Lecture V

Other angiogenic mediators VEGF family

Vascular endothelial growth factorVascular endothelial growth factor--A (VEGF)A (VEGF)

Yancopoulos, Science 2000

Receptors on endothelial

cells

VEGF-A is a major angiogenic growth factor. It acts on endothelial cells, being produced by numerous cell types, including vascular smooth muscle cells (VSMC), fibroblasts or tumor cells.

VEGFs growth factors activate the class of receptorsVEGFs growth factors activate the class of receptors::VEGFRs and neuropilinsVEGFRs and neuropilins

Gene Sequence homology Splice variants

Main activity

VEGF-A

121,145, 165,183, 189, 206

regulation of normal and

pathological angiogenesis, survival

VEGF-B

45% homology with VEGF-A

167, 186

embryonic myocardial

vascularization

VEGF-C

30% homology with VEGF-A 165 27% homology with VEGF-B 167

388

promotion of angiogenesis and

lymphangiogenesis

VEGF-D

61% homology with VEGF-C

31% with VEGF-A 165

333

regulator of lymphangiogenesis, proliferation of endothelial cells

PlGF

42% homology with VEGF-A

131, 152

modulation of pathological angiogenesis, migration of

endothelial cells and monocytes

Placenta growth factor

- expressed primarily in the placenta, especially late stages of gestation

- expression up-regulated during pathological conditions: heart ischemiamyocardial infarction, wound healing

- binds to VEGFR-1

Placental growth factor

Trends Cardiovasc Med. 2002

131 aa

152 aa

203 aa

Signal peptide

Molecular characterization of PlGF

* Biologicaly active glicoprotein acting as homodimer 46 kDa, as well as heterodimer with VEGF

* PlGF 1 – 131 a.a. secretory protein, undergoes N glycosylation

* PlGF 2 – 152 a.a. highly cationic 21 a.a. carboxyterminal (encoded in the 6th exon) enables it’s association with membrane by binding to polyanionic heparan sulphate

* PlGF 3 – the longest of all three isoforms, extra 72 a.a.(216 b.p.) insert between 4th and the 5th exon, N glycosylation

Receptor for PlGF

* Distinct pathophysiological roles of PlGF are mediated by binding to the fms-like tyrosine kinase receptor 1 Flt 1/ VEGFR1

* PlGF binds to Flt 1/ VEGFR1 with high affinity leading to its trans phosphorylation, thus activating signal transduction cascade

* PlGF does not cause trans phosphorylation and further signal transduction cascade through the KDR/ VEGFR2

Role Role of PlGF of PlGF –– effect of PlGF gene knockouteffect of PlGF gene knockout

1. Does not affect embryonic angiogenesis 2. Impaired inflammatory angiogenesis 3. Bone-marrow transplant restored inflammatory angiogenesis in

adult PlGF-ko mice 4. Synergism with VEGF, not bFGF 5. Amplify the angiogenic response to VEGF 6. Effect of PlGF in inflammation is mediated by VEGFR1, but it is not

angiogenic

Role Role of PlGF in embryo and in the adult of PlGF in embryo and in the adult

- amplification of VEGF-driven angiogenesis by PlGF

Carmeliet et al., 2003

Intramolecular crossIntramolecular cross--talk talk through flkthrough flk--1/1/fltflt--1 1 dimerization dimerization

Carmeliet et al., 2003

Synergism between Synergism between VEGF VEGF and PlGF and PlGF

1. By activating VEGFR-1, PlGF induces an intermolecular cross-talk between VEGFR-1 and VEGFR-2, what activates VEGFR-2 and enhances the response to VEGF

2. PlGF, as a subunit of the PlGF/VEGF heterodimer, stimulatesangiogenesis by inducing the formation of VEGFR-1/VEGFR-2 heterodimers, which transphosphorylate each other in intramolecular reaction

3. PlGF upregulates VEGF, thereby amplifying the synergism

Embryonic stem cells derived tumors Embryonic stem cells derived tumors

Wild type

PlGF-/-

VEGF-/-

Carmeliet et al., 2002

Impaired wound healing in PlGFImpaired wound healing in PlGF--//-- mice mice

Carmeliet et al., 2002

Vascular response Vascular response to to PlGF adenoviral gene PlGF adenoviral gene transfer transfer in the in the ear of nude ear of nude mice mice

