vascular endothelial growth factors b and c: gene
TRANSCRIPT
Vascular Endothelial Growth Factors B and C:
Gene Regulation and Gene Transfer In Vivo
Berndt Enholm
Molecular/Cancer Biology Laboratory
Haartman Institute and
Helsinki University Central Hospital
Biomedicum Helsinki
University of Helsinki
Finland
Academic Dissertation
To be publicly discussed with the permission of the Faculty of Medicine of the University
of Helsinki, in Auditorium 2, Biomedicum Helsinki, 6th of August, 2004 at 12 noon
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Hic Labor,
Hoc Opus Est
Vergilius
Aeneas, song 6. verse 129
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Supervised by:
Kari Alitalo, MD, Ph.D.
Research Professor of the Finnish Academy of Sciences
Molecular/Cancer Biology Laboratory
Biomedicum Helsinki
University of Helsinki
Reviewed by:
Ulf-Håkan Stenman, MD. Ph.D.
Department of Clinical Chemistry,
Helsinki University and Helsinki University Central Hospital
Biomedicum Helsinki
Olli Ritvos M.D., Ph.D.
Developmental and Reproductive Biology Program
Biomedicum Helsinki
University of Helsinki
Opponent:
Wolfgang Schaper MD, Ph.D.
Professor of Physiology
University of Giessen-Germany
Director of the Max Planck Institute for Physiological and
Clinical Research (W.G. Kerckhoff-Institut)
Bad Nauheim
Germany
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TABLE OF CONTENTSTABLE OF CONTENTS .........................................................................................................................................5
ABBREVIATIONS...................................................................................................................................................7
ORIGINAL PUBLICATIONS................................................................................................................................9
ABSTRACT.............................................................................................................................................................10
REVIEW OF THE LITERATURE......................................................................................................................11
THE VASCULAR SYSTEM......................................................................................................................................11THE BLOOD VASCULAR SYSTEM .........................................................................................................................11
Embryonic Development, Structure and Function ........................................................................................11Angiogenesis in the Adult ...............................................................................................................................12
MOLECULAR REGULATORS OF THE BLOOD VASCULAR SYSTEM .......................................................................14VEGF...............................................................................................................................................................14VEGF-B...........................................................................................................................................................15PlGF ................................................................................................................................................................16VEGF Homologues Encoded by Orf and Parapox Viruses ..........................................................................17VEGFR-2 and VEGFR-1 ................................................................................................................................17Neuropilins......................................................................................................................................................18Ephs and Ephrins............................................................................................................................................18Platelet Derived Growth Factors (PDGFs)...................................................................................................18Angiopoietins and Tie Receptors....................................................................................................................19
MARKERS OF THE VASCULAR SYSTEM................................................................................................................19PECAM-1 ........................................................................................................................................................19vWF..................................................................................................................................................................20VE-cadherin ....................................................................................................................................................20
PATHOLOGIC ANGIOGENESIS AND ARTERIOGENESIS..........................................................................................21Angiogenesis in Ischemic Disease..................................................................................................................21Arteriogenesis .................................................................................................................................................21Tumor Angiogenesis .......................................................................................................................................22
THE LYMPHATIC SYSTEM ....................................................................................................................................23Embryonic Development, Structure and Function ........................................................................................23
MOLECULAR REGULATORS OF THE LYMPHATIC SYSTEM...................................................................................25VEGF-C and VEGF-D....................................................................................................................................25VEGFR-3.........................................................................................................................................................26
MARKERS OF THE LYMPHATIC SYSTEM ..............................................................................................................27Lymphatic Vessel Endothelial Hyaluronan Receptor-1 (LYVE-1)................................................................27Podoplanin ......................................................................................................................................................27Prox-1..............................................................................................................................................................27
PATHOLOGIC LYMPHANGIOGENESIS....................................................................................................................28Tumor Lymphangiogenesis.............................................................................................................................28Lymphedemas..................................................................................................................................................28
THERAPEUTIC APPROACHES ................................................................................................................................30Vascular Gene Therapy ..................................................................................................................................30Viral Vectors ...................................................................................................................................................30Proangiogenic Therapy ..................................................................................................................................31Lymphangiogenic Therapy .............................................................................................................................31
AIMS OF THE STUDY .........................................................................................................................................32
MATERIALS AND METHODS...........................................................................................................................33
RESULTS AND DISCUSSION.............................................................................................................................35
I. STUDIES ON THE REGULATION OF VEGF, VEGF-B, VEGF-C AND ANG-1 MRNA EXPRESSION..................35II. PROINFLAMMATORY CYTOKINES REGULATE EXPRESSION OF VEGF-C.......................................................36III. ADENOVIRAL EXPRESSION OF VEGF-C IN THE SKIN INDUCES LYMPHANGIOGENESIS...............................39IV. VEGF-B ACTIVATES VEGFR-1, BUT PROVIDES VERY LITTLE ANGIOGENIC OR ARTERIOGENICACTIVITY IN VIVO IN COMPARISON WITH PLGF..................................................................................................40
CONCLUDING REMARKS.................................................................................................................................42
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ACKNOWLEDGEMENTS...................................................................................................................................43
REFERENCES........................................................................................................................................................44
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ABBREVIATIONS
AAV Adeno associated virusAd AdenovirusAng AngiopoietinbFGF Basic fibroblast growth factorCAM Chorioallantoic membrane assayCEP Circulating endothelial progenitorEC Endothelial cellECM Extracellular matrixEGF Epidermal growth factorFlt-1 fms-like tyrosine kinase-1Flt-4 fms-like tyrosine kinase 4FOXC2 Forkhead box C2GM-CSF Granulocyte monocyte-colony stimulating factorHA HyaluronanHIF Hypoxia inducible factorHMW-MAA High molecular weight human melanoma associated antigenHRE Hypoxia regulated elementHSPG Heparan sulphate proteoglycanHUVEC Human umbilical vein endothelial cellIL InterleukinIg ImmunglobulinIGF Insulin like growth factorLacZ β-galactosidase geneLD Lymphedema distichiasisLYVE-1 Lymphatic vessel endothelial hyaluronan receptor-1MCP-1 Monocyte chemoattractant protein-1mRNA Messenger ribonucleic acidNrp NeuropilinORF Open reading framePDGF Platelet derived growth factorPDGFR Platelet derived growth factor receptorPECAM-1 Platelet endothelial cell adhesion molecule-1Prox-1 Prospero related homeobox protein-1RTK Receptor tyrosine kinaseSLC Secondary lymphoid chemokineSH Src homology domainTek Tunica interna endothelial cell kinase-1TGF-β Transforming growth factor-βTie Tyrosine kinase with Ig and EGF homology domainsTK Tyrosine kinaseTNF-α Tumor necrosis factor-αVEGF Vascular endothelial growth factorVEGFR Vascular endothelial growth factor receptorvWF Von Willebrand factor
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ORIGINAL PUBLICATIONS
This thesis is based on the following articles, which will be referred to by their Roman
numerals.
I Enholm B, Paavonen K, Ristimäki A, Kumar V, Gunji Y, Klefström J, Kivinen L,
Laiho M, Olofsson B, Joukov V, Eriksson U, Alitalo K. Comparison of VEGF,
VEGF-B, VEGF-C and Ang-1 mRNA regulation by serum, growth factors,
oncoproteins and hypoxia. (1997) Oncogene, 14(20):2475-83.
II Ristimäki A, Narko K, Enholm B, Joukov V, Alitalo K. Proinflammatory cytokines
regulate expression of the lymphatic endothelial mitogen vascular endothelial
growth factor-C. (1998) J. Biol. Chem., 273(14):8413-8.
III Enholm B, Kärpänen T, Jeltsch M, Kubo H, Stenbäck F, Prevo R, Jackson DG,
Ylä-Herttuala S, Alitalo K. Adenoviral expression of vascular endothelial growth
factor-C induces lymphangiogenesis in the skin. (2001) Circ. Res., 88(6):623-9.
IV Enholm B, Tammela T, Paavonen K, Jeltsch M, Mustjoki S, Carmeliet P, Eriksson
U, Ylä-Herttuala S, Alitalo K. VEGF-B acitvates VEGFR-1, but provides very little
angiogenic or arteriogenic activity in vivo in comparison with PlGF. (2004).
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ABSTRACT
The unfolding of the molecular biology of the vascular system has provided the scientific
community with an array of molecular tools for investigation of the circulatory system as
well as for therapeutic intervention in a number of disorders of the vasculature. The
circulatory system consists of the cardiovascular system and the lymphatic system that
consists of lymphatic capillaries, collecting ducts, the thoracic duct and lymph nodes.
While the cardiovascular system has been thoroughly studied and its role in disorders such
as ischemic heart disease, peripheral vascular disease and in the growth of solid tumors is
well known, the lymphatic system has until recently attracted very little attention from the
scientific community. The discovery of specific growth factors and their receptors and an
array of markers has enabled investigators to study the vascular system in detail. In
particular, the Vascular Endothelial Growth Factor (VEGF) family members VEGF,
VEGF-B, Placenta Growth Factor (PlGF), VEGF-C and VEGF-D and their receptors
VEGFR-1, -2 and -3 have facilitated understanding of the vasculature and provided tools
and targets for therapeutic intervention. Of the VEGF family members, VEGF induces
angiogenesis i.e. growth of new blood vessels from pre-existing ones, whereas PlGF
primarily mediates arteriogenesis, the formation of collateral arteries from pre-existing
arterioles. VEGF-C and VEGF-D have both angiogenic and lymphangiogenic effects.
