Role of VEGF in vasculogenesis and angiogenesis
lecture II
14th March 2011
Angiogenesis and VEGF “history”
VEGF “history”
1787 ‐ Dr John Hunter first uses the term 'angiogenesis' to describe blood
vessels growth
1983 ‐ Vascular Permeability Factor (VPF), is discovered by Dr Harold Dvorak. The molecule VPF causes leaky blood vessels associated with tumors
VPF was 50 000 times more potent than histamine.
1989 ‐ One of the most important angiogenic factors, vascular endothelial
growth factor (VEGF), is discovered by Napoleone Ferrara and by Jean Plouet. It turns out to be identical to the molecule called Vascular Permeability Factor
(VPF) discovered in 1983 by Dr. Harold Dvorak.
Main proangiogenic factor
Vascular
Permeability
Factor
1983,
Dr H. Dvorak
=
Vascular
Endothelial
Growth
Factor
V
P
F
vascular permeability factor
endothelial cell survival factor
endothelial cell proliferation
endothelial cell migration
V
E
G
F
1989, Dr N. Ferrara
Dr J. Plouet
Blood vessel formation – various ways
Carmeliet, 2005; Semenza 2003
Vasculogenesis capillaries are formed from
vascular progenitor cells
Vasculogenesis begins very early after the initiation of
gastrulation in the mammalian embryo, with the formation
of the blood islands in the yolk sac and angioblast precursors in the head mesenchyme
Vasculogenesis
1. First phase
• Initiated from the generation of hemangioblasts;
2. Second phase
• Angioblasts proliferate and differentiate into endothelial cells
3. Third phase
• Endothelial cells form primary capillary plexus
In the yolk sac, these progenitors aggregate into endothelial‐lined blood islands that
then fuse to generate a primary capillary plexus. The primary capillary plexus
undergoes remodelling along with intra‐embryonic vessels to form a mature circulation
Vasculogenesis­ blood islands
YOLK SAC MESODERM
ANGIOBLASTS
Endothelium for vessels
HEMANGIOBLASTS
HEMATOPOIETIC CELLS
Developing blood cells
Vasculogenesis ­ initial vessel formation
MESENCHYME
ADVENTITIAL FIBROBLASTS
Endothelium
MEDIAL SMCs
Endothelium
Vasculogenesis – later steps
ADVENTITIAL FIBROBLASTS
Endothelium
MEDIAL SMCs
VASCULOGENESIS is prominent in
establishing the vascular beds of
liver, lung, & heart
Hemangioblast
Hemangioblast is a multipotent cell, common precursor to hematopoietic and endothelial cells. Hemangioblast was first hypothesized in 1900 by Wilhelm His. Hematopoiesis in zebrafish (Danio rerio)
Expression of the flk­1 represents the earliest marker of the developing endothelial lineage during vasculogenesis SCL transcription factor is crucial for the development
of blood cells and blood vessels
Formation of vascular network
Common vascular progenitor cells for endothelial cells and
vascular smooth muscle cells Origin of endothelial and mural cells from progenitor cells Hemangioblast
Blood cells Skeletal muscles
Angioblast
VEGF
Venous
endothelium
Arterial
endothelium
Pericytes/smooth muscles
PDGF­BB
progenitor cells of vascular smooth muscles
(various forms)
VEG
F
­
PDGF
BB
Common
vascular
progenitor
cells
(flk­1+)
Origin of endothelial and mural cells from
common vascular progenitor cell
Flk1+ cell differentiation in serum­free culture
CD31 (purple)/SMA(brown) immunostaining
a, Vehicle‐treated cells. b, PDGF‐BB. c, VEGF.
d, Simultaneous administration of VEGF and PDGF‐BB.
Vasculogenesis in adult
Vasculogenesis occurs also in adult organism
Previously, postnatal vascularization was thought to occur exclusively due to angiogenesis. However, recent investigations have shown that vasculogenesis is
involved in blood vessel formation also during postnatal live and the cells
responsible for that are EPCs.
