Angiogenesis - Objectives

ANGIOGENESIS Gerald Prager, M.D. Medical University of Vienna Department of Medicine I gerald.prager@meduniwien.ac.at Angiogenesis - Objectives Explain differences between vasculogenesis and angiogenesis and collateral vessel growth Describe the most relevant angiogenic molecules (cells, growth factors, matrix proteins, adhesion molecules) Explain the role of angiogenesis in tumorigenesis and wound healing Describe the rational behind pro-, and angiogenic therapies

Case I 35a female patient, upon self examination she discovered a tumor in the left breast Radiologist evaluate this as a benign tumor, because no blood vessels were revealed in duplex-sonography At a 6 month setup vascularization was detected Tumor was resected and it turned out to be malign; ER negativ; no lymph node, no adjuvant therapy After five years the patient suffered massiv pain from the back bone, bone metastases were detected. The patient did not respond to any radiatio- or chemotherapy. Therefore, an experimental anti-angiogenetic therapy was started. The patient responded.

Angiogenesis Skin Model 5d 10d 15d 20d Angiogenesis versus Vasculogenesis po2 VEGF

Vasculogenesis, Angiogenesis, Arteriogenesis Vasculogenesis new vessel formation Hemangioblasts VEGF family, Ang-1,2 Angiogenesis Outgrowth from pre-exisiting vessels Endothelial cells FGF-1,2,4,5; VEGF-1,2,3 Arteriogenesis Maturation ECs, SMCs, Monocytes PDGF, FGFs, Ang-1,2; MCP-1 Arteriole formation Embryonal Vasculogenesis Adult Moore MA. J Clin Invest 2002; 109:313-315.

Postnatal angiogenesis and vasculargenesis Angiogenesis Physiological angiogenesis Wound healing Ovulation Pathological angiogenesis cancer psoriasis arthritis blindness obesity asthma atherosclerosis infectious disease Menstruation Implantation insufficient or abnormal vascular remodelling heart ischemia brain ischemia neurodegeneration hypertension pre-eclampsia respiratory distress osteoporosis

EC VEGF SMC Blood Vessel Formation TF Mature blood vessel VEGF Vasculogenesis Hemangioblasts Primitive endothelial tubes TIE 2 TIE 2 PDGF Angiopoietin I SDF-1a Arteriogenesis Angiopoietin II Stroma pericytes Angiogenesis Endothelial sprouts (Hemangiomas) Arterial versus venous Thickness of SMC wall SMC have distinct origin (neural crest thoracic vessels, epicardium coronary artery, others from mesenchym) Notch pathway (Notch-1, Notch-3 and Notch-4: receptors) and (Delta-like-4, Jagged-1 and Jagged-2) is important for arterial differentiation by repressing venous differentiation. VEGF and Hedgehog act upstream and Gridlock downstream of the Notch pathway. Neuropilin-1 (a VEGF receptor) also acts as arterial differentiation initiator

Tumor angiogenesis Stetler-Stevenson JCI 1999; 103: 1237-1241 Stimuli I. Growth Factors vascular endothelial growth factor (VEGF) fibroblast growth factor (FGF-2) angiopoetin (Ang1, Ang2) platelet derived growth factor (PDGF) II. Hypoxia hypoxia inducible factor (HIF-1) III. Inflammatory Cytokines transforming growth factor (TGF-b) tumor necrosis factor (TNFa) CXCR-1, CXCR-3

Vascular Endothelial Growth Factors VEGF-A: VEGF-B: VEGF-C: VEGF-D: PlGF-1, 2: Smooth muscle cells, Keratinocytes, Epithelial cells Tumor Cells, Fibroblasts, Makrophages; Vascular permeability, Endothelial cell proliferation, Angiogenesis Striated muscle cells (heart and muscle); Angiogenesis Heart, Placenta, Tumor cells, small intestine; Lymphangiogenesis Lung, Skin; Mitogen for endothelial and fibroblasts Placenta; synergistic with VEGF-A Placenta VEGF and VEGF Receptors SMC, Keratinocytes, Epithelial, Macrophages Tumor cells small intestine Striated MC Lung, Skin PLGF1, 2 VEGF-A VEGF-B VEGF-C VEGF-D svegfr-1 Endothelial Cell RTK RTK RTK PKC VEGFR-1 (flt-1)vegfr-2 (flk-1) Splice variants: Secreted: +/- Heparin binding Membrane/Matrix bound Lymphatic- Endo thelial Cell VEGFR-3 (flt-4) Migration Migration, Proliferation, Permeability Proliferation