This study examined aspects of the biology of VEGF-B and VEGF-C in vitro and in
vivo. We showed that VEGF-C mRNA expression is controlled by serum growth factors
and inflammatory mediators, suggesting a function for VEGF-C in inflammation. VEGF-B
mRNA levels did not respond to any of the stimuli tested. In vivo, expression of VEGF-C
in the skin by adenoviral gene transfer induced the formation of lymphatic vessels de novo,
or lymphangiogenesis. The lymphangiogenic effect of VEGF-C could be used in gene
therapy aimed at reconstructing lymphatic vessels in patients suffering from disorders of
the lymphatic system such as hereditary or acquired lymphedema. Over-expression of
VEGF-B in vivo had very little effects on the vasculature compared to the two other
VEGFR-1 agonists, VEGF and PlGF.
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REVIEW OF THE LITERATURE
The Vascular System
The circulatory system consists of the heart and the blood circulation. The blood vascular
system is further divided into arterial and venous compartments. The blood vascular system
supplies the tissues with oxygen and nutrients and transports catabolic end products away
from tissues. The lymphatic system collects extravasated fluid, returns it to the blood, and
serves as an immunosurveillance organ. The growth and remodeling of the vascular system
is controlled in part through peptide growth factor signals transmitted via cell membrane
bound receptor tyrosine kinases (RTKs) that relay extracellular signals to the cytoplasm and
on to the nucleus where transcription of genes is readjusted. Perhaps the most important
groups of growth factors involved in the development and remodelling of the vascular
system are the VEGF family of growth factors (VEGF, VEGF-B, VEGF-C and VEGF-D
and their cognate receptors VEGFR1-3) and the Tie (tyrosine kinase with Ig and EGF
homology domains)-angiopoietin family (angiopoietins-1 to -4 and their cognate receptor
Tie-2, as well as Tie-1). These signalling systems are attractive targets for pro- or
antiangiogenic therapy aiming at vascular remodelling in various diseases ranging from
ischemic heart disease to suppression of angiogenesis in solid tumors.
The Blood Vascular System
Embryonic Development, Structure and Function
During embryogenesis, endothelial cells (ECs) develop from mesenchymal precursors
termed angioblasts and subsequently assemble into tubes that form a honeycomb-like
structure, the primary vascular plexus1,2(Figure 1). Recruitment of resident and circulating
angioblasts for formation of the primary vascular plexus is termed vasculogenesis.
Angioblasts originally develop from hemangioblasts, common precursors of hematopoietic
cells and ECs, that form aggregates called blood islands in the embryo (Figure 1).
Angioblasts express VEGFR-2 and angioblast differentiation is influenced by VEGF3-5 ,
however, VEGFR-1 signalling supresses angioblast differentiation6. After the formation of
the primary vascular plexus the development of the embryonic vasculature continues by
angiogenesis, the formation of new arteries and veins from the primary vascular plexus by
endothelial cell sprouting and splitting from pre-existing vessels (Figure 1). Embryonic
oxygenation initially depends on diffusion, but as tissues expand hypoxia sets in and
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triggers signalling through hypoxia inducible transcription factors (HIFs)7. Downstream
effectors of HIFs include VEGF which mediates proliferation, sprouting and migration of
ECs8.
Structurally, the mature blood vessel wall consists of an inner layer of ECs
separated by a basal lamina from a perivascular coat formed by smooth muscle cells
(SMCs) and pericytes. The perivascular support structures have an important function in
providing vascular tone and they are required for the formation of mature, stable vessels.
Furthermore, vessel enlargement and endothelial cell sprouting in angiogenesis as well as
in formation of vascular collaterals are dependent on the disruption of this coat9.
Angiogenesis in the Adult
Angiogenesis involves a tightly regulated cascade of events12. ECs respond to the mitogenic
signals from angiogenic factors such as VEGF. Blood vessels lose their pericyte coverage,
the basement membrane and the extracellular matrix (ECM) are degraded by proteases and
formed EC tubuli are subsequently covered and stabilized by deposition of a basement
membrane and recruitment of perivascular pericytes and SMCs9. SMCs are important
regulators of vascular tone and provide support for the larger vessels. Angiogenesis in
healthy adults occurs only during certain processes such as in the ovary and endometrium
during the female reproductive cycle and the placenta during pregnancy14. Angiogenesis
also occurs during hair follicle development and exercise-induced remodelling of muscles11.
Angiogenesis also happens in a variety of pathophysiological conditions including wound
healing, solid tumor growth, ischemic disease such as ischemic heart and peripheral
Figure. 1 Embryonic development of the vasculature starts with the development of angioblasts fromhemangioblast precursors, subsequent vasculogenesis and angiogenesis and finally recruitment of mural cellsfor stabilisation of the newly formed vessels (Adapted from Carmeliet 2003 and Conway 200110,11).
Hemangioblastprecursor
Angioblasts VasculogenesisAngiogenesis
Mural cellrecruitment
Maturevasculature
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disease, psoriasis, atherosclerosis, development of vascular tumors such as hemangiomas
and during pathological ocular neovascularisation15,16.
Recently the blood circulation has been suggested to contain circulating endothelial cell
progenitors (CEPs) which incorporate into newly formed vessels at sites of active
angiogenesis17-20. The contribution of CEPs to neovascularisation mimics embryonic
neovascularisation by differentiation of circulating angioblasts and is thus also termed
vasculogenesis. CEPs express a variety of endothelial cell surface markers including VEGF
receptors, platelet endothelial adhesion molecule-1 (PECAM-1) and von Willebrand factor
(vWF). CEPs are mobilised from the bone marrow by cytokines such as VEGF and
PlGF21,22. Skeletal muscle has also been suggested to contain cells with stem cell properties
that can contribute to angiogenesis, raising the possibility that tissues can contain resident
angioblasts23. CEPs are incorporated into forming blood vessels during physiological
angiogenesis such as during the female reproductive cycle24, during pathologic
angiogenesis in experimental solid tumors or in the limb muscles or the myocardium during
ischemia24-27. Furthermore, CEPs can develop a non-thrombogenic surface on endovascular
grafts after transplantation 28,29.
Figure 2. Vascular remodelling requires the interplay of different cell populations. Macrophages are recruitedto sites of active angiogenic cytokine production, followed by detachement of pericytes and SMCs andendothelial cell sprouting and/or vessel enlargenment. Remodelled blood vessels are stabilised by recruitedmural cells and failure to recruit these results in regression of the newly formed vessels (Adapted fromCarmeliet 2003 and Conway 200110,11).
Vasculogenesis
Macrophage recruitment
Rerecruitment of pericytes
= Macrophage = Hemangioblast = Endothelial cells = Mural cells
Angiogenesis Vessel regression
Remodeled, mature blood vessel
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Molecular Regulators of the Blood Vascular System
Figure 3. The angiogenic and arteriogenic members of the VEGF family of growth factors and their cognatereceptors. Extracellular immunoglobulin homology domains are represented by white circles, theextracellular domains of Nrp-1 and Nrp-2 are shown in striped boxes and tyrosine kinase (TK) domains arerepresented by black boxes.
VEGF
VEGF is the ligand for VEGFR-1 and VEGFR-2 and one of the key players in the
induction of blood vessel formation30 (Figure 3). Human VEGF is expressed as seven
different splice isoforms consisting 121, 138, 145, 162, 165, 189 and 206 amino acid
residues respectively, these differ in their binding affinity for heparin and in their
interaction with neuropilins (Nrp) 1 and 2. VEGF is an EC mitogen, motogen,
chemoattractant and survival factor that increases the permeability of blood vessels31. The
importance of VEGF in the formation of the vascular- and hematopoietic systems is
exemplified by vegf gene haploinsuffiency in mice with one inactivated vegf allele5. Mice
deficient in VEGF die early in embryonic development due to defects in angiogenesis and
hematopoiesis4. Partial inactivation of VEGF function by deletion of isoforms 164 and 188
affects postnatal angiogenesis in the heart and kidney, retinal vascular patterning and
enchondral bone formation32-34. Loss of approximately 50% of progeny suggests that the
NRP-2 NRP-2
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functions of VEGF isoforms are not redundant. Selective expression of VEGF120 resulted
in defective vascular outgrowth and patterning and selective expression of VEGF188 gave
rise to impaired arterial development35. Overexpression of VEGF in vivo induces blood
vessel formation as evidenced by adenoviral overexpression of VEGF in the skin of
mice36,37. Transcription of vegf is regulated by a variety of growth factors such as basic
Fibroblast Growth Factor (bFGF), Transforming Growth Factor-β (TGF-β) and Platelet
Derived Growth Factor (PDGF) as well as by oncogenes such as ras38. Importantly, VEGF
transcription is governed by hypoxia through interaction of hypoxia responsive elements
(HREs) in the VEGF gene promoter with HIF-1α, a transcription factor that mediates
intracellular hypoxic signaling39,40. Induction of VEGF expression by growth factors,
hypoxia and oncogenes mediates angiogenesis in solid tumors and in tissue ischemia7,41,42.
VEGF-B
VEGF-B is a ligand for VEGFR-1 and Nrp-1 (Figure 3). It can form heterodimers with
VEGF43-46(Table 1). VEGF-B is expressed as two different isoforms consisting of 167 or
186 amino acid residues47. VEGF-B167 binds heparan sulphate proteoglycans (HSPGs) and
is mostly sequestered in the ECM while VEGF-B186 is freely diffusible. In vivo, VEGF-
B167 is the predominant form and is abundantly expressed in brown fat, myocardium and
skeletal muscle48. VEGF-B may regulate the plasminogen activator activity in ECs45.