• Classic Paradigm: Angiogenesis
– Mature ECs migrate and proliferate to form new vessels
• New Model: Angiogenesis + Vasculogenesis
– Bone Marrow derived EPCs circulate to sites of neovascularization
JCI 1999;103:1231‐1236
Endothelial progenitor cells A primitive cell made in the bone marrow (or other
organs) that can enter the bloodstream and go to areas
of blood‐vessel injury to help repair the damage
Plasticity of adult stem cells
the ability to form specialized cell types of other tissues
(also called transdifferentiation) Differentiation pathways for pluripotent
bone marrow stromal cells Adult progenitor cells
Endothelial Progenitor Cells
• 1997 Asahara et al. reported putative EPCs
or angioblasts
– Cells display progenitor phenotype
– Differentiate into mature ECs in vitro
– Participate in neovascularization when injected into ischemic animal model
Science 1997;275:964-967
Endothelial Progenitor Cells
DiI AcLDL uptake
EPC
1:200 2h
Anna Grochot­Przeczek
BODIPY AcLDL uptake
Anna Grochot­Przeczek
Isolectin binding by EPC Lectin from Bandeira simplicifolia
control
isolectin
Anna Grochot­Przeczek
Immunohistochemical characterisation of
culture­expanded EPCs
2 weeks
culture
Uptake
of acetylated
low density
lipoproteins
Cytoplasmic factor VIII (von Willebrand factor)
tube formation
on Matrigel
Dzau et al., Hypertension 2005,
Markers of endothelial progenitor cells
CD34
VEGFR2
CD133 (prominin, AC133)
CD133 is absent on mature endothelial cells and monocytic cells Mechanisms of EPCs homing and differentiation
Urbich & Dimmeler, Circ Res 2004
Mobilization of vascular progenitors
• Tissue ischemia results in expression of cytokines like VEGF
• Recruites progenitor cells
• Steady state 0.01% MNCs in blood are CEPs
• Amount of circulating progenitors are
increased after trauma, infectious injuries or
tumour growth
• 24 h after injury: 12%
VEGF, VEGF­A
vascular endothelial growth factor
main regulator of angiogenesis, pro­angiogenic factor
‐ a dimeric glycoprotein
‐ belongs to a so‐called cysteine‐knot superfamily of growth factors
‐ one interchain disulfide bond
Oloffson et al., 2000
VEGF belongs to VEGF family
Endogenously expressed in mammals
VEGF-A
Encoded by the
double stranded
DNA virus, orf
Ferrara et al. 2004
VEGF
• VEGF (vascular endothelial growth factor, vascular permeability factor, vasculotropin)
– homodimeric protein, 34‐42 kDa
• produced by many types of cells
(e.g. macrophages, VSMC, fibroblasts, and cancer cells)
• expression is induced in response to hypoxia and proinflammatory cytokines
• receptors
(VEGF‐R1 and VEGF‐R2) are present mostly on endothelial cells, therefore VEGF acts specifically on endothelium (but also on neurons and
Schwann cells). • VEGF‐R1 is expressed also on monocytes and vascular smooth muscle cells –
their activation upregulates expression of metalloproteinases and increases cell
migration. VEGF
• It protects endothelial cells from apoptosis and induces their proliferation, migration, and formation of capillaries
• VEGF acts protectively on neurons
• VEGF is required for the normal development of embryonic vasculature, the
cyclic growth of blood vessels in the female reproductive tract, and the
formation of capillaries during wound repair
• however, VEGF is also involved in
abnormal angiogenesis, as seen in
proliferative retinopathies, rheumatoid arthritis, psoriasis, and malignancies
VEGF is highly conserved between species
VEGF has been found in all vertebrate species :
• fish (the zebrafish Danio rerio)
• frogs (Xenopus laevis)
• birds (Gallus gallus)
• mammals
The sequence and genomic organization of the vertebrate VEGF‐A
genes is highly conserved between fish and mammals.
