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 angiogenesis vessel mural cell cancer tissue injury others endothelial cells angiogenic factors (EC) such as VEGF ✓ disrupt cell adhesions between EC-mural cell and between EC-EC ✓ proliferate and migrate
Visualization of in vivo angiogenesis Transgenic zebrafish embryo expressing GFP in vascular endothelial cells Fli:GFP intersegmental vessel dorsal aorta
Vascular quiescence vs. Angiogenesis angiogenesis Angiopoietin-1 ( ) acts as both angiogenic inhibitor and stimulator vascular quiescence EC mural cell Angiogenic Angiogenic inhibitor stimulator
Angiopoietin-1/Tie2 receptor system ✓ Angiopoietin-1 (Ang1) is a ligand for the receptor Angiopoietin-1 tyrosine kinase Tie2, which is expressed on vascular (Ang1) endothelial cells. ✓ Ang1/Tie2 signal plays a critical role in How does Ang1/Tie2 signal play distinct roles in How does Ang1/Tie2 signal play distinct roles in developmental vascular formation. Ig-like both vascular quiescence and angiogenesis? both vascular quiescence and angiogenesis? EGF-like ✓ In adult vasculature, Ang1/Tie2 signal regulates not only vascular quiescence, but also physiological and pathological angiogenesis. FN III Tie2 vascular quiescence angiogenesis kinase EC-EC contact ↑ ↓ proliferation ↓ ↑ migration ↓ ↑
Ang1 Tie2 How? quiescent vessels angiogenic vessels ischemia cell-cell contacts (+) cell-cell contacts (-)
Ang1 induces recruitment of Tie2 at cell-cell contacts COMP-Ang1 Ang1 HUVEC COMP (Cartilage oligomeric matrix protein)
Ang1 induces recruitment of Tie2 at cell-cell contacts CHO cells expressing Tie2-GFP were stimulated with COMP-Ang1 Tie2-GFP 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 Tie2 Δ cyto- GFP in suspension. Cell expressing Tie2 Δ cyto-GFP Cell expressing GFP GFP GFP
Ang1 induces trans -association of Tie2 at cell-cell contacts Cell expressing Cell expressing Tie2 Δ cyto-GFP COMP-Ang1 Tie2 Δ cyto-HA HA GFP HA GFP
Conclusion Part1: in the presence of cell-cell contacts Tie2 is broadly expressed on plasma membrane in the absence of Ang1 Tie2 Endothelial cell Endothelial cell Ang1 stimulation Formation of Ang1-bridged Tie2 trans-association
Ang1 Tie2 How? quiescent vessels angiogenic vessels ischemia cell-cell contacts (+) cell-cell contacts (-)
Ang1 induces accumulation of Tie2 at cell-substratum contacts, which are different from focal adhesions. Sparse HUVECs were stimulated with COMP-Ang1 and Ang1.
Extracellular domain is sufficient for accumulation of Tie2 at cell-substratum interface Sparse HUVEC expressing Tie2 Δ cyto-GFP and RFP-Crk (focal adhesion marker) were stimulated with COMP-Ang1 RFP-Crk Tie2 Δ cyto (focal adhesion -GFP marker) GFP merge phase contrast
Tie2 is anchored by ECM-bound Ang1 to cell-substratum contacts Tie2-GFP-expressing CHO cells Tie2 Ang1 plated on collagen-coated Ang1 ECM COMP-Ang1 stimulation (min) 0 30 60 ECM binding assay Tie2 C-Ang1
Conclusion Part2: in the absence of cell-cell contacts Tie2 Endothelial cell ECM Ang1 binding to ECM Ang1 stimulation Anchoring of Tie2 to cell-ECM contact by Ang1 ECM-anchored Tie2
Biological significance of ECM-anchored Tie2 and trans-associated Tie2 Sparse and confluent HUVECs were stimulated with Ang1. confluent sparse ECM-anchored Tie2 Trans-associated Tie2 Erk Akt ↓ ↓ migration cell survival proliferation ↓ vascular quiescence ↓ angiogenesis
Preferential activation of Erk in sparse endothelial cells and that of Akt in confluent cells upon Ang1 stimulation confluent sparse confluent sparse sparse confluent Erk>Akt Akt>Erk ECM-anchored Tie2 trans-associated Tie2
Anchoring of Tie2 to ECM facilitates Erk pathway and induces focal complex assembly suspended attached EC EC Erk collagen Ang1/collagen vinculin staining
ECM-anchored Tie2 activates Erk pathway partly via FAK, leading to the enhanced endothelial cell migration Endothelial cell migration migration Erk integrin Tie2 FAK Ang1 ECM ECM-anchored focal complex Tie2 (integrin/ECM) Knock-down of FAK
