wyklad 2 12 03 2012
TRANSCRIPT
VascularVascular endothelialendothelial
growthgrowth factorfactor andand otherother
propro--angiogenic angiogenic factorsfactors
lecture II
12th March 2012
Oloffson et al., 2000
VEGF, VEGFVEGF, VEGF--AA
vascular endothelial growth factor
main regulator of angiogenesis, pro-angiogenic factor
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.
Angiogenesis and VEGF “history”AngiogenesisAngiogenesis andand VEGF VEGF ““historyhistory””
VascularVascular
EndothelialEndothelial
GrowthGrowth
FactorFactor
VV
EE
GG
FF
VascularVascular
PermeabilityPermeability
FactorFactor
VV
PP
FF
1983,1983,
Dr H. Dr H. DvorakDvorak
1989, 1989,
Dr N. FerraraDr N. Ferrara
Dr J. Dr J. PlouetPlouet
=
vascularvascular permeabilitypermeability factorfactor
endothelialendothelial cellcell survivalsurvival factorfactor
endothelialendothelial cellcell proliferationproliferation
endothelialendothelial cellcell migrationmigration
MainMain proangiogenicproangiogenic factorfactor
VEGF VEGF belongsbelongs to VEGF to VEGF familyfamily
EndogenouslyEndogenously expressedexpressed inin mammalsmammals
EncodedEncoded by by thethe
double double strandedstranded
DNA DNA virusvirus, , orforf
VEGFVEGF--AA
VEGFVEGF
• VEGF (vascular endothelial growth factor,
vascular permeability factor, vasculotropin)
– homodimeric protein
• 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.
VEGFVEGF
• 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 VEGF isis highlyhighly conservedconserved betweenbetween speciesspecies
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
Invertebrate VEGF/VEGFR systems have been identified in fly (Drosophila
melanogaster), nematode (Caenorhabditis elegans) and, most recently, in jellyfish
(Podocoryne carnea).
VEGF-like proteins are present in several invertebrate species
PresencePresence ofof VEGFVEGF--likelike proteinsproteins inin differentdifferent animalsanimals
• 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
Jorgensen & Mango, Nat Rev Gen 2002; Tarsitano et al. FASEB J 2006.
Control HUVEC HUVEC + VEGF HUVEC + PVF-1
O’Farrell, J Clin Invest 2001
DrosophilaDrosophila melanogastermelanogaster
respiratory (respiratory (trachealtracheal) system) 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
• 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
OrganisationOrganisation ofof VEGF VEGF genegene andand VEGF isoforms VEGF isoforms
Exons 1-5 8
Exons 1-5 86A1 A2
Exons 1-5 87
VEGF-A121
VEGF-A145
VEGF-A165
VEGF-A189
VEGF-A206
Exons 1-5 876A1 A2
Exons 1-5 876A1 A2 6B
Sequestered in ECM but releasedby cleavage
1-8 plus additional exon206VEGF-A206
Sequestered in ECM but releasedby cleavage
1-8189VEGF-A189
Sequestered in ECM but releasedby cleavage
1-5, short exon 6, 7, 8183VEGF-A183
Secreted, endogenous inhibitory form of VEGF-A165
1-5, 7, alternative exon 8165VEGF-A165b
The most abundant and biologicallyactive isoform; secreted; binds
NRP1 and NRP2
1-5, 7, 8165VEGF-A165
Binds NRP2 but not NRP1; secreted1-6, 8145VEGF-A145
Secreted1-5, 8121VEGF-A121
FeaturesCoding exonsSize(amino acid)
Isoform
VEGF isoformsVEGF isoformsVEGF isoforms
PropertiesProperties ofof VEGF isoforms 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
ExpressionExpressionofof VEGF isoformsVEGF isoforms
• Most VEGF-producing cells express VEGF121, VEGF165, VEGF189,
and often VEGF183. VEGF206 is 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 Not everyevery cells cells expressexpress
thethe same same amountsamounts ofof VEGF VEGF
HASMC HMEC-primary HMEC-1 rat Müller cells
VEGF VEGF expressionexpression in in severalseveral cellcell lineslines -- intactintact cellscells
(24 h incubation)
MBEC ~ 300-400 pg/ml
HepG2 ~ 200 pg/ml
HMEC-1 ~ 20 pg/ml
165
121
ReceptorsReceptors for VEGFfor VEGF--AA
Main receptors:
VEGFR-1 (Flt-1)
VEGFR-2 (Flk1; KDR)
Accessory receptors
Neuropilin 1 (NRP1)
Neuropilin 2 (NRP2)
Storage
heparan sulfate proteoglycans
ReceptorsReceptors for VEGFfor VEGF--AABoth 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.