Deletion of the VEGF-B gene does not result in an obvious phenotype in mice49. These
mice do however have somewhat smaller hearts and a prolonged PQ interval in
electrocardiograms and they recover less well from myocardial ischemia than wild-type
mice4950. Furthermore, one report has claimed that mice lacking VEGF-B fail to develop
pulmonary hypertension in response to hypoxia, suggesting a role for this growth factor in
hypoxic pulmonary vascular remodelling51. Strikingly, VEGF-B was also reported to
protect mice lacking VEGF-B from the development of hypoxic pulmonary hypertension in
a manner comparable to VEGF52. Lack of VEGF-B does not affect development of the
retinal vasculature and neither does it affect development of hypoxia induced retinopathy of
the newborn53.
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Table 1. Structure of VEGF family members.
Characteristic VEGF-A(Genatlasdatabase)
PlGF30,76 VEGF-B45 VEGF-C77 VEGF-D78
Gene 15.9 kb,7 exons
13.9 kb,7 exons
3.71 kb,7 exons
109.2 kb,7 exons
38.86 kb,7 exons
Protein spliceisoforms
VEGF-A121,
VEGF-A138,VEGF-A145,VEGF-A162,VEGF-A165,VEGF-A189,VEGF-A206
PlGF-1(PlGF131),PlGF-2(PlGF152),PlGF-3
VEGF-B167,VEGF-B186
PrecursorVEGF-C388
PrecursorsVEGF-D358,VEGF-D326
Chromosomelocation
6p12 14q24.3 11q13 4q34 Xp22.31
Sequencehomology
Isoformactivities aremodulated bytheir heparansulfate andneuropilinbinding;
42% sequenceidentity toVEGF-A
45% sequenceidentity toVEGF-A
30%homologousto VEGF-A165
61% sequenceidentity toVEGF-C;31% identicalto VEGF-A165
Dimerization Homodimers;heterodimerwith VEGF-Band PlGF
Homodimers,heterodimerswith VEGF-A
Homodimers;heterodimerswith VEGF-A
Homodimers;heterodimerswith VEGF-D
Homodimers;heterodimerswith VEGF-C
PlGF
PlGF and VEGF-B share properties such as VEGFR-1 and Nrp-1 binding and the capacity
to form heterodimers with VEGF (Table 1 and Figure 3). Of the three reported human
isoforms of PlGF (PlGF-1, -2 and -3)54-57 only PlGF-2 binds HSPGs58. Only the PlGF-2
isoform has been found in mice. PlGF/VEGF heterodimers bind VEGFR-2 and VEGFR-
1/VEGFR-2 heterodimers in vitro59,60. PlGF is predominantly expressed in the placenta,
heart and in the lungs61. Heterozygous deletion of the plgf gene does not result in any
apparent phenotypic change, these mice do however suffer from impaired recovery after
experimental myocardial infarcts and from impaired collateral formation during hind limb
ischemia62. Furthermore, administration of recombinant PlGF induced angiogenesis and
collateral formation after ligation of coronary or femoral arteries in wild-type mice63.
Overexpression of PlGF in the skin of transgenic mice resulted in a hypervascular
phenotype with increased inflammatory and permeability responses64,65 and local
administration of PlGF using recombinant adenoviruses or recobinant peptides induced the
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formation of enlarged, leak-resistant blood vessels by arteriogenesis63 in a macrophage-
dependent manner66. Activation of VEGFR-1 by PlGF or VEGF was reported to induce
different gene expression profiles by phosphorylation of distinct tyrosine residues in the TK
domain of VEGFR-167 and combined administration of these factors enhanced VEGF
driven angiogenesis in vitro and in vivo62,68.
VEGF Homologues Encoded by Orf and Parapox Viruses
Genes with sequence homology to VEGF were discovered in two strains of orf viruses and
one strain of pseudocowpox viruses. These viruses denoted ORFVNZ2, ORFVNZ769 and
PCPV(VR634)70 , cause highly vascularised and edematous pustular dermatitis in sheep,
goats and occasionally in humans. Two of these viral VEGFs specifically bind and activate
VEGFR-2 and Nrp-171-73 whereas one binds only VEGFR-270. Consequently,
overexpression of ORFVNZ7 VEGF in the skin of transgenic mice results in a non-
edematous angiogenic phenotype77. Although viral VEGFs do not play a role in mammalian
vascular biology, they can be used as VEGFR-2 specific agonists in experimental models
of angiogenesis in vitro and in vivo.
VEGFR-2 and VEGFR-1
VEGFR-1 is a receptor for VEGF, VEGF-B and PlGF (Figure 3). VEGFR-1 by itself
transmits only weak mitogenic signals in ECs78 but it forms heterodimers with VEGFR-2
with stronger signalling properties than both VEGFR-1 and VEGFR-279. Targeted
disruption of vegfr1 results in embryonic lethality from an excess of ECs and disturbed
organisation of ECs into blood vessels6. The promoter of vegfr1 contains HREs and unlike
for VEGFR-2 and VEGFR-3, transcription of VEGFR-1 is induced by hypoxia41. In the
embryo, VEGFR-1 is expressed in angioblasts and ECs but is downregulated later in
development6,80,81. Chemotaxis of macrophages and macrophage mediated arteriogenesis is
relayed via VEGFR-1 signalling63,66,82. Furthermore, VEGFR-1 may mobilise bone-marrow
derived stem cells that incorporate into sites of active arteriogenesis62. A alternatively
spliced soluble form of VEGFR-1, sFlt-1, inhibits VEGF function and supresses tumor
angiogenesis83. This soluble form of VEGFR-1 has also been implicated in pre-eclampsia84.
VEGFR-2 binds VEGF, VEGF-C, VEGF-D and VEGF-E and relays important signals for
endothelial cell survival as well as in the migration and proliferation of ECs in VEGF
driven angiogenesis (Figure 3). Selective activation of VEGFR-1 and VEGFR-2 has shown
that VEGFR-2 is the primary receptor transmitting VEGF signals in ECs85. VEGFR-2 is
expressed by ECs and by embryonic angioblasts during development. In the adult, VEGFR-
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2 is expressed by ECs during angiogenesis4,5,86. Disruption of the gene for VEGFR-2 in
transgenic mice results in embryonic lethality at an early developmental stage due to
defects in angiogenesis, vasculogenesis and hematopoiesis3. The expression of vegfr2 is
autoregulated: VEGF, VEGF-C and VEGF-D upregulate the expression of VEGFR-287,88
(III). Blockade of VEGFR-2 effectively blocks VEGF driven angiogenesis in experimental
tumors in mice89.
Neuropilins
The Neuropilins, Nrp-1 and Nrp-2 are receptors for semaphorins, molecules that govern
axonal guidance during neuronal development90,91. Nrp-1 and Nrp-2 are expressed by ECs
and VEGFs bind to them in an isoform specific fashion56,92,46,93,94 (Figure 3). Accordingly,
targeted disruption of Nrp1 and/or Nrp2 results in disturbed formation of the vasculature,
defects of the nervous system and, in the case of Nrp-1, lethality during embryonic
development95-98.
Ephs and Ephrins
The Ephs are transmembrane receptor TKs that direct axonal guidance during neural
development as well as migration and morphogenesis of other cells types such as ECs.
Ephs relay bidirectional signalling through interaction with ephrins, a class of cell
membrane bound ligands. In the vascular system ephrin-B2 and EphB4 form a ligand
receptor pair, whose components are specifically expressed in arteries and veins,
respectively. It has therefore been suggested that bidirectional signalling by this ligand
receptor pair relays repulsive signalling between the cell populations that eventually form
arteries and veins during embryonic develoment99. Targeted disruption of ephrin B2 or
EphB4 results in defective arterio-venous patterning of the primary vascular plexus and
consequent embryonic lethality100-102.
Platelet Derived Growth Factors (PDGFs)
The PDGFs function as paracrine growth factors during development and as mitogens for a
wide array of cells such as fibroblasts, SMCs and ECs. The eight cysteine residues of the
VEGF growth factors are conserved in the PDGFs103. Structurally, the PDGFs consist of
homo- and heterodimers formed by four different monomers: PDGF-A, PDGF-B, PDGF-C
and PDGF-D that bind their cognate RTKs PDGFR-α and PDGFR-β103. Receptor
specificity is determined by dimer composition, PDGF-AA, -AB, -BB and PDGF-CC can
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induce αα-receptor homodimers, PDGF-AB and -BB can induce αβ-receptor heterodimers
and PDGF-BB and PDGF-DD can induce ββ-receptor dimers. PdgfB and Pdgfrβ are
essential for vascular development as the deletion of either gene results in embryonic
lethality due to hemorrhage caused by failure to assemble a mural-cell coating of blood
vessels104-106. Deficiency of PDGF-A and PDGFR-α causes embryonic lethality due to non-
vascular developmental defects107-110. In the adult, PDGF signalling occurs in wound
healing and is implicated in atherosclerosis, fibrotic diseases and cancer111,112.
Angiopoietins and Tie Receptors
The vascular structures are regulated in part by a family of receptors called the Tie
receptors and their ligands, the angiopoietins113. The Tie receptors, Tie-1 and Tie-2 have
divergent functions during development; Tie-1 is required for the structural integrity of
blood vessels during development, and a defiency in Tie-1 results in hemorraghe, edema
and embryonic death at E13.5114,115. Tie-2, or tunica interna endothelial cell kinase-1 (Tek),
mediates vascular-pericyte interactions in vessel remodeling and maturation114-117. While the
ligand for Tie-1 is not known, Tek has two ligands, Ang-1 and Ang-2, which have opposite
functions, Ang-1 is an agonist and Ang-2 is an antagonist of Tek. This theory is supported
by studies in transgenic mice in which overexpression of Ang-2 has a phenotype similar to
that of mice deficient in Ang-1118,119. The role of the Tie-Ang system in angiogenesis seems
to be the maturation of newly formed blood vessels by interactions with pericytes and
SMCs. Overexpression of Ang-1 in transgenic mice or by adenoviral gene transfer blocks
vessel permeability as induced by VEGF and co-expression of VEGF and Ang-1 induces
the formation of enlarged vessels that are leak-resistant120. On the other hand, it has been
suggested that Ang-2 induces detachment of pericytes from blood vessels and thereby
facilitates endothelial cell responses to EC mitogens121. Interestingly, Ang-2 knock-out
mice have defects in the lymphatic vasculature and chylous ascites, suggesting a role for
angiopoietin-2 in lymphatic development122. The lymphatic, but not the blood vascular
phenotype of these mice can be rescued by substitution of Ang-1 in place of Ang-2122.