Fish VEGF‐A shows 68% and 69.7% amino‐acid identity with human and mouse
VEGF‐A, respectively
VEGF­like proteins are present in several invertebrate species
Invertebrate VEGF/VEGFR systems have been identified in fly (Drosophila
melanogaster), nematode (Caenorhabditis elegans) and, most recently, in jellyfish
(Podocoryne carnea). Presence of VEGF­like proteins in different animals
• In the nematode Caenorhabditis elegans four possible homologs of PDGF/VEGF receptors (VER‐1 to VER‐4) and
one ligand (PVF‐1) are known
• PVF‐1 has the ability to bind to human receptors VEGFR‐
1 and VEGFR‐2 and to induce angiogenesis in two model systems derived from vertebrates
Control HUVEC
HUVEC + VEGF
HUVEC + PVF-1
Jorgensen & Mango, Nat Rev Gen 2002; Tarsitano et al. FASEB J 2006.
Drosophila melanogaster
respiratory (tracheal) system
‐ Branching tubular system of trachea delivers oxygen to the tissues of insects.
‐ Its development shows parallels to the angiogenesis
‐ Branchless (a homolog of mammalian FGF), PVF1, PVF2, PVF3 (homologs of
mammalian VEGF/PDGF) and PVR receptor regulate the migration of early hemocytes and are necessary for formation of tracheal system.
Tracheal tree of Drosophila embryo
DB – dorsal branch; DT – dorsal trunk; GB – ganglionic branch; VB – visceral branch
O’Farrell, J Clin Invest 2001
Organisation of VEGF gene and VEGF isoforms • The human VEGF‐A gene is characterized by a highly conserved eight exon structure, • Alternative splicing of the human VEGF‐A gene gives rise to at least five different
transcripts encoding isoforms of the following lengths (in amino acids)
121, 145, 165, 189 and 206
8
VEGF-A121
8
VEGF-A145
7
8
VEGF-A165
7
8
VEGF-A189
7
8
VEGF-A206
Exons 1-5
Exons 1-5
6A1
A2
Exons 1-5
Exons 1-5
6A1
A2
Exons 1-5
6A1
A2
6B
VEGF isoforms
Isoform
Size
(amino
acid)
Coding exons
Features
VEGF-A121
121
1-5, 8
Secreted
VEGF-A145
145
1-6, 8
Binds NRP2 but not NRP1; secreted
VEGF-A165
165
1-5, 7, 8
The most abundant and biologically
active isoform; secreted; binds
NRP1 and NRP2
VEGF-A165b
165
1-5, 7, alternative exon 8
Secreted, endogenous inhibitory
form of VEGF-A165
VEGF-A183
183
1-5, short exon 6, 7, 8
Sequestered in ECM but released
by cleavage
VEGF-A189
189
1-8
Sequestered in ECM but released
by cleavage
VEGF-A206
206
1-8 plus additional exon
Sequestered in ECM but released
by cleavage
Splice variants of human VEGF
1
2-5
6a
6b
7
26 a.a.
115 a.a.
24 a.a.
17 a.a.
44 a.a.
8
6 a.a.
VEGF121
VEGF145
NRP-1
VEGF165
VEGF183
VEGF189
VEGF206
signal
peptide
VEGF-R1 VEGF-R2
HSPGs
Heparan
­Sulfate Proteoglycan
Heparan­Sulfate
Proteoglycan
After Robinson and Stringer 2001, J Cell Science 114:853-65
Properties of VEGF isoforms VEGF121 is a soluble acid polypeptide
VEGF189 and VEGF206 are highly basic and bind very strongly
to heparin, thus they are completely sequestred in extra‐
cellular matrix (ECM)
VEGF165 has intermediate properties: it is secreted, but significant
fractions remains bound to cell surface and ECM
Expression of VEGF isoforms
• VEGF is produced by virtually all cell types. It is synthesized by a number of non‐malignant cells including keratinocytes, skeletal myotubes, vascular smooth muscle cells and certain endothelial cells. It is produced in big amount by different tumor cells
• Most VEGF‐producing cells express VEGF121, VEGF165, VEGF189, and often VEGF183. VEGF145 and VEGF206 are seemingly restricted
to cells of placental origin.
