

coated plates. HMEC-1 immortalized human microvascular endothelial cells (Ades et al., 1992)

were grown on fibronectin coated plates. All endothelial cells were cultured in endothelial cell

basal medium (ECBM) with supplements provided by PromoCell. EAhy.926 cells, immortalized

hybrid cell line of HUVEC and A549 cells (Edgell et al., 1983), were grown on gelatin coated

plates in Dulbecco’s modified eagle medium (DMEM, Lonza) supplemented with 10% FBS

(PromoCell). 293gpg retroviral packaging cell line (Ory et al., 1996) was grown in DMEM with

10% FBS and tetracycline (1 #g/#l), neomycin (0.3 #g/#l), puromycin (2#g/#l). All cell types

were maintained in 5% CO2 incubator at 37° C. For immunoprecipitation (IP) and Western blot

the cells were rinsed with Dulbecco’s phosphate buffer saline (DPBS) and starved for 30 min in

ECBM without supplements (starvation medium) before addition of different stimulants,

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otherwise the cells were directly starved in starvation medium for 30min. Subsequently after

starvation, the stimulants, such as PMA, VEGF, COMP-Ang1 etc. were applied into the same

starvation medium.

Table 2 Primary cells and cell lines used.

Cells Cell type Growth Property Coating Material Medium

BEC Primary human blood

microvascular endothelial

Adherent Fibronectin ECBM

LEC Primary human lymphatic

endothelial

Adherent Fibronectin ECBM+VEGF-C

HUVEC Primary human umbilical vein

endothelial

Adherent Gelatin ECBM

HMEC-I Immortalized human

microvascular endothelial

Adherent Fibronectin ECBM

HDMEC Primary human microvascular

endothelial

Adherent Fibronectin ECBM+VEGF-C

EAhy.926 Hybridoma of HUVEC and A549 Adherent Gelatin DMEM

293gpg Retroviral packaging cell line Adherent No DMEM

A549 Adenocarcinomic human alveolar

basal epithelial cells

Adherent No DMEM

3.3. Retroviruses

For retroviral production the insert in the pMXs retroviral vector

(http://www.lablife.org/p?a=vdb_view&id=g2.ZESodFCNA.7.jO4HBZ1Syxo2U_0-) was

transfected into 293gpg retroviral packaging cells using FuGENE® 6 transfection reagent (Roche

Applied Science). Specifically, on the day before transfection cells were divided in 1:2 ratio to

obtain 50% confluent cells the next day. On the day of transfection, the cells were changed into 5

ml of transfection media (DMEM supplemented with 10% FBS, 20 mM HEPES, 0.05 mM

Glutamine and antibiotics). 30 µl of FuGENE was mixed with 600 µl of opti-MEM® I (1X)

reduced serum medium (Invitrogen) and incubated at room temperature for 5 min, and

subsequently, 10 µg of plasmid of interest was added to the mixture and incubated at room

temperature for further 30 min before application on 293gpg cells. After 5 hours of incubation

5ml of transfection media was added to the transfected cells. Media was completely replaced the

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next day, and viral supernatant was collected from the cells on day four, five and six following

transfection. After each collection of virus supernatant new media was applied to the plate. The

viral supernatant was centrifuged 13,000 X g for 3 min and filtered though a 45 µm pore size

filter, and stored at -70° C in aliquots.

The viral supernatants were tested by transduction of viruses to A549 cells (Giard et al., 1973;

Lieber et al., 1976). Specifically, virus-containing media was mixed with hexadimethrine

bromide (Polybrene, final concentration 8 µg/ml) (M&C Gene Technology), and applied on

A549 cells on coverslips. DMEM with supplements with 10% FBS was added after 5 hours and

the media was completely changed the next day. On the third day of transfection coverslips were

fixed and viral expression was tested using immunofluorescence staining. Viral transduction to

endothelial cells was performed as explained above for A549 cells.

All viral works were performed in a Biosafety level 2 laboratory. Before starting the viral work, I

was educated on the safety procedures required to work with viruses as well as handling of the

virus containing waste. All works were performed in a safety hood, lab coat and gloves were

required and the laboratory was kept locked and unauthorized personnel were not allowed to

enter.