Markers of the Vascular System
PECAM-1
PECAM-1 is a 130 kD glycoprotein member of the immunoglobulin (Ig) superfamily that
is abundantly expressed on the lateral junctions of endothelial cells as well as on the surface
of platelets and leukocytes123. PECAM-1 consists of six extracellular Ig domains, a
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transmembrane domain and a cytoplasmic domain. The ligands for PECAM-1 include
itself, CD38 and αvβ3 integrin. Phosphorylation of intracytoplasmic tyrosyl residues of
PECAM-1 mediates the recruitment of proteintyrosine phosphatases and results in
supression of TK signalling124. In angiogenesis, PECAM-1 is involved in the formation and
stabilization of endothelial cell-cell contacts and maintenance of a vascular permeability
barrier125-127. PECAM-1 also mediates transendothelial migration of monocytes and
neutrophils128.
vWF
Factor VII related antigen (FVIIIRA) or vWF is a multimeric protein produced by
endothelial cells that mediates clotting on injured endothelial surfaces. vWF stains the
endothelium of especially larger vessels whereas capillaries stain more weakly and
lymphatic ECs are negative for vWF expression129,130. Thus, vWF expression demonstrates
specificity for certain EC populations.
VE-cadherin
Cell-cell and cell-matrix adhesions are mediated by integrins, selectins and cadherins. VE-
cadherin is an EC-specific adhesion molecule localised to the adherens junctions of most
ECs populations131. Targeted deletion of VE-cadherin is lethal as mice deficient in VE-
cadherin fail to develop a proper vascular architechture132. Among vascular markers, VE-
cadherin is unique in that it is not expressed in hematopoietic precursor cells or circulating
blood cells. VE-cadherin inhibits VEGFR-2 signalling and EC growth in a density
dependent manner in vitro133,134.
Pericyte Markers
Markers for the SMCs and pericytes that coat the endothelium in blood vessels and
lymphatic vessels include α-smooth muscle actin (α-SMA), PDGF-Rβ, desmin, human
melanoma associated antigen (HMV-MAA) and its murine counterpart NG2. α-SMA is
expressed in pericytes and SMCs covering arterioles, arteries and post-capillary venules in
normal human and murine tissues135. α-SMA expression in pericytes is altered in tumors136
and can be induced by TGF-β137. Smooth muscle actin is not expressed in the pericytes that
cover capillaries138. PDGFR-β is co-expressed with the HMW-MA on pericytes and
mediates recruitment of pericytes to form blood vessels139. HMV-MAA is a proteoglycan
originally described as a molecule expressed on pericytes in tumors and healing wounds,
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and its murine counterpart NG2 is expressed by activated pericytes during both normal and
pathological neovascularisation140-142. Desmins are intermediate filaments that form part of
the cytoskeleton in striated and SMCs; mice deficient in desmin display defects in all types
of muscle tissue and die of cardiac failure143,144.
Pathologic Angiogenesis and Arteriogenesis
Blood vessel formation plays a critical role in a number of pathophysiological conditions,
most notably cardiovascular disease and cancer, the biggest causes of mortality in
developed countries. Many other disorders such as psoriasis, atherosclerosis, hemangiomas,
vascular malformations, Kaposi's sarcoma, diabetic retinopathy and arthritis are also
associated with disturbed blood vessels functions11. To date most attempts at interfering
with pathological angiogenesis have targeted solid tumors, peripheral tissue ischemia and
myocardial ischemia.
Angiogenesis in Ischemic Disease
Reduced blood perfusion of tissues results in hypoxia which triggers specific intracellular
signalling pathways and subsequent gene transcription7. Cellular hypoxia leads to inhibition
of prolyl hydroxylases that regulate the degradation of transcription factors HIF-1α to 3α7.
These factors form complexes with ARNT (HIF-1β) and bind HREs in the promoters of
hypoxia inducible genes involved in glucose transport, glycolysis, and angiogenesis.
Examples of hypoxia-regulated genes include Glut-1, Cox-2, VEGF and VEGFR-17.
Deletion of a HRE in the VEGF promoter in transgenic mice results in progressive motor
neuron degeneration presumably because of insufficient vascular perfusion of nervous
tissue and direct effects on motoneuron survival via loss of VEGF induction145. The
immature morphology of blood vessels generated in response to hypoxia-induced VEGF is
exemplified in the newborn by retinopathy and the chaotic vessel hierarchy and lack
integrity in tumor angiogenesis146. Thus, VEGF driven angiogenesis in progressive
ischemic disease may not be sufficient to restore perfusion because the formed vessels are
not functional.
Arteriogenesis
Reduction of blood flow in thromboembolic disease leads to collateral formation or
arteriogenesis, a process in which pre-existing arterioles enlarge, leading to a 10- to 20-fold
increase in blood flow147. Increased fluid shear stress induces expression of monocyte
22
chemotactic proteins and cell adhesion molecules, which recruit macrophages and initiate
arteriogenesis. The recruited macrophages release proteases that degrade the basal lamina
enabling the vessel to enlarge148. A new basal lamina is synthesized and mural cells such as
SMCs and pericytes are recruited to stabilize the remodelled vessel149. Enlargement of the
pre-existing collaterals is a process mediated by cytokines that enhance recruitment of
monocytes or prolong their life span such as monocyte chemoattractant protein-1 (MCP-1),
granulocyte monocyte-colony stimulating factor (GM-CSF), TGF-β or tumor necrosis
factor-α (TNF-α). Cytokines such as PlGF or FGFs not only recruit monocytes, but also act
directly on ECs and SMCs by inducing the growth of these cell populations. In general,
induction of vascular collaterals by cytokines in experimental animal models of myocardial
and hindlimb ischemia may restore perfusion and rescues tissue from
necrosis62,150,151.However, presumably because arteriogenesis is initiated by shear stress, the
arteriogenic process is prematurely terminated as shear stress decreases in the enlarging
vessel. Thus, compensatory conductance by arteriogenesis often remains at less than 50%
of the original conduction prior to occlusion.
Tumor Angiogenesis
Blood vessels in tumors display several distinct features that have implications for tumor
biology and treatment. Tumor angiogenesis is mediated mainly by VEGF expression in the
tumor cells152,153. Blood vessels in tumors have a chaotic structure and are leaky. As a
consequence, blood flow in the tumor is sluggish and the interstitial fluid pressure is
high154. The sluggish blood flow results in hypoxic necrosis of the inner parts of the tumor
and perpetuates VEGF production and the high interstitial fluid pressure hampers delivery
of therapeutic agents154. Since tumor growth is angiogenesis dependent, therapeutic
targeting of the tumor vasculature is an attractive alternative or adjunct to conventional
chemo- or radiotherapy155. ECs in tumors express a number of molecules that are unique to
tumor blood vessels such as VEGFR-2 and integrin αVβ3 that can be targeted for
therapeutic purposes. Furthermore, ECs are genetically stable and apparently cannot
develop drug resistance in the manner of tumor cells. To date, a number of angiogenesis
inhibitors have been tested in phase I-III clinical trials and one, the anti-VEGF blocking
antibody bevacizumab (Avastin, rhuMAb-VEGF) has shown efficacy in the treatment of
metastatic colorectal cancer and renal cancer156,157.
23
The Lymphatic System
Embryonic Development, Structure and Function
The lymphatic system is a network of vessels that maintains tissue homeostasis by
collecting extravasated plasma and returning it to the blood circulation via the lymph
nodes. The lymph also transports fatty acids absorbed in the gut and performs
immunosurveillance functions by directing leukocytes, especially antigen-presenting cells
such as Langerhans cells, and antigens to the lymph nodes. Accordingly, improper
functioning of the lymphatic system is implicated in a variety of diseases including
lymphedema, inflammation, infectious and immune disease. In cancer, malignant tumors
form distant metastases mainly by spreading through the lymphatics. The lymphatic system
was first described in the 17th century by Gasparo Aselli as he observed vessels filled with a
“milky fluid” in the stomach of a well-fed dog161. The lymphatic vessels consist of
capillaries that form collecting lymphatics which eventually empty into the venous system
Figure 4. According to the model proposed by Wigle et al158, venous ECs are initially pluripotent and committo the lymphatic phenotype after expression of Prox-1 begins. Committed venous ECs then express lymphaticmarkers such as VEGFR-3 and secondary lymphoid chemokine (SLC), and form lymphatic vessels bysprouting in response to VEGF-C159(adapted from Detmar and Oliver 2002160).
