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).
! $,! !
Figure 6.!VEGF induces the cleavage of Tie1 in BECs but not in LECs. BECs and LECs were starved for 30min in
ECBM without supplements and subsequently stimulated for 30min. PMA induced Tie1 cleavage in both cell types,
whereas VEGF induced the cleavage only in BECs but not in LECs.
! %-! !
!
!
Figure 7. VEGF-C does not induce Tie1 cleavage in BECs nor in LECs. BECs and LECs were starved for 30min in
ECBM without supplements and subsequently stimulated with 100ng/ml of VEGF-A and VEGF-C for 30min or left
unstimulated. Tie1 staining was reduced in the VEGF-A-treated BECs, whereas there was no effect on VEGF-C
treated BECs or LECs.
!The result was also further supported by Western blot analysis of the cell culture media of the
same samples. Tie1 ectodomain was immunoprecipitated from the media collected after cell
stimulation, and analyzed in SDS-PAGE. The 100kDa band represents the cleaved Tie1
ectodomain. There was a significant increase of Tie1 ectodomain accumulation in the media in
VEGF-treated BECs, but not LECs, while in both cell types PMA induced the appearance of the
Tie1 ectodomain in the cell culture media (Figure 8.c).
! %$! !
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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