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