LYVE-1
Prox-1
LYVE-1 LYVE-1
Prox-1 SLC+
VEGF-C
VEGF-R3+
VEGF-C
24
Table 2. Morphological characteristics of blood and lymphatic vessel capillaries.Blood vessels Lymphatic vessels
Anchoring filaments Absent Present
Basement membrane Present Mostly absent
Endothelial cell cytoplasm Abundant Scant
Interendothelial tight junctions Common Infrequent
Lumen Regular Irregular
Overlapping endothelial cells Absent Present
Perivascular support structures Present Scant
(Based on data from Schmid-Schonbein 1990 and Nathanson, 2003)
via the thoracic duct. The lymphatic vessels themselves are thin tubes formed by ECs; the
larger lymphatics may contain a sparse covering of pericytes or SMCs and valves similiar
to those found in veins. Ultrastructurally, lymphatic vessels differs from blood vessels in
that that they have overlapping, valve-like endothelial cell junctions, a discontinous or
absent basement membrane and anchoring filaments in the vessel wall162 (Table 2). In
mouse embryos the first lymphatic outgrowths called lymphatic primordia can be identified
at embryonic day 10.5. The lymphatic primordia develop by budding from large central
veins in the perimesonephric and jugular regions. Subsequently, these lymphatic primordia
remodel into primitive lymphatic sacs from which the lymphatics develop by
sprouting163,164. An alternative hypothesis proposes that the lymphatics develop in situ from
mesenchymal precursors, or lymphangioblasts, or by a combination of sprouting and
development in situ165,166. The former theory is supported by studies demonstrating that
expression of VEGFR-3, which is initially expressed in veins, is later restricted to
lymphatic endothelia167-169. Furthermore, the prospero related homeobox protein-1 (Prox-1)
is initially expressed in a subset of ECs in the anterior cardinal vein which subsequently
buds to form the primitive lymph sacs (Figure 4). Deletion of Prox1 gene results in lack of
the lymphatics (see below). Support for the theory on the incorporation of
lymphangioblasts into developing lymphatics is obtained from studies in the avian
chorioallantoic membrane assay and in chick-quail chimeras170. In these studies, homotopic
transplantation of Prox-1 negative tissue resulted in lymphatics composed of cells from
both species171. Lymphangiogenesis in adults is assumed to occur in concert with
angiogenesis, as has been demonstrated during wound healing in pigs129.
25
Molecular Regulators of the Lymphatic System
Figure 5. Ligand-receptor interactions in the induction of lymphangiogenesis. Receptor extracellular Ighomology domains are represented by white circles, the extracellular domains of Nrp-1 and Nrp-2 are shownas striped boxes and the TK domains are represented by black boxes.
VEGF-C and VEGF-D
VEGF-C and VEGF-D (also called C-fos induced factor or figf) share sequence homology
with VEGF (Table 1) and bind VEGFR-2 and VEGFR-3172-175. In vivo, VEGF-C and
VEGF-D are potent inducers of lymphangiogenesis176.
The expression patterns of VEGF-C and VEGFR-3 during embryogenesis, suggests that
these molecules signal in a paracrine manner177. Expression of VEGF-C is first detected in
the head mesenchyme and the developing vertebrae at E8.5. Subsequently, VEGF-C
mRNA localises to the metanephric and jugular areas at sites where the primitive lymph
sacs develop, suggesting that VEGF-C has a role in the formation of these structures177. The
expression then decreases in several tissues, remaining high in the lymph nodes178. It may
be that in adults, VEGF-C expression is mainly regulated by proinflammatory cytokines
and growth factors, suggesting a role for lymphangiogenesis in inflammatory disease. In
contrast to VEGF, VEGF-C is not induced by hypoxia (I, II).
NRP-2NRP-2
26
In lymphatic ECs VEGF-C induces migration, proliferation and survival signals in
vitro179. In vivo, overexpression of VEGF-C or VEGF-D in the epidermis of transgenic
mice induces lymphatic vessel hyperplasia while local overexpression by adenoviral gene
transfer induces sprouting lymphangiogenesis176,180 (III). Transgenic mice lacking both
Vegfc alleles fail to develop lymphatic vessels and succumb to tissue edema at E15.5-
17.5159. Loss of one vegfc allele results in a phenotype characterised by hypoplasia of the
cutaneous lymphatic vessels and lymphedema159. Sprouting of endothelial cells committed
to the lymphatic endothelial lineage in vegfc-/- mice could be induced by recombinant
VEGF-C and to a lesser degree with VEGF-D, but not VEGF indicating that development
of the lymphatic vessels is dependent on VEGF-C in a non-redundant fashion159.
VEGF-C and VEGF-D have long N-terminal and C-terminal extensions that are
proteolytically cleaved upon secretion181,182. The receptor specificity of VEGF-C and
VEGF-D is regulated by this proteolytic processing as only the fully processed secreted
forms are capable of efficient binding of VEGFR-2181,182.
In vivo, VEGFR-3 activation is sufficient to induce lymphangiogenesis while the
vascular permeability induced by VEGF-C and VEGF-D is mediated by VEGFR-2182,183.
Furthermore, overexpression of a soluble form of the extracellular domain of VEGFR-3
blocks lymphangiogenesis in transgenic mice184. Interestingly, murine VEGF-D binds only
to VEGFR-3185. VEGF-D overexpression by adenoviral gene transfer in mouse skin or in
experimental tumors induces both angiogenesis and lymphangiogenesis88,176,186.
VEGFR-3
VEGFR-3 (fms-like tyrosine kinase 4, Flt4) is a transmembrane TK consisting of six
extracellular immunoglobulin like loops and an intracellular part containing a TK domain
for interaction with intracellular signal transduction molecules187,188. In humans, alternative
splicing of the VEGFR-3 gene generates two isoforms of VEGFR-3 that differ in their C-
termini and are designated VEGFR-3s (s=short) and VEGFR-3l (long)189. Expression of
VEGFR-3 is restricted to the lymphatic ECs in the adult whereas a blood vascular ECs also
express VEGFR-3 during embryonic develoment (Figure 4). VEGFR-3 defiency results in
embryonic lethality due to defects in cardiovascular remodelling167. In ECs, VEGFR-3
relays survival signalling by activation of Akt and p42/p44 MAPK. Migration of lymphatic
ECs is also stimulated by VEGFR-3 signals179. Intriguingly, a subpopulation of CD34
positive CEPs express VEGFR-3, and some of these CEPs differentiate in vitro and start to
express lymphatic endothelial cell markers suggesting that circulating lymphangioblasts
exist and could contribute to lymphangiogenesis in some conditions190.
27
Markers of the Lymphatic System
Lymphatic Vessel Endothelial Hyaluronan Receptor-1 (LYVE-1)
LYVE-1 is receptor for hyaluronan, an ECM glycosaminoglycan and a homologue of
CD44, a hyaluronan receptor expressed in lymphocytes191,192. The function of LYVE-1 in
lymphatic vessels may be to transport hyaluronan into the lymphatic vessels for transport
to, and degradation in regional lymph nodes192. However, LYVE-1 is not restricted to the
lymphatic ECs but is also expressed in normal liver blood sinusoids, activated macrophages
and capillaries in the lung193,194.
Podoplanin
Podoplanin is a 38 kD glomerular podocyte membrane mucoprotein expressed by the
lymphatic endothelium and glomerular epithelial cells. Podoplanin is expressed in all cells
in Kaposi’s sarcoma195 and angiosarcomas 196 and is co-expressed with VEGFR-3 in the
skin and kidney. During embryogenesis, podoplanin is first expressed between E10.5 and
11.5 in ECs of the cardinal vein and in budding Prox-1 positive lymphatic ECs197.
Podoplanin seems to be a Prox-1 regulated gene198,199. Homozygous deletion of podoplanin
in mice results in abnormal development of lymphatic vessels and lymphedema197.
Overexpression of podoplanin in vascular endothelial cell cultures promotes the formation
of elongated cell extensions and significantly increased endothelial cell adhesion, migration
and tube formation197.
Prox-1
Prox-1 is a transcription factor expressed in lymphatic ECs which has a similiar pattern of
expression to that of VEGFR-3. Knock-out studies have shown that Prox-1 is essential for
the formation of the lymphatic system during embryogenesis169 as well as for the
development of the liver 200 and the lens169. Prox-1 is expressed in the subpopulation of ECs
that give rise to the lymphatic sacs201 and sprouting and migration of ECs from these sacs is
impaired in the Prox1-/- mice158. Haploinsufficiency of Prox1 leads to intestinal chylous
fluid accumulation and death shortly after birth169. Evidence for the role of Prox-1 in the
determination of lymphatic endothelial cell fate is further demonstrated by the fact that its
overexpression in blood vascular ECs results in an alteration of their gene expression
profile towards that of the lymphatic endothelial cells198,199.
28
Pathologic Lymphangiogenesis
Tumor Lymphangiogenesis
The intravasation of tumor cells into lymphatic vessels is one of the first steps in lymphatic
metastasis. Consequently, lymphangiogenesis around a solid tumor should promote
lymphatic metastasis by providing a larger target for the intravasation of tumor cells.
Overexpression of VEGF-C in experimental tumors in mice induces growth of lymphatic
vessels into the tumor stroma indicating that expression of this growth factor in tumors can
induce lymphangiogenesis202,203. Furthermore, combined overexpression of VEGF-C and
the oncogenic SV40 virus large T antigen in the pancreatic islets of transgenic mice
induced lymphangiogenesis and promoted lymph node metastasis as compared to mice
expressing only the oncogene204. Overexpression of VEGF-D, a growth factor with both
lymphangiogenic and angiogenic properties, also promoted lymph node metastasis in
experimental tumors in mice88. Interestingly, neutralisation of VEGF-C and VEGF-D in an
experimental tumor by systemic overexpression of a recombinant molecule consisting of
the extracellular part of VEGFR-3 fused to the Fc domain of immunoglobulin G (VEGFR-3
Ig) reduced the number of lymph node metastases, indicating that inhibition of tumor
lymphangiogenesis could have therapeutic potential205. Although enlarged lymphatic
vessels at the tumor edge have been reported in human cancers, functional intratumoral
lymphatic vessels have not been reported206,207. One explanation could be that the high
interstitial fluid pressure in tumors compresses lymphatic vessels. According to another
hypothesis, compressed lymphatic vessels seen in the tumor stroma did not grow there by
lymphangiogenesis but were pre-existing lymphatic vessels that were engulfed by the
expanding tumor208. However, lymph node metastasis in human cancers correlates with
tumoral VEGF-C or VEGF-D expression209,210 and although lymphatic metastasis is not
necessarily dependent on lymphangiogenesis207, tumor lymphangiogenesis has been
reported to be a prognostic indicator for cutaneous melanoma211,212 and squamous cell
cancers of the head and neck213,214.