Binding of VEGF to its Receptors VEGFR VEGF VEGF VEGFR Monocyte Tumor cell CYR61 Neutrophil Protease X binding binding? Angiostatin Ox PL Lox-1 TSP like u-par PAI-1 RTK u-par TGFβ TNF-R Gαs Gαq VEGF FGF Angiostatic Integrins GP130 Pgen EGF effects α β DEP-1 camp PI3 PI4P fyn JAK TRAF FAK - PLC K? PDK2 LRP PLCγ M6P-R SOS PI3, 4P cas pax PKA - IP 3 STAT vvo Tyrosine Ras-GTP NIK Ca ++ substrates 14-3-3 Akt? ROK Microtubuli PKCε RAF Cdc42/RAC cpl + Calcineurin Dia MEK-1/2 A 2 PAK LIMR - lipoxygenase MEKK 1 NFAT Stressfibres ERK-1/2 MEK 3/6 TAB 12 HETE P XIAP Tf,, flt, upa / upar NFκB IKK2 TAK MLCK CREB 38 others EGR-1 TFs, Myc Lamellipodia - NFκB IκB-P - Cycl D MEK-4/7 Inflammatory mediators E-2-T E2F Nab-2 - others TSG101 Antiapoptotic Migration Rb CDK - Tube-Formation 4 CDI-Kip Cell cycle ARF + - HOMO p53 E-3-L JNK MDM Rb-P - - CBP/p3oo 2 Src PPARγ Src E-2- T x

Monocyte binding Ox PL Lox-1 RTK Gαs Gαq VEGF FGF EGF Tumor cell CYR61 Protease X? Angiostatin TSP like u-par u-par Integrins DEP-1 α β PAI-1 TGFβ GP130 Pgen Neutrophil binding TNF-R camp PI3 PI4P fyn JAK TRAF FAK - PLC K? PDK2 LRP PLCγ M6P-R SOS PI3, 4P cas pax PKA - IP 3 STAT vvo Tyrosine Ras-GTP NIK Ca ++ 14-3-3 Akt? substrates ROK Microtubuli PKCε RAF Cdc42/RAC cpl + Calcineurin Dia MEK-1/2 A 2 PAK LIMR - lipoxygenase MEKK 1 NFAT Stressfibres ERK-1/2 MEK 3/6 TAB 12 HETE P XIAP Tf,, flt, upa / upar NFκB IKK2 TAK MLCK CREB 38 others EGR-1 TFs, Myc Lamellipodia - NFκB IκB-P - Cycl D MEK-4/7 Inflammatory mediators E-2-T E2F Nab-2 - others TSG101 Antiapoptotic Migration Rb CDK Tube-Formation 4 CDI-Kip Cell cycle ARF + - HOMO p53 E-3-L MDM Rb-P - E-2- JNK T - x CBP/p3oo 2 Src PPARγ Src Gene Defective Mice VEGF A -/- : Delayed differentiation of ECs Reduced sprouting Altered lumen formation LETHAL VEGF A +/- : LETHAL PlGF -/- : VEGFR-1 -/- : VEGFR-2 -/- : Embryonic development normal Altered wound healing Normal Fertility Normal EC Development Channel formation disturbed Increased EC proliferation EC development disrupted LETHAL LETHAL