Retrovirus coding for Tie1 chimera (hTie1-eGFP-hTie2) and wild type (hTie1-eGFP) were

produced and transduced to endothelial cells.

3.4. Lentiviruses

Lentiviruses coding for an shRNA against the Tie1 (A6 Tie1 shRNA) were used to suppress the

expression of Tie1 in endothelial cells and as a control, lentiviruses coding for control shRNA

(H1 scramble shRNA) were used. Kirsi Manttari from our laboratory produced these lentiviruses.

Lentiviral transduction to the endothelial cells was performed as explained above for the

retroviral transduction.

3.5. Immunofluorescence staining

All procedures were performed at room temperature. Following stimulation, the cells were fixed

with 4% paraformeldehyde (PFA) - PBS for 10 min at room temperature, washed with PBS 3 x 5

min and permeabilized with 1% Triton X-100 in PBS for 5 min, if intracellular epitopes were to

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be analyzed. The cells were blocked with 1% BSA-PBS for 5 min and incubated with primary

antibodies in 1% BSA-PBS for 30 min, washed with PBS 3 x 5 min, blocked as above and

incubated with fluorochrome conjugated secondary antibodies for 30 min. The cells were

mounted using Vectashield mounting media with DAPI (Vector Laboratories). Images were

obtained using epifocal microscope (Zeiss Axioplan 2 microscope) or using the laser scanning

confocal microscope (Zeiss LSM 5 Duo), both available at BIU (http://www.biu.helsinki.fi/).

3.6. Cell lysis and immunoprecipitation

Following stimulations, the cells were lysed with ice cold PLCLB lysis buffer (50mM Hepes, pH

7.5, 150 mM NaCl, 5% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM

Na4P207x10H2O) containing protease inhibitors (aprotinin, leupeptin, PMSF) and phosphatase

inhibitors (sodium orthovanadate, 100 mM NaF). Lysates were cleared by centrifugation at +4°

C, 14,000 X g for 10 min. Total protein concentration of the lysates was measured with BCATM

Protein Assay Kit (Thermo Scientific). The same amount of protein was always used for IP and

running of total cell lysates in Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE). The volumes of the lysates were adjusted to equal by adding PLCLB lysis buffer

containing inhibitors.

For IP, the lysates were incubated with 1 #g / ml of specific antibodies at +4o C. Then, 20 #l of

drained protein G sepharose (GE Healthcare) suspended in lysis buffer was added to the

immunocomplexes, and subsequently, incubated at +4o C for 1 hour. The immunoprecipitates

were centrifuged at 13,000 X g for 2 min, and the protein G sepharose beads were washed 4

times with PLCLB lysis buffer containing sodium orthovanadate. 5 X reducing Laemmli sample

buffer was added to the immunoprecipitates, and the immunoprecipitates were boiled for 5 min

to elute the bound protein.

3.7. SDS-PAGE and Western blot

The protein samples were separated in 7.5% SDS-PAGE (Precast gel; Bio-Rad Laboratories) in a

mini-PROTEAN® Tetra System (Bio-Rad Laboratories), and transferred to Whatman® PROTAN

nitrocellulose membrane (Perkin Elmer TM) using a Semi-Dry Transfer Cell (Bio-Rad

Laboratories). The transfer was confirmed by staining the membrane with Ponceau-S. The

membrane was blocked with 5% BSA-0.05% TBS-TWEEN to prevent nonspecific binding of

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detection antibodies. The membrane was incubated at room temperature for 1 hour, or at +4

overnight with specific primary antibodies, at room temperature for 30 min with biotinylated

anti-goat or anti-mouse secondary antibodies followed by streptavidin biotin HRP conjugate for

20 min (Amersham Biosciences), all diluted in 5% BSA-0.05% TBS-TWEEN. The membrane

was washed after each antibody incubation for 3 x 10 min using 0.5% TBS-TWEEN. After final

washes the membrane was washed 3x 10 min with TBS. The proteins were detected using the

SuperSignal® ECL West Femto Maximum Sensitivity Substrate (Thermo Scientific), and

chemiluminescence was captured by exposure of Fuji medical X-ray films (Fujifilm).