Conclusion Part3 PRESENCE OF CELL-CELL CONTACTS Ang1 stimulation Tie2 Erk vascular survival integrity quiescence Akt Ang1-bridged Tie2 trans -association ECM ABSENCE OF CELL-CELL CONTACTS Tie2 migration Ang1 angiogenesis Erk proliferation stimulation Ang1 Akt ECM Ang1-mediated Tie2 anchoring to ECM Ang1 oligomerized Ang1 Tie2 activated Tie2 Nat. Cell Biol. 10: 513-526, 2008
Distinct sets of genes are regulated by trans -associated Tie2 and ECM-anchored Tie2 Ang1 Ang1 confluent sparse sparse Ang1 stimulation for 1 h Krüppel-like factor 2 (KLF2) confluent sparse/ RNA extraction GeneChip analysis confluent
Role of KLF2 in blood vessels 1 110 267 354 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
Inhibitory effects of Ang1 and KLF2 on VEGF-induced vascular leakage KLF2 inhibits VEGF-induced vascular leakage Ang1 inhibits VEGF-induced Ad-GFP vascular leakage. Ad-KLF2 Bhattacharya et al. J. Biol. Chem. , 2005 Thurston et al. Science , 1999
Hypothesis Ang1/Tie2 signal may induce vascular quiescence through KLF2 expression. ✓ Intracellular signaling pathway involved in Ang1-induced KLF2 expression ✓ Biological consequence of Ang1-induced KLF2 expression
Trans -associated Tie2 induces KLF2 expression Real-time PCR KLF2 expression in endothelial cells from Ang1 transgenic mice Western blot analysis
Trans -associated Tie2 induces KLF2 expression through MEF2 KLF2 promoter/luc construct Knock-down of MEF2 MEF2 KLF2wt-Luc Luc -221 +1 MEF2 × KLF2mut-Luc Luc -221 +1 MEF2 MEF2: myocyte enhancer factor 2 Ang1-induced trans -associated Tie2 MEF2 KLF2
A PI3K/Akt pathway is involved in Ang1-induced KLF2 expression PI3K inhibitor trans -associated Tie2 ↓ Wortmannin PI3K ↓ Akt Knock-down ? Knock-down of Akt MEF2 KLF2
A PI3K/Akt/MEF2 signaling pathway induces KLF2 expression MEF2 × KLF2wt-Luc Luc -221 +1 MEF2 trans -associated Tie2 ↓ PI3K/Akt MEF2 KLF2
Ang1 inhibits VEGF-induced inflammation through KLF2 VEGF monocyte-EC adhesion KLF2 Ang1/Tie2 monocyte endothelial cells cell adhesion molecule (VCAM1, ICAM1 et al.) VCAM1 expression Knock-down of KLF2
Vascular quiescence regulated by Ang1/Tie2 signal Ang1 stimulation Akt PI3K PI3K PI3K PI3K Akt trans -associated Tie2 anti-inflammation apoptosis anti-angiogenesis angiogenesis Foxo1 Foxo1 MEF2 MEF2 Foxo1- KLF2 target genes Vascular quiescence oligomerized Ang1 Ang1 Tie2 Activated Tie2 J. Biol. Chem. , in press
Proposed Model angiogenic vessel quiescent vessel ischemia VEGF VEGF VEGF VEGF VEGF VEGF VEGF Ang1 Ang1 VEGF Ang1 VEGF VEGF Ang1 VEGF Ang1 Ang1 Ang1 EC Tie2 Ang1 mural cell Ang1 Ang1/Tie2 Ang1/Tie2 ↓ angiogenic signal ↓ angiostatic signal
Acknowledgments NCVC Research Institute Keio University Toshio Suda Naoki Mochizuki Keisuke Sako The University of Osaka Nobuyuki Takakura National Institute of Dental and Kazuomi Noda Craniofacial Research, NIH J. Silvio Gutkind Korea Advanced Institute for Science and Technology Tokyo Medical and Dental Gou Young Koh University LSBM, Tokyo University Masabumi Shibuya Takashi Minami Tatsuhiko Kodama University of Tsukuba Takao Hamakubo Akiyoshi Fukamizu
Filopodia extensions are actively produced by endothelial tip cells in response to angiogenic factors Stalk cell Angiogenic factors Tip cell Tip cell
Akt-Foxo1 pathway is preferentially induced by trans -associated Tie2 at cell-cell contacts nuclear export Foxo1 phosphorylation by COMP-Ang1 Akt P P P Foxo1 nucleus Nuclear export
Vascular quiescence regulated by Ang1/Tie2 signal Ang1 stimulation PI3K PI3K PI3K PI3K Akt Akt trans -associated Tie2 anti-inflammation apoptosis anti-angiogenesis angiogenesis Foxo1 Foxo1 MEF2 MEF2 KLF2 Vascular quiescence oligomerized Ang1 Ang1 Tie2 Activated Tie2 J. Biol. Chem. , in press
Ang1 induces trans -association of Tie2 in vitro in vitro Tie2 trans -association assay COMP-Ang1 +/- COMP-Ang1 B ead B ead HA HA Tie2-Fc Tie2-HA Tie2-Fc Tie2-HA
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