Regulation of VEGF Expression Growth factors: PDGF FGF-4 IGF-1 HGF KGF TGF-β Tumor supressor genes: vhl p53 Inflammatory Cytokines: IL-1 TNFα VEGF NO Hypoxia HIF-1 VEGF NOS die aktivierte Endothelzelle Die Angiogenese ist ein entscheidener Mechanismus der Pathogenese maligner Erkrankungen und ist in den letzten Jahren als therpeutisches Ziel ins Zentrum des Interesses gerückt. Ein besseres Verständnis der zugrundeliegenden Mechanismen ist Grundvorraussetzung für effektive therapeutische Ansätze. Schritte der aktivierten Endothelzelle im Rahmen der Angiogenese: 1. Lösung der Zell-Zell-, sowie der Zell-Matrix- Migration Proliferation Tube formation Kontakte Specific matrix Laminin ERK 1-2 DEP-1 upar downregulation αvβ3/5 upa PAI-1 FN upar Matrix degradation bfgf VEGF Ang 2 Tumor 2. Transmigration durch die Basalmembran in umliegende Matrix 3. Zell-Proliferation 4. Zell-Polarisation zur Formierung Kapillarähnlichen Strukturen 5. Zellüberleben: Während all diesen Schritten sind Endothelzellen pro-apoptotischen Einflüssen ausgesetzt. Zellüberleben stellt daher eine Grundvorraussetzung dar.

Effects of VEGF on ECs Blood Stream MATRIX Zhhh.. Blood Stream Endothelial Cell

VEGF MATRIX Blood Stream Endothelial Cell VEGF MATRIX Blood Stream Endothelial Cell

Zhhh.. Adhesion Proteolysis Blood Stream Endothelial Cell Zhhh.. Adhesion Proteolysis Blood Stream Endothelial Cell

Steps of angiogenesis 1. EC proliferation 2. EC migration / invasion 3. EC survival 4. Capillary-like tube formation 5. Vessel maturation (pericytes) provided by

Angiopoietins Bind With Tie2 Receptor Tie2 is a tyrosine kinase receptor that can be found in vascular endothelial cells Ligand binding of angiopoietin-1 (Ang1) promotes Tie2 receptor dimerization Ang2 exerts antagonistic functions on Ang1/Tie2 signaling Pericytes Ang1 Endothelial Cells Ang2 Tie2 Thomas M, Augustin HG. Angiogenesis. 2009;12:125 137. Provided as an educational resource. Do not copy or distribute. Provided as an educational resource. Do not copy or distribute. provided by

Angiopoetin-1, Tie2 receptor Stabilize vessels Inhibits endothelial permeability Angiopoetin-2, Tie2 receptor Impairs vessel maturation and stabilization PDGF-BB Recruits smooth muscle cells FGF-2 Mediates EC migration, proliferation TGF-beta Stimultes extracellular matrix production

II. Hypoxia Hypoxia activates hypoxia-inducible transcription factor (HIFs) HIFs induce expression of VEGF, NOS, PDGF, Ang2 + myocardial infarction, stroke - blindness in premature newborns, diabetic III. Inflammation Monocytes, platelets, macrophages, other leukocytes: Release of VEGF, Ang1, FGF2, TGF-b, PDGF, TNFa

Blood Vessel Formation Inflammation EC TF MCP-1 Shearstress ICAM-1 MCP-1 TNF Mature blood vessel SMC Monocyte Arteries/Arteriols Molecular properties of angiogenic endothelial cells

Molecular properties of angiogenic endothelial cells Tumor angiogenesis Migration Proliferation Tube formation DEP-1 Specific matrix Laminin ERK 1-2 upar downregulation αvβ3/5 upa Non specific matrix PAI-1 FN upar Matrix degradation bfgf VEGF Ang 2 Tumor

endothelial cell response A. Proteolytic enzymes urokinase/plasminogen system Matrix metalloproteinases (MMPs) B. Adhesion molecules integrin adhesion receptors (alphavbeta3) A. Proteolytic System VEGF induces an increase in D-Dimer concentrations in supernatants 3 D-dimer mg/l 2,5 2 1,5 1 0,5 control VEGF 10µM control 0 10µM 5min 15min 30min 60min 120min VEGF D-dimer concentrations in supernatants Prager GW, et al. Blood (2004 ); 103(3): 955-962 10µM VEGF, 60min D-dimer