• VEGF165 is most abundantly expressed, but VEGF189 is a major isoform in lungs, and both VEGF165 and VEGF189 predominate in
heart. Furthermore, the relative levels of VEGF isoforms may vary
during development or in response to cytokine stimulation.
Not every cells express
the same amounts of VEGF VEGF expression in several cell lines ­ intact cells
(24 h incubation)
HASMC
HMEC­primary HMEC­1
HUVEC
rat Müller cells
165 121
Receptors for VEGF­A
Main receptors: VEGFR‐1 (Flt‐1) VEGFR‐2 (Flk1; KDR) Accessory receptors
Neuropilin 1 (NRP1)
Neuropilin 2 (NRP2)
Storage
heparan sulfate proteoglycans
VEGF­A belongs to VEGF family
Receptors for VEGF-A
Both VEGF receptors have 7 immunoglobulin‐like domains in the extracellular domains, a single transmembrane region and a consensus tyrosine kinase sequence that is
interrupted by a kinase‐insert domain. Growth factors and receptors of the VEGF family
VEGF121
VEGF145
VEGF165
VEGF189
VEGF-B
PlGF-1
PlGF-2
VEGF121
VEGF145
VEGF165
VEGF-C
VEGF-D
VEGF-E
VEGF-C
VEGF-D
TK
Sema-III
Sema-E
Sema-IV
VEGF165
PlGF-2
VEGF-B
VEGF-E
Heparan-Sulfate Neuropilin-1
Proteoglycan
TK
VEGF-R1
VEGF145
VEGF165
VEGF189
VEGF206
VEGF-B167
VEGF-E
PlGF-2
VEGF-R2
Sema-E
Sema-IV
VEGF165
Neuropilin-2
VEGF-R3
After Neufeld et al.. 1999, FASEB J 13:9-22
Expression of VEGF receptors
‐ endothelial cells: VEGFR‐1, VEGFR‐2, co‐receptors
‐ other cells: monocytes
vascular smooth muscle cells?
tumor cells? hematopoietic stem cells neuronal cells
Expression of VEGF receptors is not restricted to ECs
Zachary et al., 2003
Significance of VEGF and VEGF receptors
has been recognized by targeting
disruption of those genes in mice Knockout of VEGF is lethal in heterozygous form Yolk sac of E10.5 VEGF+/+ and VEGF +/– mouse embryos
Ferrara & Alitalo, Nature Med. 2000
Effect of knockout of VEGF receptors
VEGFR­1
Flt1‐/‐ mice die in utero between days 8.5 and 9.5
‐ EC develop but do not organize into vascular channels
‐ excessive proliferation of angioblasts
VEGFR­2
Flk1‐null mice die between day 8.5 and 9.5
Lack of vasculogenesis and failure to develop blood islands
and organized blood vessels
Semaphorin receptors – Np­1 and Np­2 ‐ form complexes with type A plexins
‐ complexes serves as signaling receptors for class‐3 semaphorins
‐ involved in axonal guidance
Np­1 and Np­2 in angiogenesis
‐ binds VEGF165, VEGF‐B, PlGF‐2
‐ knockout of Np‐1 – lethal at E12.5
‐ overexpression of Np1‐ excessive capillary formation, dilated blood vessels
extensive hemorrhage
‐ no visible abnormalities in Np‐2 knockout mice, but Np‐2‐/‐ Np1+/‐ are lethal
‐ double knockouts Np‐1‐/‐Np‐2‐/‐ died in utero at E8.5, completely avascular
yolk sacs
Why effect of knockouts of VEGF receptors is different?