3.8. Hypoxia treatment

BECs grown on coverslips in complete ECBM growth medium were transferred into the Invivo2

400 hypoxia chamber (Ruskinn Technology) and incubated at 1% O2 (hypoxia) for 16 hours or

maintained at normoxic conditions at 37° C. After 16 hours the cells were fixed and stained for

Ang2 and Tie2.

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4. Results

4.1. PMA-induced Tie1 cleavage varies between different endothelial cells

Previously, it has been shown that PMA induces proteolytic cleavage of Tie1 on the surface of

endothelial cells, specifically on HUVECs and HMEC-1 cells. We tested if Tie1 cleavage differs

between different endothelial cell lines or primary endothelial cells. When the primary

endothelial cells BECs, LECs, HUVECs and HDMECs as well as immortalized endothelial

HMEC-I cells were starved for half an hour in ECBM starvation media and treated with 50 ng/ml

PMA for 30 min, the extracellular domain of Tie1 was cleaved in the PMA-treated, but not

control samples, as evidenced by staining of fixed cells with an anti-Tie1 antibody, which

recognizes the extracellular domain of Tie1 (Figure 5). Furthermore, the Tie1 signal was almost

completely lost in all endothelial cell types when treated with PMA. On the contrary, there was

no effect on Tie1 in EA.hy926 cells even when the cells were treated with 100 ng/ml PMA for 1

hour. This indicated that PMA-induced Tie1 cleavage is a general phenomena in endothelial

cells, however, not all endothelial cells appear to be able to respond to PMA by inducing Tie1

cleavage. The specific molecular mechanisms responsible for Tie1 cleavage have not yet been

found out, and it is possible that some of the components required for the Tie1 cleavage are not

present in EA.hy926 cells.

! $*! !

Figure 5. PMA causes Tie1 ectodomain cleavage in endothelial cells. Different endothelial cells: HMEC-1,

HUVEC, HDMEC, LEC and BEC cells were starved for 30 min, stimulated with 50 ng/ml of PMA for 30 min or left

unstimulated, and stained with antibodies against the extracellular domain of Tie1. There was a significant decrease

of Tie1 ectodomain in PMA treated cells, but not in control cells or in EA.hy926 cells treated with 100 ng/ml of

PMA for 1 hour.

! $+! !

4.2. VEGF differentially stimulates Tie1 cleavage in BECs and LECs

It has been reported that VEGF also induces Tie1 ectodomain clevage, but with lower

efficiciency than PMA (Yabkowitz et al., 1999). We decided to study the effect of VEGF on

different endothelial cells as VEGF is a physiologial ligand, whereas PMA induces pleiotropic

effects on cells. Two different types of endothelial cells, BECs and LECs, were challanged with

VEGF, or PMA or left untreated. Interestingly, VEGF induced the cleavage of Tie1 in the BECs,

but not in the LECs. There was a significant loss of Tie1 immunostaining in the VEGF-treated

BECs; on the other hand there was hardly any detectable effect on the LECs. Such differences

have not previously been reported (Figure 6).We also tested the effect of VEGF-C on the LECs

and BECs, but it did not induce Tie1 cleavage in either of cell types (Figure 7). PMA, however,

induced Tie1 cleavage in both types of endothelial (Figure 6).

The results from the immunofluorescence staining were confirmed by Western blotting. The cells

were treated with PMA, VEGF or left unstimulated. Before cell lysis, the growth media was

collected. Tie1 was captured from the cell lysates by immunoprecipitating with antibodies

recognizing the extracellular domain of Tie1. The IPs were separated in SDS-PAGE, and Tie1

was detected by immunoblotting with the same antibody used for IP. In VEGF-treated BECs, but

not in LECs, we found a significant decrease in the upper 135 kDa Tie1 band, which represents

the fully matured glycosylated form of Tie1. In contrast, there was a complete loss of the upper

135kDa form in both PMA-treated BECs and LECs. The lower 125kDa band which represents

the immature unglycosylated form of Tie1 remained unaffected in all samples (Figure 8.a).

! $,! !