GrowthGrowth factorsfactors andand receptorsreceptors ofof thethe VEGF VEGF familyfamily
VEGFVEGF--R1R1 VEGFVEGF--R2R2 VEGFVEGF--R3R3
HeparanHeparan--SulfateSulfateProteoglycanProteoglycan
NeuropilinNeuropilin --11 NeuropilinNeuropilin --22
After Neufeld et al.. 1999, FASEB J 13:9-22
VEGF121VEGF121VEGF145VEGF145VEGF165VEGF165VEGF189VEGF189VEGFVEGF--BBPlGFPlGF--11PlGFPlGF--22
VEGF121VEGF121VEGF145VEGF145VEGF165VEGF165VEGFVEGF--CCVEGFVEGF--DDVEGFVEGF--EE
VEGFVEGF--CCVEGFVEGF--DD
VEGF145VEGF145VEGF165VEGF165VEGF189VEGF189VEGF206VEGF206VEGFVEGF--B167B167VEGFVEGF--EEPlGFPlGF--22
SemaSema--IIIIIISemaSema--EESemaSema--IVIVVEGF165VEGF165PlGFPlGF--22VEGFVEGF--BBVEGFVEGF--EE
SemaSema--EESemaSema--IVIVVEGF165VEGF165
TK
TK
ExpressionExpression ofof VEGF VEGF receptorsreceptors
- endothelial cells: VEGFR-1, VEGFR-2, co-receptors
- other cells:
monocytes
vascular smooth muscle cells
tumor cells
hematopoietic stem cells
neuronal cells
SignificanceSignificance ofof VEGF VEGF andand VEGF VEGF receptorsreceptors
hashas beenbeen recognizedrecognized by by targetingtargeting
disruptiondisruption ofof thosethose genesgenes inin mice mice
Ferrara & Alitalo, Nature Med. 1999
KnockoutKnockout ofof VEGF VEGF isis lethallethal inin heterozygousheterozygous form form
Yolk sac of E10.5 VEGF+/+ and VEGF +/– mouse embryos
EffectEffect ofof knockoutknockout ofof VEGF VEGF receptorsreceptors
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
VEGFR-1
VEGFR-2
Flk1-null mice die between day 8.5 and 9.5
Lack of vasculogenesis and failure to develop blood islands
and organized blood vessels
SemaphorinSemaphorin receptorsreceptors –– NpNp--1 1 andand NpNp--2 2
- form complexes with type A plexins
- complexes serves as signaling receptors for class-3 semaphorins
- involved in axonal guidance
Np-1 and Np-2 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
Functions of the VEGF receptors familyFunctions of the VEGF receptors family
Found only in lymphatic endothelial cells
Associated with lymph node metastasis
VEGFR-3
Mediates the majority of VEGF angiogenic effectsVEGFR-2
Crucial to embryonic angiogenesis
Does not appear to be critical in pathogenic
angiogenesis
VEGFR-1
The The VEGFRsVEGFRs differ in their downstream signaling effectsdiffer in their downstream signaling effects
Effects mainly in lymphatic cellsVEGFR-3
Proliferation
Migration
Survival
Angiogenesis
VEGFR-2
Possible “decoy receptor” effect
Induction of other factors
VEGFR-1
EffectsReceptor
MechanismsMechanisms ofof antianti--apoptoticapoptotic VEGF VEGF signalingsignaling
Zachary, Cardiovasc Res 2001
Phosphotydyloinositol
3 kinase
Focal
adhesion
kinase
AKT = PKB
MechanismsMechanisms ofof chemotacticchemotactic VEGF VEGF signalingsignaling
Zachary, Cardiovasc Res 2001
MechanismsMechanisms ofof mitogenicmitogenic VEGF VEGF signalingsignaling
Zachary, Cardiovasc Res 2001
Extracellular signal-regulated kinases
Diacyloglicerol+
Inositol 1,4,5 -trisphosphate
Proteins with
src homology
(SH) 2 domain
phosphatydylinositol
4,5 biphosphate
Phospholipase C
VEGF VEGF levellevel hashas to be to be tightlytightly
regulatedregulated duringduring developmentdevelopment
andand postnatalpostnatal lifelife
Embryonic development is disrupted by modest
increase in VEGF gene expression
Miquerol L, Langille BL, Nagy A.Development, 2000: 127:3941-6
2-3 fold overexpression is deletorious to embryonic development
Enlarged hearts
Embryos died between E12.5 and E14.5
A A andand B. B. NoteNote prominent prominent tissuetissue edemaedema
andand newnew bloodblood vesselvessel formationformation. .
