angiostasis and angiogenesis regulated by angiopoietin1-tie2 … · 2009. 7. 1. · japan-mexico...
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Japan-Mexico Workshop on “Pharmacology” and “Nanobiology”Feb. 25, 2009; Universidad Nacional Autönoma de Mëxico, Mexico City
Angiostasis and Angiogenesis Regulated by Angiopoietin1-Tie2 Receptor System
Shigetomo Fukuhara
Department of Structural Analysis, National Cardiovascular Center Research Institute
Vascular uiescence and Angiogenesisquiescent
vessel
mural cell
endothelial cells(EC)
angiogenesis
✓disrupt cell adhesions between EC-mural
cell and between EC-EC
✓proliferate and migrate
angiogenic factors such as VEGF
cancertissue injuryothers
Fli:GFP
Transgenic zebrafish embryo expressing GFP in vascular endothelial cells
dorsal aorta
intersegmental vessel
Visualization of in vivo angiogenesis
Angiogenic stimulator
Angiogenic inhibitor
Vascular quiescence vs. Angiogenesis
Angiopoietin-1 ( ) acts as both angiogenic inhibitor and stimulator
angiogenesis
vascular quiescenceEC
mural cell
Angiopoietin-1/Tie2 receptor system✓ Angiopoietin-1 (Ang1) is a ligand for the receptor tyrosine kinase Tie2, which is expressed on vascular endothelial cells.
✓ Ang1/Tie2 signal plays a critical role in developmental vascular formation.
✓ In adult vasculature, Ang1/Tie2 signal regulates not only vascular quiescence, but also physiological and pathological angiogenesis.
Tie2
Ig-like
EGF-like
FN III
kinase
Angiopoietin-1(Ang1)
vascular quiescence angiogenesis
EC-EC contactproliferation
migration
↑↓↓
↓↑↑
How does Ang1/Tie2 signal play distinct roles in How does Ang1/Tie2 signal play distinct roles in both vascular quiescence and angiogenesis?both vascular quiescence and angiogenesis?
Tie2
Ang1
quiescent vessels angiogenic vessels
cell-cell contacts (+) cell-cell contacts (-)
How?
ischemia
Ang1 induces recruitment of Tie2 at cell-cell contacts
COMP (Cartilage oligomeric matrix protein)
COMP-Ang1 Ang1
HUVEC
Ang1 induces recruitment of Tie2 at cell-cell contacts
CHO cells expressing Tie2-GFP were stimulated with COMP-Ang1
GFP
Tie2
-GFP
Accumulation of Tie2 at cell-cell contacts requires Tie2 expression
in adjacent cells.
Ang1 may induce trans-association of Tie2 at cell-cell contacts
Ang1 induces aggregation of 293F cells expressing Tie2Δcyto-GFP in suspension.
GFP
Cell expressing Tie2Δcyto-GFP
Cell expressing GFP
GFP
GFP
Cell expressing Tie2Δcyto-GFP
GFP
Cell expressing Tie2Δcyto-HA
HA
HA
COMP-Ang1
Ang1 induces trans-association of Tie2 at cell-cell contacts
Formation of Ang1-bridged Tie2 trans-association
Ang1 stimulation
Tie2
Tie2 is broadly expressed on plasma membrane in the absence of Ang1
Endothelial cell Endothelial cell
Conclusion Part1: in the presence of cell-cell contacts
Tie2
Ang1
quiescent vessels angiogenic vessels
cell-cell contacts (+) cell-cell contacts (-)
How?
ischemia
Ang1 induces accumulation of Tie2 at cell-substratum contacts, which are different from focal adhesions.
Sparse HUVECs were stimulated with COMP-Ang1 and Ang1.