Figure 6.!VEGF induces the cleavage of Tie1 in BECs but not in LECs. BECs and LECs were starved for 30min in

ECBM without supplements and subsequently stimulated for 30min. PMA induced Tie1 cleavage in both cell types,

whereas VEGF induced the cleavage only in BECs but not in LECs.

! %-! !

!

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Figure 7. VEGF-C does not induce Tie1 cleavage in BECs nor in LECs. BECs and LECs were starved for 30min in

ECBM without supplements and subsequently stimulated with 100ng/ml of VEGF-A and VEGF-C for 30min or left

unstimulated. Tie1 staining was reduced in the VEGF-A-treated BECs, whereas there was no effect on VEGF-C

treated BECs or LECs.

!The result was also further supported by Western blot analysis of the cell culture media of the

same samples. Tie1 ectodomain was immunoprecipitated from the media collected after cell

stimulation, and analyzed in SDS-PAGE. The 100kDa band represents the cleaved Tie1

ectodomain. There was a significant increase of Tie1 ectodomain accumulation in the media in

VEGF-treated BECs, but not LECs, while in both cell types PMA induced the appearance of the

Tie1 ectodomain in the cell culture media (Figure 8.c).

! %$! !

Figure 8. The effect of PMA and VEGF on Tie1 ectodomain cleavage in LECs and BECs. LECs and BECs were

stimulated with VEGF or PMA for 30min or left unstimulated, followed by immunoprecipitation of Tie1 from the

cell lysates and growth media. (a) Reduction in the level of the upper 135kDa band (full length Tie1) was detected in

VEGF-treated BECs, whereas in the LECs Tie1 levels remained unchanged. PMA caused a complete loss of the

upper Tie1 band in both BECs and LECs (d) shows quantification of the 135kDa Tie1 band in different samples,

quantification was done using Fiji software. (b) To control equal protein concentration in the samples, HSC-70 was

blotted from total cell lysates. (c) Media was immunoprecipitated with anti-Tie1 antibody and detected by

immunoblotting with the same anti-Tie1 ab recognizing the Tie1 extracellular domain. Increased levels of the 100

kDa (cleaved Tie1 ectodomain) were detected in both PMA treated LECs and BECs, while VEGF induced the

increase in Tie1 ectodomain levels only in BECS but not LECs.

! %%! !

4.3. Cleavage-resistant Tie1 mutant

We next thought to investigate if it was possible to create a Tie1 form that was resistant to

proteolysis. To this, we compared the peptide sequence of Tie1 to that of Tie2, and predicted the

secondary structures using PHYRE web server (http://www.sbg.bio.ic.ac.uk/~phyre/)

(collaboration with Dr. Veli-Matti Leppänen, Molecular/Cancer Biology Laboratory, Helsinki).

We decided to replace amino acids 740-760 in Tie1, containing the site for proteolysis, with the

corresponding amino acids 732-746 from Tie2. We then cloned this Tie1 mutant in pMXs

retroviral vector (cloning done by Dr. Seppo Kaijalainen, Molecular/Cancer Biology Laboratory,

Helsinki), and confirmed it by sequencing. The Tie1-Tie2 chimeric protein encoding retrovirus

was produced and used to infect BECs. To analyze the proteolytic properties of the Tie1-Tie2

chimera, the BECs were transduced with retrovirus coding chimeric Tie1 (hTie1-eGFP-hTie2)

and challenged with 50 ng/ml PMA for 30 min or left unstimulated. As a positive control the

BECs were transduced with wild type Tie1 (Tie1-eGFP) and treated similarly as the hTie1-

eGFP-hTie2 mutant. PMA induced the ectodomain cleavage of wild type Tie1, whereas hTie1-

eGFP-hTie2 was not cleaved (Figure 9). We also noticed that the hTie1-eGFP-hTie2 was not

optimally expressed on the cell surface, possibly due to secretory problem.

Figure 9. The effect of the cleavage site mutation on Tie1 ectodomain shedding. a) BECs were transduced with

retrovirus coding wildtype hTie1 with eGFP tag (hTie1-eGFP). Following 30 min starvation the transduced cells

were treated with PMA or left unstimulated and stained as indicated. The Tie1 extracellular domain was completely

depleted in PMA treated cells. b) BECs were transduced with retrovirus coding chimeric hTie1-Tie2 with eGFP tag

(hTie1-eGFP-hTie2). Similarly, as in a) the cells were treated with PMA after starvation and stained as indicated.

