other angiogenic mediators vegf familybiotka.mol.uj.edu.pl/zbm/handouts/2006_05_angiogeneza.pdf ·...
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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
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