VEGF receptors – role in angiogenesis
Affinity of VEGF to VEGFR1 –
16‐114 pM
Affinity of VEGF
to VEGFR2 –
0.4‐1 nM
Effects of knockouts of VEGF genes and VEGF receptors genes
Functions of the VEGF receptors family
VEGFR‐1
Crucial to embryonic angiogenesis
Does not appear to be critical in pathogenic angiogenesis
VEGFR‐2
Mediates the majority of VEGF angiogenic effects
VEGFR‐3
Found only in lymphatic endothelial cells
Associated with lymph node metastasis
The VEGFRs differ in their downstream signaling effects
Receptor
Effects
VEGFR‐1
Possible “decoy receptor” effect
Induction of other factors
VEGFR‐2
Proliferation
Migration
Survival
Angiogenesis
VEGFR‐3
Effects mainly in lymphatic cells
Angiogenic and vasculoprotective functions of VEGF
‐ vascular permeability functions
‐ endothelial cells survival factor
‐ endothelial cell proliferation
‐ endothelial cell migration
‐ inhibition of thrombosis
Angiogenic and vasculoprotective functions of VEGF
Mechanisms of anti­apoptotic VEGF signaling
Focal
adhesion
kinase
Phosphotydyloinositol
3 kinase
AKT = PKB
Zachary, Cardiovasc Res 2001
Mechanisms of mitogenic VEGF signaling
phosphatydylinositol
4,5 biphosphate
Phospholipase C
Proteins with
src homology
(SH) 2 domain
Diacyloglicerol+
Inositol 1,4,5 ‐trisphosphate
Extraxellular signal‐regulated kinases
Zachary, Cardiovasc Res 2001
Mechanisms of chemotactic VEGF signaling
Zachary, Cardiovasc Res 2001
VEGF level has to be tightly
regulated during development
and postnatal life
Embryonic development is disrupted by modest
increases in VEGF gene expression
Miquerol L, Langille BL, Nagy A.
Development, 2000: 127:3941-6
2­3 fold overexpression is deletorious to embryonic development
Enlarged hearts
Embryos died between E12.5 and E14.5
Too high and unbalanced expression of VEGF after gene delivery using adenoviral vectors
A and B. Note prominent tissue edema
and new blood vessel formation. C. Note also a prominent leakage of
plasma protein complexes from
locally hyperpermeable ear vessels.
The effect of different
isoforms of VEGF on angiogenesis
Vessel formation and sprouting angiogenesis in
embronyic bodies in response to the different VEGF ligands
Formation of peripheral vascular plexus in two‐dimensional EB cultures, visualized by anti‐CD31 immunostaining (red), was induced by 1 nmol/L VEGF‐A165, but not by VEGFA165b,VEGF‐A121, VEGF‐A145, or vehicle.
Kawamura et al. Cancer Res. 2008
Vessel formation and sprouting angiogenesis in
embronyic bodies in response to the different VEGF ligands
Vascularization of Matrigel plugs in nude mice. Plugs were fixed and stained to detect CD31 on endothelial cell (red) and α‐SMA (ASMA) on pericytes (green) by immunofluorescent detection.
Kawamura et al. Cancer Res. 2008
VEGF­A165b
inclusion of VEGF‐A165b led to invasion of endothelial cells
into the Matrigel, but the cells failed to organize into vessels
and also failed to attract pericytes
endothelial cells
pericytes
Kawamura et al. Cancer Res. 2008
VEGF­A121
In the VEGF‐A121–containing Matrigel plugs, occasional
vessel structures were seen, which lacked branch
points and a pericyte coat
endothelial cells
pericytes
Kawamura et al. Cancer Res. 2008
VEGF­A145
In VEGF‐A145–containing Matrigel plugs, branching, pericyte‐clad vessels were seen but to a much lesser extent
than in the VEGF‐A165–containing Matrigel plugs
endothelial cells
pericytes
Kawamura et al. Cancer Res. 2008
VEGF­A165
Inclusion of VEGF‐A165 induced abundant vascularization of
the Matrigel with richly branched, pericyte‐covered vessels
endothelial cells
pericytes
!VEGF165 is the crucial isoform!