The PMA could not induce the cleavage of hTie1-eGFP-hTie2.

! %&! !

4.4. PMA induces the secretion of Ang2 in endothelial cells

Ang2 is secreted by endothelial cells in an autocrine fashion (Augustin et al., 2009; Veikkola et

al., 2003) and Ang2 secretion can be stimulated with PMA (Augustin et al., 2009). Confluent

cultures of BECs were starved for 30 min and stimulated with PMA 50ng/ml. Cell surface Tie1

was stained in the fixed cells, and Ang2 after permeabilization of the cells. Along with the Tie1

cleavage, PMA induced Ang2 secretion, and interestingly, Ang2 could be detected in the

endothelial cell-cell junctions in the PMA-treated, but not control cells (Figure 10). Furthermore,

we also detected translocation of Tie2 into the cell-cell junctions in PMA treated cells, while in

the control cells Tie1 was uniformly localized in the cells.

Increased Tie2 signal in the cell-cell contacts was seen in the PMA-treated samples but when the

cells were pre-treated with a monoclonal antibody against Ang2, Tie2 translocation to cell

junctions was prevented (Figure 11). These results suggest that PMA induces the secretion of

Ang2 and ultimately translocation of Tie2 to the cell-cell junctions.

We next suppressed Tie1 expression and analyzed the effect of PMA on the endothelial cells.

BECs were transfected with lenti viruses coding for shRNA against Tie1 (A6 Tie1-shRNA) or

control shRNA (H1 scramble-shRNA) and after two days stimulated with PMA. PMA had

similar effects on Tie1 and scramble shRNA transfected cells. We could see punctate Tie2

staining in the cell-cell contacts in Tie1 shRNA treated cells, suggesting that PMA induced Ang2

secretion and Tie2 activation, and these occurred irrespective of Tie1 (Figure 12).

! %'! !

!

Figure 10. PMA induces Ang2 secretion and its accumulation at cell-cell contacts. BECs were starved for 30 min in

ECBM without supplements, stimulated with PMA for 30 min or left untreated and stained as indicated. Ang2 could

be seen localized in the cell junctions.

! %(! !

Figure 11. PMA induces Ang2 secretion, which further induces Tie2 accumulation at cell junctions. BECs were

starved for 30 min and treated either with PMA for 30 min, 2 #g/ml anti-Ang2 antibody for 45 min or with the

combination of anti-Ang2 antibody and PMA (15 min anti-Ang2 followed by 30 min PMA) or left untreated and

stained as indicated. Tie2 translocation to cell junctions was prominent in PMA-treated cells, whereas it was uniform

on the cell surface in other treatments.

! %)! !

!

Figure 12. Tie1 is not required for PMA-induced Ang2 secretion and Tie2 translocation to cell junctions. BECs

were transfected with control shRNA (H1 scramble-shRNA) or shRNA against Tie1 (A6 Tie1-shRNA) using lenti

viruses. The virus transfected BECs were challenged with 50 ng/ml of PMA for 30min or left unstimulated and

stained as indicated. There was a clear traslocation of Tie2 into the cell-cell contacts in both control shRNA and

Tie1-shRNA trasfected cells.

! %*! !

4.5. Hypoxia-induced autocrine Ang2 secretion results in Tie2 translocation to cell-cell

junctions

To investigate if autocrine Ang2 results in the deposition of Tie2 in endothelial cell-cell junctions

in more physiological conditions, we decided to culture BECs under hypoxia conditions. It is

known that Ang2 mRNA expression increases during hypoxia, resulting in increased intracellular

and secreted Ang2 protein level (Pichiule et al., 2004), but the direct consequences on endothelial

Tie2 activation have not been studied. The confluent cultures of BECs in complete growth

medium were kept in the hypoxic chamber maintaining 1% oxygen for 16 hours or incubated in

the normal oxygen concentration in the 5% CO2 incubator for same time. After 16 hours, the

cells were fixed and stained for Tie2. We found increased Tie2 accumulation in the cell-cell

junctions in the cells exposed to hypoxic conditions (1% oxygen) compared to the cells kept

under normoxia (Figure 12). This result elaborated that hypoxia stimulates the release of Ang2

stored in the endothelial Weibel-Palade bodies. The secreted Ang2 further binds to the

endothelial Tie2 receptors, which translocate to cell-cell junctions and form the Tie2-Tie2 trans

complexes.

