Angiostasis and Angiogenesis Regulated by Angiopoietin1-Tie2 - - PowerPoint PPT Presentation

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 Angiogenesis

quiescent 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

cancer tissue injury

• thers

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 quiescence

EC 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

• nly vascular quiescence, but also physiological and

pathological angiogenesis. Tie2

Ig-like EGF-like FN III kinase

Angiopoietin-1 (Ang1) vascular quiescence angiogenesis

EC-EC contact proliferation 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

30 60 COMP-Ang1 stimulation (min) Tie2 C-Ang1

Ang1 ECM Tie2 Ang1

ECM Tie2 Endothelial cell Ang1 stimulation Ang1 binding to ECM Anchoring of Tie2 to cell-ECM contact by 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 ↓ migration proliferation ↓ angiogenesis Akt ↓ cell survival ↓ vascular quiescence

Preferential activation of Erk in sparse endothelial cells and that of Akt in confluent cells upon Ang1 stimulation

sparse

ECM-anchored Tie2

Erk>Akt

confluent

trans-associated Tie2

Akt>Erk

confluent sparse sparse confluent

attached suspended

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 integrin FAK

Erk migration

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

Erk survival integrity ECM

PRESENCE OF CELL-CELL CONTACTS

• ligomerized Ang1

Tie2 activated Tie2

ABSENCE OF CELL-CELL CONTACTS

Tie2 Ang1 Ang1-mediated Tie2 anchoring to ECM

Erk

Akt ECM Ang1 stimulation

angiogenesis

migration proliferation Ang1

Conclusion Part3

• Nat. Cell Biol. 10: 513-526, 2008

sparse confluent sparse/ confluent

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 Zn

1 110 267 354

Summary of KLF2 target genes

anti-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

+1 Luc MEF2 MEF2

• 221

+1 Luc MEF2

×

KLF2wt-Luc KLF2mut-Luc

Knock-down of MEF2

MEF2: myocyte enhancer factor 2

KLF2 MEF2 Ang1-induced trans-associated Tie2

Trans-associated Tie2 induces KLF2 expression through MEF2

KLF2 MEF2 trans-associated Tie2 ↓ PI3K ↓ Akt

?

Knock-down Knock-down of Akt Wortmannin PI3K inhibitor

A PI3K/Akt pathway is involved in Ang1-induced KLF2 expression

• 221

+1 Luc MEF2 MEF2

KLF2wt-Luc

×

KLF2 MEF2 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 PI3K PI3K Akt PI3K PI3K MEF2 MEF2 Foxo1 Foxo1 Ang1 stimulation trans-associated Tie2

• ligomerized Ang1

Ang1 apoptosis angiogenesis anti-inflammation anti-angiogenesis

Vascular quiescence

Foxo1- target genes

Proposed Model

Ang1 VEGF VEGF VEGF Ang1 Ang1 VEGF

ischemia

VEGF

angiogenic vessel

Ang1 VEGF VEGF VEGF Ang1 Ang1 VEGF

Ang1/Tie2 ↓ angiogenic signal

VEGF

quiescent vessel

Ang1

VEGF

Ang1 Ang1

Ang1/Tie2 ↓ angiostatic signal

mural cell EC

Tie2

Naoki Mochizuki Keisuke Sako Kazuomi Noda NCVC Research Institute Gou Young Koh Korea Advanced Institute for Science and Technology Takashi Minami Tatsuhiko Kodama Takao Hamakubo LSBM, Tokyo University

Acknowledgments

Toshio Suda Keio University Nobuyuki Takakura The University of Osaka Masabumi Shibuya Tokyo Medical and Dental University Akiyoshi Fukamizu University 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 cell

Tip cell Stalk cell

Foxo1 phosphorylation by COMP-Ang1

Akt-Foxo1 pathway is preferentially induced by trans-associated Tie2 at cell-cell contacts

Akt Foxo1

P P P

nucleus

nuclear export Nuclear export

Vascular quiescence regulated by Ang1/Tie2 signal

• J. Biol. Chem., in press

Akt

Tie2 Activated Tie2

PI3K PI3K

KLF2

MEF2 MEF2 Akt PI3K PI3K Foxo1 Foxo1

apoptosis angiogenesis

Ang1 stimulation trans-associated Tie2

• ligomerized Ang1

Ang1 anti-inflammation anti-angiogenesis

Vascular quiescence

Tie2-Fc Tie2-HA HA

Bead

Ang1 induces trans-association of Tie2 in vitro

Bead

Tie2-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-HA binding

HA

Tie2 VE-cad 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 activation

sparse confluent

Tie2 pTie2 Tie2 pTie2