0 days 14 days 28 days

Luttuin et al., Nature Med. 2002

Nature Med. 2001

Role Role of PlGF in recruiment of progenitor cells of PlGF in recruiment of progenitor cells

Different cells express Different cells express VEGF VEGF receptorsreceptors

VEGFR-1

ENDOTHELIAL CELLS; osteoblasts, monocyte/macrophages,pericytes, placental trophoblasts, renal mesangial cells, some hHematopoietic cells

VEGFR-2

ENDOTHELIAL CELLS; neuronal cells, osteoblasts,pancreatic ductal cells, retinal progenitor cells, megakaryocytes, some hematopoietic cells

VEGFR-3

Present on all endothelia during development, but in the adult becomes restricted to lymphatic EC and certain fenestratedblood vascular EC. Up-regulated on blood vascular EC in tumors

Inhibition of PlGF signaling results in:

- reduced mobilization of bone-marrow-derived myeloid progenitors into the peripheral blood

- impaired infiltration of Flt-1 expressing leukocytes in inflammed tissues

- defective activation of myeloid cells

-Inhibition of the atherosclerotic plaque growth

AntiAnti--inflammatory effect of antiinflammatory effect of anti--Flt1 (VEGFR1)Flt1 (VEGFR1)

AntiAnti--FltFlt--1 1 inhibits vascularisation inhibits vascularisation ((Matrigel plugMatrigel plug) )

Luttuin et al., Nature Med. 2002

Luttuin et al., Nature Med. 2002

VEGF/VEGFR-2 axis – important for physiological (developmental) and pathological angiogenesis

PlGF/VEGFR-1axis – specifically crucial for pathological angiogenesis

VEGFVEGF--B B

-a ligand for VEGFR-1

-Two isoforms: VEGFB-167 (binds to heparan sulphate) and VEGFB-186

- may contribute to the embryonic myocardial vascularization

- may play a role in pathological angiogenesis (eg synovial inflammation was reduced in VEGFb knockout mice )

Li &Erriskon, 2001

Heparin –binding domainVEGF homology domain

unique VEGFB186 domain

Silk domain

Functions and activity of Functions and activity of VEGFVEGF--BB

- binds to VEGFR-1 - knockouts are not lethal - activates Akt kinase (like VEGF-A) - increases eNOS expression - induced angiogenesis in subcutaneously injected Matrigel –

- angiogenesis was blocked by L-NAME and ant-VEGFR1

Seems to be required for coronary artery development

Lymphatic Lymphatic system system Gasparo Assellius – 1627

Functions:

1. Collection of fluids extravasated from the vessels 2. Fat uptake in the gut 3. Immune functions

4. Role in pathology: lymphaedema (insuficient) tumor growth and metastatis (overgrowth of

lymphatic vessels)

Vascular Vascular system system and lymphatic and lymphatic systemsystem

Jones N et al., Nature Rev Mol cell Biol. April 2001

Karkkainen et al., Nature Cell Biol 2002

Development of lymphatic Development of lymphatic system system

- from embryonic vein - from lymphangioblasticprecursors in the tissues

Rafii S & Skobe M, Nature Med. February 2003

Segregation of blood and lymphatic vessels Segregation of blood and lymphatic vessels

Development of the lymphatic Development of the lymphatic system system

Blood vessels lymph vessels

VEGFR-3LYVE-1

VEGFR-1VEGF-2Tie-2Nrp-1CD31CD31

RK Jain & TP Padera, Science 2003: 299: 209-210.