! %+! !

Figure 12. Hypoxia induces Ang2 secretion in endothelial cells. The confluent cultures of BECs grown in complete

growth medium were kept in the hypoxic chamber maintaining 1% oxygen level or maintained in the normal oxygen

concentration in the cell culture incubator for 16hr and stained as indicated. We could see increased Tie2

accumulation in the cell-cell junctions of cells kept in hypoxic condition when compared to cells grown in normoxia.

!

! %,! !

5. Discussion

During development, the VEGF-VEGFR system induces the initial assembly of the vasculature,

while the Ang-Tie system is required after the initial assembly for vascular remodeling and

maturation (Augustin et al., 2009). It has been shown that activation of the Ang-Tie siganaling

pathway regulates endothelial cell survival, migration, sprouting and permeability via activation

of different downstream signaling pathways. In vivo, the Ang-Tie system regulates vascular

remodeling, maturation, inflammation and permeability. The knockout mouse models have

shown that dysfunctional Ang-Tie signaling results in impaired angiogenesis and

lymphangiogenesis, while the Ang-tie system also contributes to tumor progression. However,

the understanding of the Ang-Tie signaling system at the molecular level is far from complete.

Specifically, the functions of Ang2 and Tie1 have remained elusive. Here we have studied some

aspects of the Ang2 and Tie1 signaling.

It has been previously shown that Tie1 on the endothelial cell surface undergoes metalloprotease-

mediated proteolytic processing (Yabkowitz et al., 1999; Marron et al., 2007). We found that

PMA induced the ectodomain cleavage of Tie1 in all primary endothelial cells investigated, but

not in EA.hy926 cells, indicating that the Tie1 ectodomain cleavage is differentially regulated in

different endothelial cell types. Importantly, VEGF appeared to regulate cell surface expressed

Tie1 in blood, but not in lymphatic endothelial cells. It should be noted that both BECs and LECs

express VEGFR-2, the receptor for VEGF. Both cell types also express VEGFR-3, the receptor

for VEGF-C, but VEGF-C did not induce the cleavage of Tie1 in either cell types. This means

that in certain situations, e.g. during sprouting angiogenesis, where VEGF is present, Tie1 might

be cleaved from blood vessels, while it would not be cleaved in lymphatic vessels. We, and

others (Marron et al., 2007), have hypothesized that Tie1 might modulate the response of

endothelial cells to angiopoietins. In vivo work shows that blood and lymphatic vessels respond

differentially to Ang1 and Ang2. Ang1 deficient mice have abnormal vasculature phenotype and

impaired heart development exhibiting death between E9.5 to E12.5 (Suri et al., 1996; Jeansson

et al., 2011). Ang1 deletion also results in higher overall number of vessels, increased vessel

diameter and unorganized vascular network (Jeansson et al., 2011). Ang1 is dispensible in

healthy adult vasculature, but is critical during microvascular injury and wound healing

(Jeansson et al., 2011). The Ang2 knockout mice on the other hand, have persistent vascular

! &-! !

defect although the embryonic vascular development is not perturbed, implying its requirement

during postnatal vascular remodeling. Although angiopoietins are not required for the initiation

of lymphatic vascular development, Ang2 seems to have a critical role in subsequent lymphatic

vascular remodeling and maturation and Ang2 knockout mice exhibit defective lymphatic

vasculature. Introduction of Ang1 to such Ang2 deficient mice in the Ang2 locus has been shown

to rescue the lymphatic, but not blood vascular defects (Gale et al., 2002), highlighting the

differential role of Ang1 and Ang2 in blood and lymphatic vasculatures. Our results suggest that

differential cleavage of Tie1 in the LECs and BECs might contribute to the differential

Ang1/Ang2 signaling in lymphatic and blood vessels, respectively.