Lymphedemas
Lymphedemas are disorders of the lymphatic system in which lymphatic drainage is
impaired with resultant accumulation of fluid and swelling of the limbs or the scrotum. In
these disorders an exudate of protein rich fluid accumulates in the subcutis. This leads to
subcutaneous fibrosis, impaired immune responses and degeneration of the adipose tissue.
29
As a result lymphedema patients suffer from recurrent infections and impaired wound
healing.
Lymphedemas are classified as primary, congenital lymphedemas and secondary or
acquired lymphedemas. Primary lymphedemas develop at birth or during adolescence and
rarely occur in adults. The lymphatic vessel structure in individuals with primary
lymphedemas is disturbed. Congential hereditary lymphedema or Milroy’s disease is
characterised by hypoplastic superficial lymphatic vessels215 while the lymphatics in
Meige’s disease, or late-onset congenital lymphedema appear enlarged. Recently, the
genetic defects underlying two forms of primary lymphedema have been characterised. In
some patients with Milroy`s disease missense mutations in the TK domain of VEGFR-3
inhibit the function of this receptor with resultant lymphedema216. In lymphedema
distichiasis, (LD) mutations in the FOXC2 gene have been identified217. FOXC2 is a
member of the forkhead/winged helix family of transcription factors that have a variety of
functions during embryonic develoment218. Other hereditary congenital lymphedemas with
putative, as yet uncharacterised genetic defects, include Turner`s syndrome and cholestasis-
lymphedema.
Secondary lymphedema is caused by iatrogenic trauma such as irradiation,surgery
or infection. Iatrogenic trauma is exemplified by lumpectomy in breast cancer patients, of
which 6-30% suffer from postoperative lymphedema219. The most common cause of
secondary lymphedema is filariasis that is caused by the parasitic worms Wucheriya
bancroftii or Brugia malayi with a prevalence of over 120 million cases worldwide.
30
Therapeutic Approaches
Vascular Gene Therapy
The molecular networks that regulate the vascular system provide attractive targets for
therapy for various diseases. Most attempts at amplifying or inhibiting angiogenic
signalling utilises systemic administration of recombinant proteins or transfer of gene
constructs that overexpress a growth factor or an angiogenesis inhibitor. Generally, gene
transfer in vivo requires the use of vectors such as cationic liposomes or recombinant
viruses but naked plasmid DNA can also be taken up by muscle tissue with resultant
expression of the transferred gene85. Gene transfer by cationic liposomes is relatively
inefficient and results only in short term gene expression220. By contrast recombinant
viruses efficiently transfer genes which are then expressed for a prolonged period of time221.
Viral Vectors
Recombinant adenoviruses infect both dividing and non-dividing cells that express the
adenovirus vector receptors CAR, αVβ3 and αVβ5 integrin222,223. In immunocompetent
hosts, adenoviral gene transfer results in transient expression of the transferred gene for 1-2
weeks. The lack of chromosomal integration of the transferred DNA as well as the transient
nature of adenoviral gene expression make recombinant adenoviruses relatively safe and
they have therefore been widely used in clinical trials. Long term expression of transferred
genes can be achieved by recombinant retroviruses, adeno-associated (AAV) viruses and
lentiviruses. Retroviruses have mainly been used for ex-vivo gene therapy of bone marrow
or muscle cells in therapy of diseases such as severe combined immune deficiency and
hemophilia224,225. AAVs efficiently infect skeletal muscle, smooth muscle cells of blood
vessels and the myocardium, making it an attractive vector for gene therapy of
cardiovascular diseases226,227, both AAVs and lentiviruses readily infect the central nervous
system and the liver228. Vectors that integrate into the host genome raise a number of safety
issues, and cases of malignant transformation of target cells in humans have been
described229. Furthermore, prolonged expression of genes transferred via AAVs can
produce unwelcome effects such as hemangiomas after transfer of angiogenic growth
factors230. Thus, the clinical use of these vectors may require the development of vector
systems that can be regulated exogenously.
31
Proangiogenic Therapy
Although cells can respond to hypoxia by expressing angiogenic cytokines such as VEGF,
angiogenesis or collateral blood vessel formation is rarely sufficient to restore normal
perfusion. For this reason, attempts have been made to boost collateral vessel formation by
infusion or local expression in vivo of angiogenic cytokines231. The induction of
angiogenesis or arteriogenesis has been attempted in cardiovascular disease in man by
employing various growth factors administered either as recombinant proteins or by local
overexpression using gene therapy 232-236. Vascular growth factors that have proceeded to
phase II and phase III pro-angiogenic clinical trials include VEGF, FGF-2, GM-CSF and
FGF-4220. The efficacy of recombinant growth factor therapy for myocardial infarction or
peripheral vascular disease varies from neglible to improved function as measured by an
exercise tolerance test while adenoviral gene transfer of FGF-4 or VEGF showed efficacy
in improving myocardial and peripheral perfusion in patients with ischemic disease220.
In addition to adminstration of recombinant growth factor peptides or recombinant
viruses, the recent discovery of CEPs has raised the possibility of using these cells in the
treatment of disorders of the vascular system19. CEPs enriched ex vivo have been used to
treat patients suffering from acute myocardial infarction or lower limb ischemia resulting in
improved blood perfusion of the ischemic tissues237,238,239,240.
Lymphangiogenic Therapy
Lymphangiogenic gene therapy aims to correct congenital or acquired defects of the
lymphatic system by induction of lymphangiogenesis in vivo using lymphangiogenic
growth factors such as VEGF-C or VEGF-D241. Induction of lymphangiogenesis with
recombinant growth factors or by gene transfer of lymphangiogenic genes has restored
lymphatic function in experimental animal models of lymphedema. Mutations in the
VEGFR-3 gene cause the Chy phenotype in mice; this is characterised by an absence
subcutaneous lymphatic vessels and accumulation of chylous ascites after birth242. The Chy
genotype is analogous to that of Milroy`s disease in humans as mutations in the VEGFR3
gene cause both syndromes. Overexpression of VEGF-C in the skin of the Chy mice results
in the formation of functional lymphatic vessels, suggesting that high enough levels of
growth factor can compensate the impaired function of VEGFR-3 and that pre-existing
lymphatic vessels are not required for lymphangiogenesis in these mice243. Moreover,
administration of recombinant VEGF-C or VEGF-D protein can reconstitute the lymphatic
circulation in a rabbit ear model of lymphedema244,245. However, the efficacy of VEGF-C
and VEGF-D in the treatment of lymphedema in humans remains to be demonstrated.
32
AIMS OF THE STUDY
I. Study of the regulation of VEGF-C and VEGF-B mRNA by oncogenes, tumor promoter,
hypoxia and various cytokines compared to VEGF.
II. Characterisation of the induction of VEGF-C by pro-inflammatory cytokines
III. Experiments to explore if gene transfer and subsequent over-expression of VEGF-C can
induce lymphangiogenesis in vivo.
IV. Comparison of the effects of two VEGFR-1 agonists, VEGF-B and PlGF, on the mouse
vasculature in vivo.
33
MATERIALS AND METHODS
The materials and methods used in I-IV are accounted for below and described in detail in
the indicated publications.
Methods Used inAdenoviral infection of cell cultures III, IVCell culture I, II, III, IVDNA cloning and subcloning I, III, IVGeneration of recombinant adenoviruses III, IVGeneration of stable cell lines I, IVGeneration of transgenic mice IVImmunofluorescence IVImmunohistochemistry III, IVImmunoprecipitation II, III, IVMorphometry IIINorthern blotting I,II, III, IVNucear run-on IIPCR IVPreparation of mouse tissues III, IVReceptor stimulation and ligand binding III, IVSouthern blotting IVTransfection of cells I, II, III, IVWhole-mount immunohistochemistry IV
Cell line Description Source Used in293EBNA Epstein Barr (EBNA)
nuclear antigenATCC III,IV
Ba/F3
expressing humanembryonic kidney cellsMouse Pre-B lymphocytes Dr. T. Mäkinen IV
C6 Rat glioblastoma cells ATCC IHeLa Human adenocarcinoma ATTC IVHT-1080 Human fibrosarcoma ATCC IHUVEC Human umbilical vein EC Dr. A. Ristimäki I, IIIMR-90 Human fibroblast ATCC I, IINIH3T3 Mouse fibroblasts ATCC I
34
Growth factor Description Source Used inEGF Human epidermal growth factor R&D IIL-1β Human interleukin-1β R&D IIPDGF-B Human Platelet Derived Growth Factor-B R&D IPlGF Human Placenta Growth Factor-2 R&D IVPMA Phorbol 12-myristate 13-acetate Sigma I, IITGF-β Human transforming growth factor-β R&D ITNF-α Human tumor necrosis factor-α R&D ΙΙ
Antigen Antibodies Source Used inLYVE-1 Rabbit antiserum against mouse LYVE-1 Dr. D.Jackson III, IV
Rabbit antiserum against mouse LYVE-1 Dr. T.Petrova IVPCNA Mouse mAb against human PCNA ZYMED IIICD45 Rat mAb against mouse CD45 Pharmingen IVSMA Rat mAb against mouse SMA Sigma III, IVVEGF Rat mAb against mouse VEGF R&D III, IVVEGF-B Rabbit Ab against human VEGF-B Dr.B.Olofsson IVPlGF Rat mAb against human PlGF R&D IVPECAM-1 Mouse mAb against mouse PECAM-1 Pharmingen III, IVVEGF-C Rabbit Ab against human VEGF-C Dr. V.Joukov I, IVF4/80 Rat mAb against mouse F4/80 Serotec IV
35
RESULTS AND DISCUSSION
I. Studies on the regulation of VEGF, VEGF-B, VEGF-C and Ang-1 mRNA expression
The regulation of mRNA expression provides important clues to the biologic functions and
molecular regulators of angiogenesis. For this reason we compared the transcriptional
responses of VEGF-C, VEGF-B, Ang-1 and VEGF to a variety of stimuli such as serum
growth factors, hypoxia and oncogenes.
