Angiostasis and Angiogenesis Regulated by Angiopoietin1-Tie2 Receptor System

Transcription

1 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

2 quiescent vessel Vascular uiescence and Angiogenesis angiogenesis mural cell endothelial cells (EC) angiogenic factors such as VEGF cancer tissue injury others disrupt cell adhesions between EC-mural cell and between EC-EC proliferate and migrate

3 Visualization of in vivo angiogenesis Transgenic zebrafish embryo expressing GFP in vascular endothelial cells Fli:GFP intersegmental vessel dorsal aorta

4 Vascular quiescence vs. Angiogenesis Angiopoietin-1 ( ) acts as both angiogenic inhibitor and stimulator angiogenesis EC vascular quiescence mural cell Angiogenic inhibitor Angiogenic stimulator

5 Angiopoietin-1/Tie2 receptor system Angiopoietin-1 () is a ligand for the receptor tyrosine kinase Tie2, which is expressed on vascular endothelial cells. Angiopoietin-1 () /Tie2 signal plays a critical role in developmental vascular formation. How does /Tie2 signal play distinct roles in both vascular quiescence and angiogenesis? In adult vasculature, /Tie2 signal regulates not only vascular quiescence, but also physiological and pathological angiogenesis. vascular quiescence angiogenesis Tie2 Ig-like EGF-like FN III kinase EC-EC contact proliferation migration

6 How? Tie2 quiescent vessels angiogenic vessels ischemia cell-cell contacts (+) cell-cell contacts (-)

7 induces recruitment of Tie2 at cell-cell contacts COMP- HUVEC COMP (Cartilage oligomeric matrix protein)

8 induces recruitment of Tie2 at cell-cell contacts CHO cells expressing Tie2-GFP were stimulated with COMP- GFP Tie2-GFP Accumulation of Tie2 at cell-cell contacts requires Tie2 expression in adjacent cells. may induce trans-association of Tie2 at cell-cell contacts

9 induces aggregation of 293F cells expressing Tie2Δcyto- GFP in suspension. Cell expressing GFP Cell expressing Tie2Δcyto-GFP GFP GFP

10 induces trans-association of Tie2 at cell-cell contacts Cell expressing Tie2Δcyto-GFP COMP- Cell expressing Tie2Δcyto-HA GFP HA GFP HA

11 Conclusion Part1: in the presence of cell-cell contacts Tie2 is broadly expressed on plasma membrane in the absence of Tie2 Endothelial cell Endothelial cell stimulation Formation of -bridged Tie2 trans-association

12 Tie2 How? quiescent vessels angiogenic vessels ischemia cell-cell contacts (+) cell-cell contacts (-)

13 induces accumulation of Tie2 at cell-substratum contacts, which are different from focal adhesions. Sparse HUVECs were stimulated with COMP- and.

14 Extracellular domain is sufficient for accumulation of Tie2 at cell-substratum interface Sparse HUVEC expressing Tie2Δcyto-GFP and RFP-Crk (focal adhesion marker) were stimulated with COMP- Tie2Δcyto -GFP RFP-Crk (focal adhesion marker) GFP merge phase contrast

15 Tie2 is anchored by ECM-bound to cell-substratum contacts ECM Tie2 ECM binding assay Tie2-GFP-expressing CHO cells plated on collagen-coated Tie2 COMP- stimulation (min) C-

16 Conclusion Part2: in the absence of cell-cell contacts Endothelial cell Tie2 ECM binding to ECM stimulation Anchoring of Tie2 to cell-ecm contact by ECM-anchored Tie2

17 Biological significance of ECM-anchored Tie2 and trans-associated Tie2 Sparse and confluent HUVECs were stimulated with. sparse confluent ECM-anchored Tie2 Trans-associated Tie2 Erk migration proliferation angiogenesis Akt cell survival vascular quiescence

18 Preferential activation of Erk in sparse endothelial cells and that of Akt in confluent cells upon stimulation sparse confluent confluent sparse sparse Erk>Akt confluent Akt>Erk ECM-anchored Tie2 trans-associated Tie2

19 Anchoring of Tie2 to ECM facilitates Erk pathway and induces focal complex assembly suspended attached EC EC Erk collagen /collagen vinculin staining

20 ECM-anchored Tie2 activates Erk pathway partly via FAK, leading to the enhanced endothelial cell migration Tie2 migration integrin FAK Erk Endothelial cell migration ECM ECM-anchored Tie2 focal complex (integrin/ecm) Knock-down of FAK

21 Conclusion Part3 PRESENCE OF CELL-CELL CONTACTS stimulation Tie2 vascular quiescence ECM survival integrity Erk Akt -bridged Tie2 trans-association ABSENCE OF CELL-CELL CONTACTS Tie2 angiogenesis migration proliferation Erk stimulation Akt ECM -mediated Tie2 anchoring to ECM oligomerized Tie2 activated Tie2 Nat. Cell Biol. 10: , 2008