However, the exact molecular mechanism regulating the proteolytic processing of Tie1 and

subsequent effects on Tie2 signaling are yet to be uncovered. Furthermore, the biological

significance of Tie1 cleavage is still to be found out. We envisioned that by creating cleavage-

resistant Tie1 mutants we might learn more about Tie1 function. To this, we analyzed one such a

mutant, and showed that it was expressed on endothelial cell surface, but was not cleaved.

However, our designed Tie1 chimera did not show optimal expression pattern in the endothelial

cells, as much of the produced Tie1 chimera seemed to be retained in vesicles inside the cells

instead of being expressed on the cell surface. Thus, additional mutations should be tested to

obtain a chimeric Tie1, which retains good expression (work ongoing). The cleavage resistant

Tie1 is useful for investigating specific effects of the Tie1 cleavage on endothelial cells.

It has been shown that Ang1 and Ang2 induce the translocation of Tie2 to the cell-cell or cell-

matrix contacts, depending on the presence of contacting cells (Fukuhara et al., 2008; Saharinen

et al., 2008). In the cell-cell contacts, angiopoietins induce homomeric Tie2 complexes in trans

across the cell-cell junctions. These complexes also include Tie1 and VE-PTP, and the Ang1-

Tie2 complex further activates the eNOS-signaling pathway (Saharinen et al., 2008). However,

the function of autocrine Ang2 in inducing junctional Tie2 activation has not been previously

analyzed.

Our results showed that along with the Tie1 cleavage, PMA induced Ang2 secretion and Tie2

translocation to the cell junctions in endothelial cells, and this could be blocked using a function-

blocking anti-Ang2 antibody. The PMA-induced Tie2 translocation occurred irrespective of Tie1

cleavage, as we saw the translocation of Tie2 to the cell-cell junctions in cells, where Tie1 was

downregulated via shRNA lentivirus. We also tried to look for the effect of Tie1 cleavage on

! &$! !

Tie2 phosphorylation but were unable to come with the consistent results, hence not mentioned

here.

Our results also showed that during hypoxia, Ang2 secretion is increased, which induces the

formation of an Ang2-Tie2 complex at endothelial cell-cell junctions. This is the first time when

autocrine Ang2 has been found to stimulate Tie2 translocation to cell junctions, and that this

occurs in hypoxic endothelial cells. The results suggest that autocrine Ang2 has a role in the

modulation of endothelial cell-cell junctions. It can be speculated that increased Ang2 secretion

by the endothelial cells of the hypoxic tumor blood vessels results in the formation of Ang2-Tie2

complexes in endothelial cell junctions. In such vessels, Ang2 would replace the Ang1-Tie2

complexes that are thought to stabilize the quiescent stage of the vessels, and via modulation of

cell-cell contacts eventually facilitate VEGF-driven neoangiogenesis. It will be interesting to

study, if hypoxia, via increased Ang2, will result in tyrosine phosphorylation of Tie2, or if the

effects of autocrine Ang2 are mediated by other means than Tie2 activation. Future work is also

required to investigate the signals that autocrine Ang2 induces in hypoxic endothelial cells via

Tie2.

! &%! !

6. Acknowledgement

It would not have been possible to complete the project without help and support of many people.

I am obliged to my supervisor Docent Pipsa Saharinen for providing me an opportunity to work

in her project. I would like to express my deepest gratitude to her for believing on me and for her

kindness, support, motivation and invaluable guidance and supervision.

I am really grateful to Kirsi Mänttäri and Anita Lampinen from our lab for their kindness,

constant support and guidance. I really appreciate their willingness to help me. I have to admit

that it would be very hard for me to work without them. I would also like to extend my special

thanks to the people from Prof. Kari Alitalo’s group for their unconditional help, support and

advice.

I would like to express my sincere gratitude towards my friends Satish Adhikari and Ramesh

Baral for their moral support and encouragement in my good and bad times.

Finally and very importantly I would like to thank my beloved parents and brother for their

endless love, support, understanding and motivation throughout my study period.

! &&! !

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