Sparse HUVEC expressing Tie2Δcyto-GFP and RFP-Crk (focal adhesion marker) were stimulated with COMP-Ang1
GFP
Tie2Δcyto-GFP
RFP-Crk(focal adhesion
marker)
Extracellular domain is sufficient for accumulation of Tie2 at cell-substratum interface
merge phase contrast
Tie2 is anchored by ECM-bound Ang1 to cell-substratum contacts
ECM binding assay
Tie2-GFP-expressing CHO cells plated on collagen-coated
0 30 60COMP-Ang1 stimulation (min)
Tie2
C-Ang1
Ang1
ECM
Tie2
Ang1
ECM
Tie2Endothelial cell
Ang1 stimulationAng1 binding to ECM
Anchoring of Tie2 to cell-ECM contactby Ang1
ECM-anchored Tie2
Conclusion Part2: in the absence of cell-cell contacts
Biological significance of ECM-anchored Tie2 and trans-associated Tie2
sparse confluent
ECM-anchored Tie2 Trans-associated Tie2
Sparse and confluent HUVECs were stimulated with Ang1.
Erk↓
migrationproliferation
↓angiogenesis
Akt↓
cell survival↓
vascular quiescence
Preferential activation of Erk in sparse endothelial cellsand that of Akt in confluent cells upon Ang1 stimulation
sparse
ECM-anchored Tie2
Erk>Aktconfluent
trans-associated Tie2
Akt>Erk
confluent
sparse
sparse
confluent
attachedsuspended
Anchoring of Tie2 to ECM facilitates Erk pathway and induces focal complex assembly
collagen Ang1/collagen
EC EC
vinculin staining
Erk
ECM-anchored Tie2
focal complex(integrin/ECM)
ECM
Tie2
Ang1
integrinFAK
Erkmigration
Knock-down of FAK
Endothelial cell migration
ECM-anchored Tie2 activates Erk pathway partly via FAK, leading to the enhanced endothelial cell migration
Tie2
Ang1 stimulation
Ang1-bridged Tie2 trans-association
vascular quiescence Akt
Erksurvivalintegrity
ECM
PRESENCE OF CELL-CELL CONTACTS
oligomerized Ang1 Tie2 activated Tie2
ABSENCE OF CELL-CELL CONTACTS
Tie2
Ang1
Ang1-mediated Tie2 anchoring to ECM
Erk
Akt
ECM
Ang1stimulationangiogenesis migration
proliferation
Ang1
Conclusion Part3
Nat. Cell Biol. 10: 513-526, 2008
spar
seco
nflu
ent
spar
se/
conf
luen
t
confluent sparse
Ang1 Ang1
Ang1 stimulation for 1 h
RNA extraction
GeneChip analysis
Distinct sets of genes are regulated by trans-associated Tie2 and ECM-anchored Tie2
Krüppel-like factor 2 (KLF2)
Role of KLF2 in blood vessels
Dekker et al. Am. J. Pathol., 2005
Role of KLF2 in vascular quiescence!
KLF2 is a zinc finger family of transcription factor.
KLF2 expression is induced by laminar shear stress in endothelial cells,
but not induced in the region of disturbed blood flow.
KLF2 has anti-inflammatory, anti-thrombotic and anti-angiogenic effects.
✓✓
✓
transactivation transrepression Zn Zn Zn1 110 267 354
Summary of KLF2 target genesanti-inflammatory: VCAM-1↓, E-selectin↓, eNOS↑anti-thrombotic: PAI-1↓, TF↓, TM↑, eNOS↑, tPA↑
vasodilatory: ET-1↓, CNP↑, eNOS↑, ASS↑
anti-angiogenic: VEGFR2↓, SEMA3F↑
KLF2 inhibits VEGF-induced vascular leakage
Ad-KLF2
Inhibitory effects of Ang1 and KLF2 on VEGF-induced vascular leakage
Ang1 inhibits VEGF-induced vascular leakage.
Thurston et al. Science, 1999
Bhattacharya et al. J. Biol. Chem., 2005
Ad-GFP
Ang1/Tie2 signal may induce vascular quiescence through KLF2 expression.