22 Distinct sets of genes are regulated by trans-associated Tie2 and ECM-anchored Tie2 confluent sparse stimulation for 1 h sparse Krüppel-like factor 2 (KLF2) RNA extraction confluent sparse/ confluent GeneChip analysis

23 Role of KLF2 in blood vessels transactivation transrepression Zn Zn Zn 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. 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 Role of KLF2 in vascular quiescence! Dekker et al. Am. J. Pathol., 2005

24 Inhibitory effects of and KLF2 on VEGF-induced vascular leakage KLF2 inhibits VEGF-induced vascular leakage Ad-GFP inhibits VEGF-induced vascular leakage. Ad-KLF2 Bhattacharya et al. J. Biol. Chem., 2005 Thurston et al. Science, 1999

25 Hypothesis /Tie2 signal may induce vascular quiescence through KLF2 expression. Intracellular signaling pathway involved in -induced KLF2 expression Biological consequence of -induced KLF2 expression

26 Trans-associated Tie2 induces KLF2 expression Real-time PCR KLF2 expression in endothelial cells from transgenic mice Western blot analysis

27 Trans-associated Tie2 induces KLF2 expression through MEF2 KLF2 promoter/luc construct MEF2 KLF2wt-Luc Luc MEF2 KLF2mut-Luc Luc MEF2 MEF2: myocyte enhancer factor 2 Knock-down of MEF2 -induced trans-associated Tie2 MEF2 KLF2

28 A PI3K/Akt pathway is involved in -induced KLF2 expression PI3K inhibitor trans-associated Tie2 PI3K Wortmannin Akt Knock-down? MEF2 KLF2 Knock-down of Akt

29 A PI3K/Akt/MEF2 signaling pathway induces KLF2 expression MEF2 KLF2wt-Luc MEF2 Luc trans-associated Tie2 PI3K/Akt MEF2 KLF2

30 inhibits VEGF-induced inflammation through KLF2 monocyte VEGF KLF2 /Tie2 monocyte-ec adhesion endothelial cells cell adhesion molecule (VCAM1, ICAM1 et al.) VCAM1 expression Knock-down of KLF2

31 Vascular quiescence regulated by /Tie2 signal stimulation MEF2 Akt PI3K trans-associated anti-inflammation Tie2 anti-angiogenesis KLF2 PI3K Foxo1 Akt Vascular quiescence apoptosis angiogenesis Foxo1- target genes oligomerized Tie2 Activated Tie2 J. Biol. Chem., in press

32 quiescent vessel Proposed Model angiogenic vessel ischemia VEGF VEGF VEGF VEGF VEGF VEGF VEGF VEGF VEGF VEGF VEGF EC Tie2 mural cell /Tie2 angiostatic signal /Tie2 angiogenic signal

33 Acknowledgments NCVC Research Institute Naoki Mochizuki Keisuke Sako Keio University Toshio Suda The University of Osaka Nobuyuki Takakura Kazuomi Noda Korea Advanced Institute for Science and Technology Gou Young Koh LSBM, Tokyo University Takashi Minami Tatsuhiko Kodama Takao Hamakubo National Institute of Dental and Craniofacial Research, NIH J. Silvio Gutkind Tokyo Medical and Dental University Masabumi Shibuya University of Tsukuba Akiyoshi Fukamizu

34 Filopodia extensions are actively produced by endothelial tip cells in response to angiogenic factors Stalk cell Angiogenic factors Tip cell Tip cell

35 Akt-Foxo1 pathway is preferentially induced by trans-associated Tie2 at cell-cell contacts Akt P P P Foxo1 nucleus nuclear export Foxo1 phosphorylation by COMP- Nuclear export

36 Vascular quiescence regulated by /Tie2 signal stimulation MEF2 PI3K Akt trans-associated anti-inflammation Tie2 anti-angiogenesis KLF2 PI3K Foxo1 Akt Vascular quiescence apoptosis angiogenesis oligomerized Tie2 Activated Tie2 J. Biol. Chem., in press

37 induces trans-association of Tie2 in vitro in vitro Tie2 trans-association assay +/- COMP- COMP- Bead HA Bead HA Tie2-Fc Tie2-HA Tie2-Fc Tie2-HA

38 Multimerization of is required for trans-association of Tie2 at cell-cell contacts GCN4- (dimer) MAT- (tetramer) COMP- (pentamer) Bead Tie2-Fc binding Tie2-HA HA Localization of Tie2 at cell-cell contacts by mutants VE-cad Tie2

39 Biological significance of ECM-anchored Tie2 and transassociated Tie2 Sparse and confluent HUVECs were stimulated with COMP-. confluent sparse ECM-anchored Tie2 Tie2 activation sparse Tie2 ptie2 Trans-associated Tie2 confluent Tie2 ptie2 Endothelial cell-cell contacts do not affect Tie2 activation by.