VEGFR-3Prox-1SLC

Prox-1LYVE-1

ProxProx--1 1 –– a master regulator a master regulator of the lymphatic vasculature phenotype of the lymphatic vasculature phenotype Prospero-like protein

Prox-1 up-regulates lymphangiogenic genes

Knockouts of Prox-1:

+/- - appear normal at birth, but die within 2-3 days after birth

-/- - die at E14.5-15.5 due to combination of several phenotypic alterations, including lack of lymphatic vessel formation

Rafii S & Skobe M, Nature Med. February 2003

Segregation of blood and lymphatic vessels Segregation of blood and lymphatic vessels

Markers for the lymphatic vessels

Gale & Yancopoulos, 1999

VEGF-C - like VEGF, stimulates the migration of EC and increase vascular permeability and EC proliferation (but at higherconcentrations than VEGF)

- binds to VEGFR-3 (FLT4) and VEGFR-2; binds also to Nrp-2

- its expression does not seem to be regulated by hypoxia, but is by pro-inflammatory cytokines

- regulate physiological and pathological-blood vessel growth in vivo

- regulate growth of lymphatic vessels

- overexpression of VEGF-C in skin keratinocytes leads to dermal-lymphatic vessel hyperplasia

- knockouts – results in defects in intestinal and cutaneous lymphatic vessels

VEGFRVEGFR--3 3 and lymphangiogenesis and lymphangiogenesis

VEGFR-3 is initially expressed in all venous endothelial cells

Only Prox-1-expressing cells start the sprouting process and simultaneously upregulate other lymphatic markers, such as podoplanin and LYVE-1

Signaling by VEGFR-3 alone is sufficient to induce lymphangiogenesis

Knockout of VEGFR-3 Lethal at E 9.5 - enlarged vessel lumens, cardiovascular failure

Proteolytic activations of VEGFsProteolytic activations of VEGFs

McColl et al., APMIS 2004

Solubilisation Release from ECM

Binding to Neuropilin-1

Binding to VEGFR-2 and increased affinity to VEGFR-3 and Neuropilin-1

Binding to VEGFR-2 and increased affinity to VEGFR-3 and neuropilin-1

VEGF-A VEGF-B186 VEGF-C VEGF-D

Processing of VEGF-C

VEGF-C acquires the capacity to bind VEGFR-2

- binding activity of processed VEGF-D to VEGR-2 increasesby 290-fold

Processing signifcantly enhances activity ofProcessing signifcantly enhances activity of VEGFVEGF--CC andand DD

1. In Vegfc-/- mice, endothelial cells commit to the lymphatic lineage but do not sprout to form lymph vessels.

2. Sprouting was rescued by VEGF-C and VEGF-D but not by VEGF, indicating VEGF receptor 3 specificity.

3. The lack of lymphatic vessels resulted in prenatal death due to fluid accumulation in tissues, and Vegfc+/- mice developed cutaneous lymphatic hypoplasia and lymphedema.

VEGF-C is the paracrine factor essential for lymphangiogenesis, and show that both Vegfc

alleles are required fornormal lymphatic development.

Effect of VEGF-C gene knockout

Karkkainen MJ, Nature Immunol 2004

VEGFVEGF--D D c-FOS induced growth factor (FIGF)

- 61% of aa identity to VEGF-C

- binds VEGFR-3 and VEGFR-2 (but mice VEGF-D does not bind to VEGFR2)

- also proteolytically processed in a similar way like VEGF-C

- stimulates EC proliferation

- is lymphangiogenic when overexpressed in skin keratinocytes

- little is known about VEGF-D expression in physiological conditions

VEGFVEGF--D D knockoutknockout

?

Karkkainen et al., Nature Cell Biol 2002

VEGFVEGF--C C and and VEGFVEGF--D D signalingsignaling

VEGFR-3

Present on all endothelia during development, but in the adult becomes restricted to lymphatic EC and certain fenestratedblood vascular EC. Up-regulated on blood vascular EC in tumors

Functions of Functions of VEGFVEGF--C C and and VEGFVEGF--D D in tumors in tumors

Functions of Functions of VEGFVEGF--C C and and VEGFVEGF--D D in tumors in tumors

- produced by tumor and stromal cells

- signal through VEGFR-2 – leads to angiogenesis and tumor growth

- signal through VEGFR3- on lymphatic endothelial cells – promotes metastasis

- modulate also immune functions: VEGF-C – is chemotactic for macrophages – express VEGFR-3macrophages also express and secrete VEGF-C and VEGF-D

VEGF-A and VEGF-D induces strong capillary enlargement in skeletal muscle

Rissanen et al., Circ Res 2003

Regulation of VEGFs expression Regulation of VEGFs expression

McColl et al., APMIS 2004