Vascular endothelial growth factor

lecture II

9th March 2015

Angiogenesis and VEGF “history”

1983 - Vascular Permeability Factor (VPF), is discovered by Dr Harold Dvorak. The VPF molecule 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

V E G F

1989, Dr N. Ferrara Dr J. Plouet

vascular permeability factor endothelial cell survival factor endothelial cell proliferation endothelial cell migration Oloffson et al., 2000

Blood vessel formation – various ways

Carmeliet, 2005; Semenza 2003

VEGF belongs to VEGF family Endogenously expressed in mammals VEGF-A

Encoded by the double stranded DNA virus, orf

VEGF • 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.

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

VEGF-like 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 VEGF-like 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 VEGFR1 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.

VEGF belongs to VEGF family VEGF-A

Organisation of VEGF gene The human VEGF-A gene is characterized by a highly conserved eight exon structure

Organisation of VEGF gene and VEGF isoforms • 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

Nrp-1 HBD – heparin binding domain

Woolard J et al., Canc Res 2008

Not every cells express the same amounts of VEGF VEGF expression in several cell lines - intact cells (24 h incubation) HASMC

HMEC-primary HMEC-1

rat Müller cells 165 121

MBEC ~ 300-400 pg/ml HepG2 ~ 200 pg/ml HMEC-1 ~ 20 pg/ml

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 or binds to ECM; binds to 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

Matrix-bound VEGF isoforms are released by proteinases Matrix-bound VEGF isoforms are released by proteinases:  heparinases and matrix metalloproteinases (MMPs) produce VEGF113 (Lee et al., 2005)

 plasmin generates an active VEGF fragment of 110 NH2-terminal residues, named VEGF110 (Keyt et al., 1996)

VEGF 113 is bioactive

Lee et al., 2005

Plasmin cleavage of human VEGF

.

Endothelial cell proliferation in response to VEGF isoforms

Keyt et al., 1996

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 or binds to ECM; binds to 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

• The particular splicing event in the terminal exon creates two whole families of isoforms: the classic pro-angiogenic VEGFxxx (through proximal splice-site selection and more recently described antiangiogenic VEGFxxxb isoforms (through distal splice-site selection)

Peiris-Pages, Cell Adh Migr, 2012

basic properties

• Exon 8a encodes - Cys-Asp-Lys-Pro-Arg-Arg

neutral properties, loss of disulfide bond, conformational changes lack of binding the VEGFR-2 co-receptor Nrp-1

• Exon 8b encodes – Ser-Leu-Thr-Arg-Lys-Asp

Peiris-Pages, Cell Adh Migr, 2012

• particularly, the tyrosine 1054 is not phosphorylated following VEGF165b-VEGFR2 binding Peiris-Pages, Cell Adh Migr, 2012

Peiris-Pages, Cell Adh Migr, 2012

VEGF165b is anti-angiogenic

Mice that received injections of A375 melanoma cells transfected with VEGF165 grew larger tumors more quickly than VEGF165b. Mice that received injections of equal amounts of both transfected cell types grew at an intermediate rate.

Woolard J et al., Canc Res 2004

VEGFxxx and VEGFxxxb – sister families? More than 50 000

9 -> VEGFxxxb 37 -> VEGF165b

Do anti-angiogenic VEGF (VEGFxxxb) isoforms exist? A cautionary tale. Harris S, Craze M, Newton J, Fisher M, Shima DT, Tozer GM, Kanthou C. PLoS One. 2012;7(5):e35231.

Detection of VEGF-A(xxx)b isoforms in human tissues. Bates DO, Mavrou A, Qiu Y, Carter JG, Hamdollah-Zadeh M, Barratt S, Gammons MV, Millar AB, Salmon AH, Oltean S, Harper SJ. PLoS One. 2013 Jul 31;8(7):e68399 We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is.