Kawamura et al. Cancer Res. 2008
How to assess the role of different
VEGF isoforms, if the knockout of the
gene is lethal?
Conditional knockouts of genes
This strategy is based on a tissue‐specific inactivation of the gene of interest. This can be achieved by means of a Cre recombinase, that catalyzes site‐specific
recombination of DNA between loxP sites. A Cre recombinase is an enzyme that deletes the DNA fragment located
between the two recombinase‐specific (LoxP) sites. A mouse bearing the
recombinase‐specific sites (introduced by homologous recombination in
Embryonic Stem cells) is bred with a mouse expressing the recombinase
(generated by homologous recombination or transgenesis). The tissue‐specific
expression of the recombinase allows the inactivation of the gene of interest
only in the tissue where the recombinase is expressed. Cre­driven conditional expression of genes
Transgenic animals, in which the target gene is flanked
by Lox sequences, must also express Cre recombinase
Thus, they have to be cross‐bred with mice expressing
Cre. The expression of Cre can be: 1.
Tissue specific – Cre gene is driven by the tissue
specific promoter, eg. heart, liver etc. 2. Conditionally induced – Cre gene is driven by the
inducible promoter, eg. tetracycline‐induced or IFN‐α
induced
VEGF is required for growth and survival in neonatal mice One allele of VEGF deleted in exon 3
Gerber et al., 1999
VEGF is required for growth and survival in neonatal mice 1. 38% mortality at day 7 in mice without VEGF (its synthesis was blocked from day 3);
2. Liver changes ‐ smaller hepatocytes, immature sinusoids, increased
extramedullary hematopoiesis and almost complete absence of
Flk‐1 positive endothelial cells; 3. Similar effects as after targeted knockouting of VEGF were obtained
when mice were treated with anti‐VEGF antibodies
Targeting of VEGF isoform­specific alleles
Stalmans et al., JCI 2002
Impaired retinal vascular development
in VEGF120/120 and VEGF188/188 mice Stalmans et al., JCI 2002
Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms
The present study investigates
the distinct role of the different
VEGF isoforms in retinal vascular
development. Retinal vascular
development was normal in
VEGF164/164 mice. In contrast, VEGF120/120 mice exhibited
severe vascular defects, with
impaired venous and severely
defective arterial vascular
development in the retina. VEGF188/188 mice had normal
venous development, but aborted
arterial outgrowth. Stalmans et al., JCI 2002
Effect of conditional knockout of VEGF164 on myocardial angiogenesis
Capillary density increases 300% in
VEGF+/+ hearts (filled bars) but not in
VEGF120/120 hearts (stippled bars).
There were fewer α‐actin stained
coronary vessels per section in
VEGF120/120 hearts (stippled bars) than in VEGF+/+ hearts (filled bars).
Carmeliet et al., 1999
Viability of VEGF­isoform mice VEGF120/120 – half neonates died shortly after births because of
cardiorespiratory distress; the other died within 2 weeks after birth, in part due to impaired myocardial
angiogenesis resulting in cardiac failure
VEGF164/164 – were normal
VEGF188/188 – half of embryos died in utero
‐ surviving gain less weigth, were less fertile
Take­home messages
VEGF (VEGF‐A) is a key mediator of vasculogenesis, angiogenesis
and arteriogenesis
VEGF is generated in the form of several isforms, being the results of alternative
splicing
The most common and the most active and crucial isoform is VEGF165
VEGF exerts its activity by binding to its receptors: VEGFR1, VEGFR2 and co‐
receptors: neuropilin 1 & 2. VEGFR2 is the key receptor, mediating the majority of actions of VEGF. VEGFR1 is a decoy receptor, playing an important role in modulating VEGF activity during development