Angiogenesis. Physiological angiogenesis. Wound healing Ovulation

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1 Physiological angiogenesis 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

2 Angiogenesis in Cancer 5d 10d 15d 20d

3 Angiogenesis versus Vasculogenesis po2 VEGF

4 Blood Vessel Formation EC VEGF SMC TF Mature blood vessel VEGF Vasculogenesis Hemangioblasts Primitive endothelial tubes TIE 2 TIE 2 PDGF Angiopoietin I Arteriogenesis Angiopoietin II Stroma Angiogenesis Endothelial sprouts (Hemangiomas)

5 Embryonal Vasculogenesis (VG) Adult Moore MA. J Clin Invest 2002; 109:

6 Blood Vessel Formation Arteriogenesis EC TF MCP-1 Shearstress ICAM-1 MCP-1 TNF Mature blood vessel SMC Monocyte Arteries/Arteriols

7 Vasculogenesis, Angiogenesis, Arteriogenesis Vasculogenesis Hemangioblasts VEGF family, Ang-1,2 mature blood vessels Angiogenesis Endothelial cells FGF-1,2,4,5; VEGF-1,2,3 Capillary tube formation Arteriogenesis ECs, SMCs, Monocytes PDGF, FGFs, Ang-1,2; MCP-1 Arteriole formation

8 Tumor angiogenesis Stetler-Stevenson JCI 1999; 103:

9 Postnatal angiogenesis - vasculogenesis - endothelial cells (Ecs) ~ > 70% - stem cell derived ECs ~ < 30%

10 Postnatal angiogenesis and vasculargenesis

11 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

12 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

13 VEGF signaling

14 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

15 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 VEGF Hypoxia HIF-1 VEGF VEGF VEGF NOS NO

16 Effects of VEGF on ECs

17 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

18 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

19 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

20 VEGF in der Gentherapie kardiovaskulärer Erkrankungen Re-endothelialisierung von geschädigten Gefäßen durch VEGF165 cdna Kollateralbildung im ischämischen Muskel durch VEGF165 cdna VEGF165 cdna induziert Angiogenese im Myocard von Patienten mit myocardialer Ischämie Gefahr: Bildung von Haemangiomen und keine Arteriogenese

21 Gαs camp PKA CBP/p3oo Ox PL Lox-1 RTK Gαq VEGF FGF EGF PLC CREB IP 3 Ca ++ Src PKCε PI3 K SOS Tyrosine substrates 2 Calcineurin lipoxygenase NFAT PLCγ NFAT Gene transcription 12 HETE PPARγ EGR-1 Ö Nab-2 cpla PI4P PI3, 4P Ras-GTP RAF MEK-1/2 ERK-1/2 TFs, Myc Cycl D P LRP NAK-1 PDK2 Gene transcription u-par ILK Integrins α FAK cas Akt P 3 8 β p50 p65 NF-κB CYR61 Src pax Antiapoptotic genes DEP-1 FADD Procaspase 8 Cdc42/RAC Inflammatory mediators apoptosis MEKK1 ¾ PAK TNFα Ö GCK XIAP TAB MEK 4/7 ¾ TAK JNK MEK 3/6 TNFR1 P TRADD TRAF2 NI K p50 p65 NF-κB LTbR PKC P P IKK1 IKK2 p IkB p50 p65 NF-κB GCK NI K PKR NEM O PAI-1 TNFR2 ELAM VCAM Rantes IL-1, IL-6, IL-8 inos COX-2 TRAF2 TRAF1 ubiquitination degradation in proteasome Tissue factor

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