The mRNA levels of VEGF and VEGF-C in starved cultured cells increased in
response to FCS, and to a lesser degree in response to PDGF and TGF-β, both principal
component growth factors in FCS. As lymphangiogenesis has been described in wounds,
these data suggest that VEGF-C could be induced in response to serum growth factors in
wounds and subsequent lymphangiogenesis could clear extravasated plasma in granulation
tissue. Interestingly, Ang-1 mRNA levels decreased in response to FCS suggesting that
reduced levels of this cytokine may be required for vessel de-stabilisation and angiogenesis
in wound healing. However, according to recent reports on wound healing in mice, Ang-1
and Tie-1 expression is not altered during wound healing whereas Ang-2 and Tie-2
expression is strongly elevated246.
Activation of oncogenes increases the VEGF mRNA levels in cell culture providing
a link between tumor progession and tumor angiogenesis. Activation of an inducible
recombinant ras gene construct in cultured cells raised the VEGF mRNA levels while
VEGF-B and VEGF-C mRNA levels remained unaffected. Interestingly, several human
tumors express VEGF-C in vivo and show proliferation and growth of lymphatic vessels at
the tumor edge. VEGF-C in tumors could also be regulated in an indiarect manner by
inflammatory cells. Ras activation did not affect VEGF-C mRNA levels suggesting that the
Grb-Shc-Sos-Ras pathway is not involved in the regulation of VEGF-C expression.
However, phorbolester (PMA) induced an increase in VEGF-C mRNA levels presumably
through interaction with an AP-1 site in the VEGF-C gene promoter247. Induction of VEGF
mRNA levels by hypoxia is one of the key features of this gene as it governs the induction
of angiogenesis in tissue ischemia and during solid tumor growth30. VEGF responds to
hypoxia via HIF-1 induced activation of transcription and stabilisation of the mRNA`s 3´-
prime untranslated region248. In contrast, neither VEGF-C nor VEGF-B mRNA levels were
induced by hypoxia thus rendering VEGF the only hypoxia regulated growth factor in the
family as neither PlGF nor VEGF-D transcription is directly regulated by hypoxia249. Our
studies further indicated that VEGF-B mRNA has a very long half-life, over 8 hours,
VEGF-C mRNA has a half-life of approximately 3.5 hours and VEGF has a half-life of 1
36
hour. The mRNA half-lives of VEGF-C and VEGF were prolonged in the prescence of
serum, resulting in higher mRNA levels. The lack of induction of VEGF-B mRNA is
consistent with its very long half-life.
II. Proinflammatory Cytokines Regulate Expression of VEGF-C
The effect of the pro-inflammatory cytokines TNF-α and interleukin-1 (IL-1β) on VEGF,
VEGF-B, VEGF-C and Ang-1 mRNAs was studied in cultured human lung fibroblasts and
HUVECs in vitro. Incubation with recombinant IL-1β induced transcription of VEGF and
VEGF-C while Ang-1 transcription was decreased and VEGF-B transcription remained
unaffected. The level of secreted, partially processed VEGF-C also increased in resoponse
to TNF-α and IL-1β. The increased VEGF-C mRNA levels induced by TNF-α and IL-1β
were mainly the result of increased transcription as evidenced by nuclear run-on data. Thus,
transcriptional regulation of VEGF-C mRNA differs from that of VEGF in that VEGF
mRNA levels are regulated both by transcriptional activation and post-transcriptional
stabilisation mediated by RNA protein interactions in the 3`-UTR of the VEGF gene250. The
promoter of the VEGF-C gene contains a putative NF-κB binding sites247 that could
mediate the activation VEGF-C mRNA transcription as has been reported for
proinflammatory cytokines251. Induction of VEGF-C mRNA levels by proinflammatory
cytokines was inhibited by dexamethasone but not by indomethacine suggesting that
prostanoids do not mediate the effects of proinflammatory cytokines on VEGF-C mRNA
transcription. In contrast, induction of VEGF mRNA transcription by proinflammatory
cytokines is mediated indirectly by prostanoids252. However, a recent report demonstrated
that prostaglandin E2 mediated COX-2 activation increased VEGF-C mRNA transcription
indicating that prostanoids in some cells can induce VEGF-C driven lymphangiogenesis253.
Lymphangiogenesis in breast cancer was shown to correlate with COX-2 expression and
prostaglandin E2 mediated activation of COX-2 was shown to increase transcription of
VEGF-C mRNA in vitro253. Furthermore, both VEGF-C and COX-2 immunoreactivity in
clinical samples of breast cancer correlated with prognosis suggesting a role for prostanoid
driven lymphangiogenesis in mammary carcinomas253. VEGF-C mRNA transcription
wasalso induced in HUVECs in response to proinflammatory cytokines. In HUVECs,
VEGFR-2 transcription was increased by IL1-β. The regulation of VEGF-C mRNA
transcription by proinflammatory cytokines indicates that VEGF-C could regulate
lymphatic vessel function during inflammation, reflecting the role of the lymphatics in the
control of immune function and lymphocyte trafficking. Accordingly, VEGF-C is highly
expressed in arthritc joints in patients with rheumatoid arthritis in the absence of frank
lymphangiogenesis suggesting that either the lymphahangiogenic response is insufficient in
37
this disorder or that VEGF-C participates in other functions254,255. Recent reports have
shown that inflammatory reactions in the tracheal mucosa or in rejected human kidney
transplants are accompanied by abundant lymphangiogenesis (Tuomas Tammela, personal
communication) and that reactive lymphadenitis is mediated by VEGFR-3 signalling
(Peter Baluk, Donald McDonald, personal communication)256. In a rabbit cornea model of
inflammatory angiogenesis and lymphangiogenesis, depletion of the bone marrow or local,
selective depletion of macrophages blocked lymphangiogenesis, demonstrating that
inflammatory cells can mediate the formation of lymphatic vessels257. The angiogenic and
lymphangiogenic response was blocked by VEGF inhibitors indicating that VEGF induces
both angiogenesis and lymphangiogenesis in this model. However, recruited macrophages
expressed abundant amounts of VEGF-C and VEGF-D, suggesting that the
lymphangiogenic effect of VEGF is indirect and mediated by inflammatory cytokines that
induce the expression of lymphangiogenic cytokines in the inflammatory infiltrate.
38
Figure 6. Adenoviral overexpression of VEGFs or ß-galactosidase (LacZ) in vivo in the skin reveals the effectof these growth factors on the blood vessels and lymphatic vessels. While VEGF induces sproutingangiogenesis (A-C, arrows in C), VEGF-C is a selective lymphangiogenic growth factor (G-I). In contrast,VEGF-B has no apparent vascular effects in the skin (D-F, open arrowheads in G). Scalebar = 200 µm.
39
III. Adenoviral Expression of VEGF-C in the Skin Induces Lymphangiogenesis
The formation of lymphatic vessels or lymphangiogenesis occurs mainly during
embryogenesis and has been described in adults only during wound healing and
inflammation129,256,257. However, the induction of lymphangiogenesis could have therapeutic
value in the treatment of congenital, infectious, traumatic or iatrogenic lymphedema241.
Focal induction of lymphangiogenesis can be achieved by gene therapy and is a requisite
for successful growth factor therapy of lymphedemas. To explore, these therapeutic
concepts, recombinant adenoviruses encoding VEGF-C were constructed and used for
transfer of the VEGF-C gene into the skin of mice. Detection of lymphatic vessels by
immunohistochemistry for the lymphatic endothelial marker LYVE-1 combined with
detection of the proliferating cell nuclear antigen showed that VEGF-C induced abundant
lymphangiogenesis at two weeks after injection. Over-expression of VEGF-C induced the
expression of VEGFR-2 and VEGFR-3 in blood vessels raising the interesting possibility
that this growth factor could have vascular effects in vivo as well. However, VEGF-C did
not significantly affect the blood vessels as compared to VEGF, which induced intense
angiogenesis (Figure 6).