C. C. NoteNote alsoalso a prominent a prominent leakageleakage ofof
plasmaplasma protein protein complexescomplexes fromfrom
locallylocally hyperpermeablehyperpermeable earear vesselsvessels..
TooToo high high andand unbalancedunbalanced expressionexpression ofof VEGF VEGF
afterafter genegene deliverydelivery usingusing adenoviraladenoviral vectorsvectors
ConditionalConditional knockoutsknockouts ofof genesgenes
This strategy is based on a tissue-specific or conditionally-induced 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.
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
CreCre--drivendriven conditionalconditional expressionexpression ofof genesgenes
Two independent approaches to inactivate the angiogenic protein
VEGF in newborn mice were employed:
1. inducible, CreloxP- mediated gene targeting
2. administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric
protein.
Partial inhibition of VEGF achieved by inducible gene targeting
resulted in increased mortality, stunted body growth and
impaired organ development, most notably of the liver.
Administration of mFlt(1-3)-IgG, which achieves a higher degree of
VEGF inhibition, resulted in nearly complete growth arrest and
lethality.
VEGF is required for growth and survival in neonatal mice
Gerber et al., 1999
Gerber et al., 1999
VEGF VEGF isis requiredrequired for for growthgrowth andand survivalsurvival inin neonatalneonatal mice mice
One allele of VEGF
deleted in exon 3
VEGF VEGF isis requiredrequired for for growthgrowth andand survivalsurvival inin neonatalneonatal mice 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 a soluble VEGF receptor chimeric protein.
Kidneys from mFlt(1-3)-IgG-treated animals weresmaller, had a granularsurface appearance andshowed punctuate areas
of hemorrhage.
Hearts from control andmFlt(1-3)-IgG-treated
animals: heartsfrom mFlt(1-3)-IgG-
treated animals weresignificantly smaller.
Gerber et al., 1999
TheThe effecteffect ofof differentdifferent
isoforms isoforms ofof VEGF on VEGF on
angiogenesisangiogenesis
VesselVessel formationformation andand sproutingsprouting angiogenesisangiogenesis inin
embronyicembronyic bodiesbodies inin responseresponse to to thethe differentdifferent VEGF isoformsVEGF isoforms
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
VesselVessel formationformation andand sproutingsprouting angiogenesisangiogenesis inin
embryonicembryonic bodiesbodies inin responseresponse to to thethe differentdifferent VEGF VEGF ligandsligands
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
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
VEGF-A165bVEGF-A165b
endothelial cells pericytes
Kawamura et al. Cancer Res. 2008
VEGF-A121VEGF-A121
endothelial cells pericytes
Kawamura et al. Cancer Res. 2008
In the VEGF-A121–containing Matrigel plugs, occasional
vessel structures were seen, which lacked branch
points and a pericyte coat
VEGF-A145VEGF-A145
endothelial cells pericytes
Kawamura et al. Cancer Res. 2008
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
Inclusion of VEGF-A165 induced abundant vascularization of
the Matrigel with richly branched, pericyte-covered vessels
endothelial cells pericytes
Kawamura et al. Cancer Res. 2008
VEGF-A165VEGF-A165
!VEGF165 is the crucial isoform!
the role of single VEGF isoforms was studied in
retinal vascular development - mice selectively
expressing single isoforms were created
OrganisationOrganisation ofof mousemouse VEGF VEGF genegene
Exons 1-5 8
Exons 1-5 87
VEGF-A120
VEGF-A164
VEGF-A188Exons 1-5 876A1 A2
ImpairedImpaired retinalretinal vascularvascular developmentdevelopment
inin VEGFVEGF120/120120/120 andand VEGFVEGF188/188188/188 mice mice
Stalmans et al., JCI 2002
ArteriolarArteriolar andand venularvenular patterningpatterning inin retinasretinas ofof mice mice
selectivelyselectively expressingexpressing VEGF isoformsVEGF 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
Site-specific removal of VEGF exons 6 and 7 in embryonic stem cells using the
Cre/LoxP system was achieved
The authors used targeted ES cells to generate VEGF+/120 mice, which seemed
normal and healthy.
Neonates expressing exclusively VEGF120 (VEGF120/120) were recovered at birth at
a normal Mendelian frequency: of 120 neonates, 26% were VEGF+/+, 51% were
VEGF+/120 and 24% were VEGF120/120.