Hypothesis
Intracellular signaling pathway involved in Ang1-induced KLF2 expression
✓
✓ Biological consequence of Ang1-induced KLF2 expression
Western blot analysis
Real-time PCR
KLF2 expression in endothelial cells from Ang1 transgenic mice
Trans-associated Tie2 induces KLF2 expression
KLF2 promoter/luc construct
-221 +1Luc
MEF2
MEF2
-221 +1Luc
MEF2×
KLF2wt-Luc
KLF2mut-Luc
Knock-down of MEF2
MEF2: myocyte enhancer factor 2
KLF2MEF2
Ang1-induced trans-associated Tie2
Trans-associated Tie2 induces KLF2 expression through MEF2
KLF2MEF2
trans-associated Tie2↓
PI3K↓
Akt
?Knock-down
Knock-down of Akt
Wortmannin
PI3K inhibitorA PI3K/Akt pathway is involved in Ang1-induced KLF2 expression
-221 +1Luc
MEF2
MEF2KLF2wt-Luc ×
KLF2MEF2
trans-associated Tie2↓
PI3K/Akt
A PI3K/Akt/MEF2 signaling pathway induces KLF2 expression
KLF2
endothelial cells
monocyte
cell adhesion molecule (VCAM1, ICAM1 et al.)
VEGF
Ang1/Tie2
VCAM1 expression
monocyte-EC adhesion
Knock-down of KLF2
Ang1 inhibits VEGF-induced inflammation through KLF2
Vascular quiescence regulated by Ang1/Tie2 signal
J. Biol. Chem., in press
Tie2 Activated Tie2
KLF2
Akt PI3KPI3K AktPI3KPI3K
MEF2MEF2 Foxo1Foxo1
Ang1 stimulation
trans-associated Tie2
oligomerized Ang1Ang1
apoptosisangiogenesis
anti-inflammationanti-angiogenesis
Vascular quiescenceFoxo1-
target genes
Proposed Model
Ang1
VEGFVEGF
VEGF
Ang1
Ang1
VEGF
ischemia
VEGF
angiogenic vessel
Ang1VEGF
VEGF
VEGF
Ang1
Ang1
VEGF
Ang1/Tie2↓
angiogenic signal
VEGF
quiescent vessel
Ang1VEGF
Ang1
Ang1
Ang1/Tie2↓
angiostatic signal
mural cell
EC
Tie2
Naoki MochizukiKeisuke Sako
Kazuomi Noda
NCVC Research Institute
Gou Young Koh
Korea Advanced Institute for Science and Technology
Takashi MinamiTatsuhiko KodamaTakao Hamakubo
LSBM, Tokyo University
Acknowledgments
Toshio SudaKeio University
Nobuyuki TakakuraThe University of Osaka
Masabumi Shibuya
Tokyo Medical and Dental University
Akiyoshi FukamizuUniversity of Tsukuba
J. Silvio Gutkind
National Institute of Dental and Craniofacial Research, NIH
Filopodia extensions are actively produced by endothelial tip cells in response to angiogenic factors
Angiogenic factors
Tip cellTip cell
Stalk cell
Foxo1 phosphorylation by COMP-Ang1
Akt-Foxo1 pathway is preferentially induced by trans-associated Tie2 at cell-cell contacts
Akt
Foxo1P P P
nucleus
nuclear export
Nuclear export
Vascular quiescence regulated by Ang1/Tie2 signal
J. Biol. Chem., in press
Akt
Tie2 Activated Tie2
PI3KPI3K
KLF2MEF2MEF2
AktPI3KPI3K
Foxo1Foxo1apoptosisangiogenesis
Ang1 stimulation
trans-associated Tie2
oligomerized Ang1Ang1
anti-inflammationanti-angiogenesis
Vascular quiescence
Tie2-Fc Tie2-HA
HABead
Ang1 induces trans-association of Tie2 in vitro
BeadTie2-Fc Tie2-HA
HA
COMP-Ang1
COMP-Ang1+/-
in vitro Tie2 trans-association assay
Multimerization of Ang1 is required for trans-association of Tie2 at cell-cell contacts
GCN4-Ang1(dimer)
MAT-Ang1(tetramer)
COMP-Ang1(pentamer)
Bead
Tie2-Fc Tie2-HAbinding
HA
Tie2
VE-c
ad
Localization of Tie2 at cell-cell contacts by Ang1 mutants
Biological significance of ECM-anchored Tie2 and trans-associated Tie2
sparse confluent
ECM-anchored Tie2 Trans-associated Tie2
Sparse and confluent HUVECs were stimulated with COMP-Ang1.
Endothelial cell-cell contacts do not affect Tie2 activation by Ang1.
Tie2 activationsparse confluent
Tie2 pTie2
Tie2 pTie2