Ternary complex (upar upa serpin) binds to LDL-R like molecules: pro-upa PAI upa LDL-Rfamily upa-receptor PAI-1: plasminogen activator inhibitor-1 upa: urokinase LDL-R: low density lipoprotein-receptor Redistribution of urokinase receptor (upar) upon VEGF stimulation control VEGF165 (50ng/ml, 2h) 10 μm upar phospho-fak

Internalization of the tetrameric complex and recycling of upar RAP upa upar pro upa PAI LDL-Rfamily Matrigel plugs after 1 week in vivo assay is used to test relevance of in vitro findings +VEGF + VEGF and bfgf

upar redistribution supports VEGF-induced endothelial cell migration in vivo invaded cells per unit 140 120 100 80 60 40 20 wild type ** ** 0 control RAP VEGF RAP+ VEGF invaded cells per unit 140 120 100 80 60 40 20 upar -/- 0 control RAP VEGF RAP+ VEGF * * wild type upar -/- control VEGF RAP+VEGF M M M M M M ** * total cell number endothelial cells p<0.05 p<0.005 in vivo matrigel plug angiogenesis assay Proteolytic cleavage products Proteolysis generates also cleavage products that regulates angiogenesis (examples: Angiostatin, mini-plasminogen)

Angiostatin is produced by hydrolysis of plasminogen either by MMP-3, MMP-7, MMP-9 or upa, whereby they contain 3-5 kringle domains (K1-3, K1-4, and K1-5).

B: Integrin Adhesion Receptors 18 alpha chains 8 beta chains integrin integrin talin talin

Integrin activation (Inside out Signaling) Src Family Kinases (?Fyn) PI3-Kinase Ca 2+ PLCγ DAG ADAP? Talin PKC αiibβ3 activation integrin integrin talin talin

Platelets in Initiation of Thrombus Formation Fibrinogen GP IIb/IIIa complex ADAMTS13 Platelet Fibrinogen Expression an der Oberfläche von aktivierten Thrombocyten: Tf3=PS; Tf1=GfV; GfVIII +vwf; TF auf zirkulierenden Mikropartikeln Von Willebrand factor integrin outside-in signaling is mediated via CD98hc matrix integrin α β CD98hc PI3kinase FAK p130cas src Rho-GTPases cell survival cell migration cell proliferation

Migration During endothelial cell migration integrins have to release at the trailing end to be redistributed to the leading edge, where they bind to the extracellular matrix 2. Integrin distribution control VEGF 5µm VEGF + RAP - blue: beta-1 - green: beta-3 - red: upar 5µm 5µm Prager-GW et al., submitted

1.Angiogenesis in wound healing Keratinocyte + + + Platelets Collagen Stroma cells TGFβ + HGF TGFβ TNF IL-1 + Ang 1 TGFβ Monocyte VEGF MCP-1 VEGF upa PDGF TGFβ + +/- flk1+ + FGF VEGF EC Matrix Thrombin FN upa Fibrin GAG Integrin shift PAI-1 FGF Pericytes Derive from perivascular progenitor cells (c-kit+, Sca-1+, VEGFR-1+) and are mobilzed upon PDGF-BB stimulation. Integrin Alpha-4 / VCAM interaction

2. Tumor Angiogenesis

Angiogenesis is pivotal for tumor growth (Judah Folkmann ) HGF VEGF PDGF PlGF TGF-β FGFs kindly provided by Hoffmann-La Roche Ltd

Chaotic and mosaic vessels in tumours Carmeliet P. et al, Nature Medicine (2003)