About half the VEGF120/120 neonates died within a few hours after birth because of
bleeding in several organs
Dissection of VEGF120/120 mice showed they had enlarged hearts, irregular heart
beats and dysmorphic, weak heart contractions
EffectEffect ofof conditionalconditional knockoutknockout ofof VEGF164 VEGF164
andand VEGF188 on VEGF188 on myocardialmyocardial angiogenesisangiogenesis
CarmelietCarmeliet et al., 1999et al., 1999
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).
No difference in angiogenic potentials of various VEGF isoforms
VEGF121
VEGF165
controlcontrol
VEGF121
VEGF165
Matrigel assay Spheroid assay
Jozkowicz and Dulak
Actions of nitric oxide,
carbon monoxide and
hydrogen sulfide- toxic pollutants
or physiological regulators?
Nitric oxide synthases
eNOS - endothelial (constitutive) NOS (NOS III) nNOS - neuronal (constitutive) NOS (NOS I)iNOS - inducible (NOS II)
L-arginine
O2
.NO
L-citrulline
NOS
cofactors
inhibitors of NOS isoforms
L- NAME - L-NG-Nitroarginine methyl ester
L-NMMA - L-NG-monomethyl arginine citrate
InvolvementInvolvement ofof nitricnitric oxideoxide inin angiogenic angiogenic activitiesactivities ofof VEGF isoforms VEGF isoforms
Time [sec]
NO
co n
cen t
r ati o
n[ n
mo l
/L]
100
200
300
400
500
05 10 15 20 25 30
VEGF121
VEGF165
VEGF
Release of NO by VEGF-stimulatedendothelial cells is strongerin case of VEGF121 isoform
Józkowicz, Dulak et al., Growth Factor, 2004
0
2
4
6
8
10
12
14
16
control 121 121L-NAME
121
D-NAME
165 165L-NAME
165
D-NAME
cGM
P[fm
ol/m
l]
#
*
#
*
Synthesis of cGMP by VEGF-stimulatedendothelial cells is higher
in case of VEGF121 isoform
A. VEGF121 eNOS activity MigrationAssemblyCapillary sprouting
Proliferation
B. VEGF165 eNOS activityeNOS expression
MigrationAssembly
ProliferationCapillary sprouting
Properties of VEGF121 and VEGF165 isoforms
H2S is generated endogenously in mammalian tissues
Hydrogen sulfide (H2S), a well known toxic gas, is generated endogenously inmammalian tissues from L-cysteine by pyridoxal-5’-phosphate-dependent enzymes, including
• cystathionine β-synthase (CBS)
• cystathionine γ-lyase (CSE),
• cysteine aminotransferase (CAT)
• cysteine lyase
The expression of H2S-generating enzymes is tissue specific.
• CBS is highly expressed in the hippocampus and cerebellum in the mammalianbrain
• CSE is predominant in the vasculature in both vascular smooth muscle cellsand endothelial cells
Angiogenesis begins fromlocal matrix degradation inpre-existing blood vessels, possibly induced by H2S.
Endothelial cell proliferationand migration and capillarysprout formation are activatedby H2S.
Newly formed capillaries mayfuse into bigger functionalvessels and H2S may be involved in this process.
Mechanisms of angiogenesis and
the effect of hydrogen sulfide
HH22S S
Hydrogen sulfide promotes angiogenesis in vivo
Wang et al., Clin Exp Pharmacol Physiol. 2010
representative post-mortemangiograms after femoral arteryocclusion in the rat. There was more collateral vessel formation inthe ischaemic left hindlimb of ratstreated with 100 umol ⁄ kg per dayNaHS than in the control.
The arrows indicate the site ofligation of the femoral artery.The arrowhead indicates the typicalappearance of collateral vessels.
Hydrogen sulfide promotes angiogenesis in vivo
Changes in total burn wound area over time. Four animals for CSE wild-type group and five for CSE knockout mice were used.
Papapetropoulos et al., PNAS 2009
Heme Heme oxygenaseoxygenase pathwaypathway
Heme oxygenase
Ferritin
tissue injury
Hemeproteins
Fe2+ tissue injury
bilirubin protectionbiliverdin
CO
protection
soluble guanylyl cyclase
cGMPGTP
protection
Heme
p38
BvR
VEGF (VEGF-A) is a key mediator of vasculogenesis, angiogenesis
and arteriogenesis
TakeTake--homehome messagesmessages
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
NO, CO and H2S are important mediators in angiogenesis