VEGFxxxb - summary • first VEGFxxxb was discovered in human renal cortex tissue in 2002 (VEGF165b) • VEGFxxx and VEGFxxxb are generated from the same transcript and their relative amounts are dependent on alternative splicing • anti-angiogenic VEGFxxxb represent a predominant proportion of the total VEGF protein found in different tissues (one exception – placenta – tissue where angiogenesis takes place under the normal, physiological conditions) • In contrast to VEGFxxx, the anti-angiogenic VEGFxxxb are down-regulated in tumors and other pathologies associated with abnormal neovascularization • VEGFxxxb inhibits VEGFR2 signaling phosphorylation of tyrosine residues

by

inducing

differential

The role of VEGF111 in tumorigenesis

Mineur et al., J Cell Biol. 2007

Receptors for VEGF-A Main receptors: VEGFR-1 (Flt-1) VEGFR-2 (Flk1; KDR) Accessory receptors

Neuropilin 1 (NRP1) Neuropilin 2 (NRP2) Storage heparan sulfate proteoglycans

VEGF-A 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.

The Biology of Cancer by R Weinberg

Ferrara et al. 2004

Significance of VEGF receptors has been recognized by targeting disruption of those genes in mice

Effect of knockout of VEGF receptors VEGFR-1 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-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

Semaphorin receptors – Nrp-1 and Nrp-2 - transmembrane glycoprotein, coreceptor to a tyrosine kinase receptor for both VEGF and semaphorins - Nrp1 plays versatile roles in angiogenesis, axon guidance, cell survival, migration, and invasion

Nrp-1 and Nrp-2 in angiogenesis

- knockout of Nrp-1 – lethal at E12.5 - overexpression of Nrp1- excessive capillary formation, dilated blood vessels extensive hemorrhage - no visible abnormalities in Nrp-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 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

VEGF signaling

VEGF-Receptor associated protein

? – indirect mechanisms Shb adaptor protein

Angiogenic and vasculoprotective functions of VEGF

Qiagen

Mechanisms of anti-apoptotic VEGF signaling

Phosphatydyloinositol 3 kinase

AKT = PKB

Zachary, Cardiovasc Res 2001

Mechanisms of chemotactic VEGF signaling

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

Ca2+ influx from internal stores

Extracellular signal-regulated kinases

Zachary, Cardiovasc Res 2001

VEGF level has to be tightly regulated during development and postnatal life

Knockout of VEGF is lethal in heterozygous form

Yolk sac of E10.5 VEGF+/+ and VEGF +/– mouse embryos Ferrara & Alitalo, Nature Med. 1999

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

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.

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 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. Transgenic animals, in which the target gene is flanked by LoxP 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

Two independent approaches to inactivate the angiogenic protein VEGF in newborn mice were employed: 1. inducible (after administration of IFN-α), 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.

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 VEGFR-2 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.

Gerber et al., 1999

Kidneys from mFlt(1-3)IgG-treated animals were smaller, had a granular surface appearance and showed punctuate areas of hemorrhage.

Hearts from control and mFlt(1-3)-IgG-treated animals: hearts from mFlt(1-3)-IgGtreated animals were significantly smaller.

Gerber et al., 1999

The effect of different isoforms of VEGF on angiogenesis

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

Vessel formation and sprouting angiogenesis in embryoid bodies in response to the different VEGF 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 VEGF-A165b, VEGF-A121, VEGF-A145, or vehicle. Kawamura et al. Cancer Res. 2008

Vessel formation and sprouting angiogenesis in Matrigel plugs in nude mice

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

VEGF-A165b 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

VEGF-A121 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

VEGF-A145 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

VEGF-A165 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

the role of single VEGF isoforms was studied in retinal vascular development mice selectively expressing single isoforms

Impaired retinal vascular development in VEGF120/120 and VEGF188/188 mice

Stalmans et al., JCI 2001

Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms

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 2001

Heparin-binding and Nrp-1-binding VEGF-A isoforms are critical for survival: mice enginereed to express exclusively the non-heparin binding and non-Nrp-1 binding VEGF120 have diminished vascular branching during embryonic development and die from postnatal angiogenesis defects shortly after birth

Take-home messages VEGF (VEGF-A) is a key mediator of angiogenesis VEGF is generated in the form of several isoforms, 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 coreceptors: 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