Anti-VEGF strategies Bevacizumab (AVASTIN): only effective in combination with chemotherapy (colon, breast, kidney, lung). wild type PAI-1 -/- Invasive behavior of malignant mouse keratinocytes (PDVA cells), 2 wk after implantation. Immunofluorescence labeling of malignant keratinocytes and vessels 2 wk after implantation Khalid Bajouet al., JCB 2001

downregulation of PAI-1 loss of PAI-1 suppresses pathological angiogenesis in tumors, ocular and other disorders, while adenoviral PAI- 1 gene transfer reverted this phenotype targeting MMPs Cancer growth and angioenesis were also impaired in mice lacking components of the MMP system (i.e., MMP-2 and MMP-9, while overexpression of MT-MMP-1 produced highly vascular tumors) MMP-system Pro-MMP-2 binds to MT1-MMP to become activated MT1-MMP is associated with integrins (alphavbeta3) TIMP-2 inhibits MMP-2 Active MMP-2 cleaves laminin collagen IV

Synthetic/semi synthetic inhibitor Endogenous inhibitors Biological antagonists Vascular targeting Antiangiogenic therapeutic strategies Substance/Approach Carboxiamidotriazole (NCI) CM101 Marimastat (British Biotech) Pentosan polysulfats TNP470 (Takeda/Abbott) Thalidomide (Grünewald) Angiopoietin-2 (Regeneron) Angiostatin (EntreMed) Endostatin IL-12 (Roche, Genetics Inst.) Interferon-α Platelet factor-4 αvβ3 integrin antagonists VEGF inhibitors VEGF receptor blockers Soluble receptors Regional TNF-α therapy Antibody targeting Vascular gene therapy Comment Ca channel blocker, phase I Analog of group B streptococcus toxin (polysaccharide) binds to tumor endothelium, induces inflammation Metalloproteinase inhibitor, inhibits endothelial and tumor cell invasion, phase II (pancreatic, lung, brain) Inhibits heparin-binding growth factors, phase I Analog of antibiotic fumagillin, inhibits endothelial cell migration and proliferatin phase III (breast, Kaposi s sarcoma, cervical) Polycyclic teratogen, antiangiogenic mechanism unknown, phase II (brain, breast, prostata) Interferes with blood vessel maturation Plasminogen fragment, antiangiogenic mechanism unknown Collagen XVIII fragment, antiangiogenic mechanism unknown Induces IP-10 phase I Decreases FGF production, phase III (infant hemangiomas) Inhibits endothelial cell proliferation Mab LM609 and mab 9G2.1.3, induce EC apoptosis Humanized neutralizing antibody, antisense oligonucleotides Small receptor tyrosine kinase antagonists Angiogenesis inhibition with soluble VEGF-R1 or soluble Tie-2 Isolated limb perfusion to target in transit metastases Use of mono- and bispecific antibodies to target angiogenic EC (VEGF-receptors, endoglin) to deliver specific angio- and/or tumor activity Transfer of dominant-negative receptors or suicide genes under the control of angiogenic EC specific promoters pro upa VEGF FGF EGF Cell-confluence + DEP LRP RAP upa PAI upar RTK SOS Ras-GTP PLCγ RAF PMA PKC MEK-1/2 ERK-1/2 MEK-3/6 PD098059 c-fos c-jun jun D src upar p38

2. Angiogenesis in other diseases a. Angiogenesis and obesity Angiogenesis is related to obesity Prae-Adipocytes migrate to sites of neovascularization and Adipose tissue is highly angiogenic (VEGF, FGF-2, leptin)

b. Diabetic Retinopathia Hypoxia-induced VEGF expression leads to neo-vascularization

c. Inflammatory Bowel Disease (IBD) CD40 CD40L (TNF) induces VEGF expression Ang2: TNFa and VEGF signaling is increased Ang1/Tie2: downregulated (stability and maturation of vessels) VEGF: VEGF expression correlates with disease activity Mediators of angiogenesis in IBD Matrix Metalloproteinases (MMP): degradation of basal membrane MMP-3, MMP-9 Urokinase (upa) /plasminogen system: upa activates MMPs and plasminogen (fibrinolysis) Integrins: adhesion of leukocytes, endothelial cell migration. Antiapoptosis.