The partially overlapping receptor binding profiles of VEGF, VEGF-C and VEGF-
D have raised the question of how specific these factors are for lymphangiogenesis or
angiogenesis. Studies on the effect of overexpression of VEGF-C and VEGF-D in normal
tissues and in tumors indicate that VEGF-C is mainly lymphangiogenic while VEGF-D is
both angiogenic and lymphangiogenic180,258. Adenoviral overexpression of VEGF-C in the
skin of nude mice induced lymphangiogenesis and enlargement of blood vessel but no
sprouting angiogenesis180,258. Overexpression of VEGF-C in experimental tumors in mice
resulted in a robust lymphangiogenic, but only a slight angiogenic response259,204. The
angiogenic response in tumors overexpressing VEGF-C could be reduced by blocking
VEGF-R2260. However, while some authors report only lymphangiogenic effects of VEGF-
C in the chick chorioallantoic membrane assay (CAM) and in the mouse cornea assay,
others also detect angiogenesis in these assays, depending on the developmental stage of
the CAM261,262,263,264. Furthermore, VEGF-C exerts angiogenic effects after gene transfer
with recombinant plasmids in ischemic rabbit hindlimbs further indicating that VEGF-C
has angiogenic properties265. The receptor binding profiles of VEGF-C and VEGF-D are
determined by proteolytic processing as only the fully processed 21 kD forms of VEGF-C
and VEGF-D bind efficiently to VEGFR-2182. In vitro, VEGFR-2 relays the angiogenic
effects of VEGF and the mature, protelytically processed form of VEGF-C. Thus the
proteolytic processing of VEGF-C and VEGF-D as well as the upregulation of VEGFR-2
and VEGFR-3 on blood vessels in response to VEGF, VEGF-C and VEGF-D could explain
40
the angiogenic effects of VEGF-C and VEGF-D266. Plasmin proteolytically cleaves the N-
and C-terminal extensions of the immature forms of VEGF-C and VEGF-D thus regulating
their affinity for VEGFR-2 and VEGFR–3267. The presence of plasmin in the
microenvironment could determine the angiogenic and/or lymphangiogenic effects of
VEGF-C and VEGF-D in vivo. For specific, VEGFR-3 mediated lymphangiogenesis a
mutant form of VEGF-C that only activates VEGFR-3 can be used268. Selective activation
of VEGFR-3 circumvents potential side-effects of VEGFR-2 activation such as peripheral
edema269,270.
The lymphangiogenic effect of VEGF has been investigated in a number of studies.
Adenoviral overexpression of VEGF in the skin has yielded conflicting results as one study
has reported induction of giant lymphatic vessels in response to VEGF271 while others have
failed to detect any such effects180. Adenoviral overexpression of VEGF in muscle or
recombinant VEGF administered to the CAM or to the mouse cornea failed to induce
lymphangiogenesis176,263,272. Furthermore, studies in murine embryonic explants of VEGF-C
knock-out mice showed that VEGF-C, but not VEGF has the capacity to induce migration
of endothelial cells commited to the lymphatic endothelial lineage159. VEGF was reported
to induce the formation of “giant lymphatics” or "lymphangioma-like structures" after
adenoviral gene transfer of VEGF in the skin of nude mice271, an effect that we failed to
detect (Figure 6). However, the murine form of VEGF164 was used in the study by Nagy et
al and recent collaborative work between Dr. Nagy and Dr. Tuomas Tammela has shown
that the lymphangiogenic effect of murine VEGF164 and human VEGF165 differ in this
assay (Tuomas Tammela, personal communication).
IV. VEGF-B Activates VEGFR-1, But Provides Very Little Angiogenic or Arteriogenic
Activity In Vivo in Comparison with PlGF
The effect of VEGFR-1 agonists on the vasculature of mice was studied in vivo by
overexpression of two VEGFR-1 agonists, VEGF-B and PlGF by adenoviral gene transfer
in the skin of transgenic mice. While the effects of VEGF, VEGF-C and VEGF-D have
been extensively characterised in vivo, the functions PlGF and VEGF-B have remained
elusive. Recently, PlGF was reported to induce the formation of vascular collaterals in
experimental myocardial infarctions and lower limb ischemia in mice63 by amplification
VEGF signalling62,67. Thus, VEGFR-1 agonists are potentially useful in therapy aiming to
restore blood perfusion in tissue ischemia273. Overexpression of PlGF in the skin of
transgenic mice64,77 resulted in a vascular phenotype with enlarged tortous blood vessels,
morphologically similar to those seen after adenoviral overexpression of PlGF in the skin63.
In contrast, our findings demonstrate that over-expression of neither isoform of VEGF-B is
41
able to induce vascular remodelling in a manner similar to PlGF in the skin of transgenic or
nude mice (Figure 6). Whole mount immunohistochemistry to detect ECs, pericytes and
macrophages revealed arteriogenic and angiogenic effects of PlGF and VEGF respectively.
In contrast, none of the cell populations mentioned above responded to VEGF-B. As
vascular collateral formation, or arteriogenesis, is dependent on macrophage recruitment,
failure of VEGF-B to induce arteriogenesis in skeletal muscle could result from an inability
of VEGF-B to recruit macrophages when compared to PlGF or VEGF. The failure of
VEGF-B to induce remodelling of the vasculature is not dependent on prolonged exposure
of the growth factor as neither overexpression in vivo for six weeks, nor constitutive
overexpression in the skin or heart of transgenic mice resulted in a modified vascular
phenotype.
Clues to the biological function of VEGF-B may be provided by reports according
to which VEGF-B is highly expressed in striated and heart muscle and brown fat as well as
in numerous tumors274,275,276 (I). Thus, the biologic function of VEGF-B may be linked to
energy metabolism or tumor angiogenesis. Interestingly, transgenic mice deficient in
VEGF-B have subtle myocardial defects and impaired recovery after experimental
myocardial infarcts suggesting that the regeneration of the coronary circulation could at
least in part be dependent on VEGF-B50. According to one published study these mice
should also display reduced angiogenic responses in collagen induced arthritis, suggesting a
role for VEGF-B in inflammatory angiogenesis277. Finally, according to another isolated
study, overexpression of VEGF-B in vivo by injection of recombinant plasmids in a mouse
model of hindlimb ischemia induced angiogenesis and VEGF-B may thus have subtle
angiogenic effects in vivo278. However, our comparison of the vascular effects of VEGF-B
and PlGF overexpression in vivo indicates that the arteriogenic potential of VEGF-B is
modest at best.
The failure to detect a biological role for VEGF-B could depend on a number of
factors. One may be the species specificity of the factors and models used. For example,
human VEGF-B might not be optimal in mice. The study of cardiovascular physiology in
man requires animal models in which normal and pathological cardiovascular physiology
resembles that of humans as closely as possible. As the effects of VEGF-B in vivo have to
date been exclusively studied in mice using human recombinant VEGF-B, a proper study of
angiogenic growth factors in vivo should ultimately be conducted in relevant animal
models such as in pigs or dogs using corresponding recombinant human, porcine or canine
growth factors. Taken together, the above studies suggest that the angiogenic effect of
VEGF-B may be heart-specific.
42
CONCLUDING REMARKS
The discovery and characterisation of the molecular networks governing vascular
remodelling in adults has raised the possibility of using various components of these
networks for therapeutic purposes. Vascular growth factors such as the VEGF family and
their cognate receptors form a palette of tools that can be used for induction of blood- or
lymphatic vessel growth which could be useful for treatment of peripheral ischemic
disease, myocardial ischemia and disorders of the lymphatic system. Furthermore,
inhibition of angiogenesis and lymphangiogenesis may become incorporated into emerging
treatments for cancer.
Determination of the optimal therapeutic use of vascular cytokines requires
thorough understanding of their underlying biological functions. Intensive efforts over the
past ten years have clarified the function of a number of the cytokines that regulate the
vasculature. VEGF family members can now be used for induction of angiogenesis,
arteriogenesis and lymphangiogenesis while the angiopoietins could be necessary for
proper disassembly/assembly of the perivascular support structures. Many of the above
mentioned growth factors have shown promise in pre-clinical trials and a few, such as
VEGF have proceeded to bona fide clinical trials. However, successful use of vascular
cytokines for therapeutic purposes requires the development of refined gene transfer
techniques which allow for exogenous regulation and efficient over-expression of the
relevant genes at any site of an ongoing patho-physiological process.
43
ACKNOWLEDGEMENTS
This study was carried out at the Molecular/Cancer Biology Laboratory, the
Molecular/Cancer Biology Research Program at Biomedicum Helsinki and the Dept. of
Pathology, Haartman Institute, University of Helsinki.
I would like to thank professors Eero Saksela and Olli Jänne for providing excellent
research facilities in the Haartman Institute and Biomedicum Helsinki, respectively. My
sincere thanks go to my supervisor professor Kari Alitalo who gave me the opportunity to
work in his laboratory at the very forefront of international biomedical science. The
reviewers of this thesis professor Ulf-Håkan Stenman and docent Olli Ritvos are
acknowledged for their great insights and constructive criticisms of this thesis. I would like
to thank all past and present members of the Molecular/Cancer Biology Laboratory for
fruitful collaborations over the years. I would also like to thank all co-authors, especially
Ulf Eriksson, Seppo Ylä-Herttuala, Frej Stenbäck and Ari Ristimäki for their positive
attitude towards scientific collaboration with a junior scientist. Technical support at the
M/CBL is top-notch and Tapio Tainola, Sanna Lampi, Riikka Ekman, Alun Parsons,
Angela Flynt, Paula Hyvärinen and Mari Helanterä are kindly acknowledged for their help
in all matters technical over the years.
My parents Monica and Sten and my brother Robin have always provided generous
support of all kinds for which I am deeply thankful. Finally, I want to thank my wife Susa
and our son Oskar for those most important things in life.
Finska Läkare Sällskapet played a crucial role in the funding of this study and the support
of that society is greatly acknowledged. This work was also supported by the Finnish
Cancer Organizations, the Finnish Medical Foundation, the Helsinki University Central
Hospital (Helsinki and Uusimaa Hospital Group, Special State Grants), Helsinki University
Chancellor’s Office, the Paulo Foundation, the K. Albin Johansson Foundation, the Oskar
Öflund Foundation and the Maud Kuistila in Memoriam Foundation.
44
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