Role of VEGF in angiogenesis

Transcription

1 Lecture II Role of VEGF in angiogenesis

2 Knockout of VEGF is lethal in heterozygous form Ferrara & Alitalo, Nature Med. 2000

3 Three ways of formation of blood vessels angioblast bfgf VEGF capillary Vasculogenesis capillaries are formed from vascular progenitor cells VEGF, Ang-2 Angiogenesis formation of new blood vessels from pre-existing vessels Ang1, bfgf, MCP-1, PDGF Arteriogenesis formation of mature blood vessels; differentiation into veins and arteries

4 Blood vessel formation various ways Carmeliet, 2005; Semenza 2003

5 Angiogenesis - Basement Membrane Breakdown Angiogenic Stimulus (VEGF) Smooth Muscle Cells Basement Membrane Matrix metalloproteinases Endothelium

6 Angiogenesis - Endothelial Cell Migration VEGF Smooth Muscle Cells Nascent Vascular Sprouts Basement Membrane Endothelium

7 Angiogenesis - Endothelial Cell Proliferation VEGF Smooth Muscle Cells Sprout Elongation Basement Membrane Endothelium

8 Angiogenesis - Capillary Morphogenesis VEGF Smooth Muscle Cells New Lumen Formation Basement Membrane Endothelium

9 Angiogenesis - Vascular Maturation VEGF Smooth Muscle Cells SMC, pericyte recruitment Basement Membrane Endothelium

10 VEGF, VEGF-A vascular endothelial growth factor main regulator of angiogenesis, pro-angiogenic factor - a dimeric glycoprotein - belongs to a so-called cysteine-knot superfamily of growth factors - one interchain disulfide bond Oloffson et al., 2000

11 VEGF-A belongs to VEGF family endogenously expressed in mammals encoded by the double stranded DNA virus, orf

12 VEGF is highly conserved between species VEGF has been found in all vertebrate species : fish (the zebrafish Danio rerio) frogs (Xenopus laevis) birds (Gallus gallus) mammals The sequence and genomic organization of the vertebrate VEGF-A genes is highly conserved between fish and mammals. Fish VEGF-A shows 68% and 69.7% amino-acid identity with human and mouse VEGF-A, respectively VEGF-like proteins are present in several invertebrate species Invertebrate VEGF/VEGFR systems have been identified in fly (Drosophila melanogaster), nematode (Caenorhabditis elegans) and, most recently, in jellyfish (Podocoryne carnea).

13 Organisation of VEGF gene and VEGF isoforms The human VEGF-A gene is characterized by a highly conserved eight exon structure, Alternative splicing of the human VEGF-A gene gives rise to at least five different transcripts encoding isoforms of the following lengths (in amino acids) 121, 145, 165, 189 and 206 Exons VEGF-A 121 Exons 1-5 6A1 A2 8 VEGF-A 145 Exons VEGF-A 165 Exons 1-5 6A1 A2 7 8 VEGF-A 189 Exons 1-5 6A1 A2 6B 7 8 VEGF-A 206

14 Organisation of VEGF gene and VEGF isoforms Bates et al., Cancer Res 2004

15 Isoform Size Coding exons Features (amino acid) VEGF-A , 8 Secreted VEGF-A , 8 Binds NRP2 but not NRP1; secreted VEGF-A , 7, 8 The most abundant and biologically active isoform; secreted; binds NRP1 and NRP2 VEGF-A165b , 7, alternative exon 8 Secreted, endogenous inhibitory form of VEGF-A165 VEGF-A , short exon 6, 7, 8 Sequestered in ECM but released by cleavage VEGF-A Sequestered in ECM but released by cleavage VEGF-A plus additional exon Sequestered in ECM but released by cleavage

16 Splice variants of human VEGF a.a. 115 a.a. 6a 24 a.a. 6b 17 a.a a.a. 8 6 a.a. VEGF121 VEGF145 VEGF165 NRP-1 VEGF183 VEGF189 VEGF206 signal peptide VEGF-R1 VEGF-R2 HSPGs After Robinson and Stringer 2001, J Cell Science 114:853-65

17 Properties of VEGF isoforms VEGF 121 is a soluble acid polypeptide VEGF 189 and VEGF 206 are highly basic and bind very strongly to heparin, thus they are completely sequestred in extracellular matrix (ECM) VEGF 165 has intermediate properties: it is secreted, but significant fractions remains bound to cell surface and ECM

18 Proteolytic processing of VEGF-A

19 Expression of VEGF isoforms VEGF is produced by virtually all cell types. It is synthesized by a number of non-malignant cells including keratinocytes, skeletal myotubes vascular smooth muscle cells and certain endothelial cells. It has also been isolated from conditioned media of a number of cell lines including bovine pituitary follicular cells, guinea pig tumor, NB41 neuroblastoma and rat glioma cells. Most VEGF-producing cells express VEGF 121, VEGF 165, VEGF 189, and often VEGF 183. VEGF 145 and VEGF 206 are seemingly restricted to cells of placental origin. VEGF 165 is most abundantly expressed, but VEGF 189 is a major isoform in lungs, and both VEGF 165 and VEGF 189 predominate in heart. Furthermore, the relative levels of VEGF isoforms may vary during development or in response to cytokine stimulation. VEGF 121, VEGF 145 and VEGF 165 induce proliferation and migration of endothelial cells.

20 Not every cells express the same amounts of VEGF VEGF expression in several cell lines - intact cells (24 h incubation) HASMC HMEC-primary HMEC-1 HUVEC rat Müller cells

21 Receptors for VEGF-A Main receptors: VEGFR-1 (flt-1) VEGFR-2 (Flk1;KDR) Accessory receptors Neuropilin 1 (NRP1) Neuropilin 2 (NRP2) Storage heparan sulphate proteoglycans

22 Vascular endothelial growth factor-a (VEGF) VEGF-A is a major angiogenic growth factor. It acts on endothelial cells, being produced by numerous cell types, including vascular smooth muscle cells (VSMC), fibroblasts or tumor cells. Receptors on endothelial cells Yancopoulos, Science 2000

23 VEGF-A belongs to VEGF family

24 Receptors for VEGF-A Both VEGF receptors have 7 immunoglobulin-like domains in the extracellular domains, a single transmembrane region and a consensus tyrosine kinase sequence that is interrupted by a kinase-insert domain.

25 Growth factors and receptors of the VEGF family VEGF121 VEGF145 VEGF165 VEGF189 VEGF-B PlGF-1 PlGF-2 VEGF121 VEGF145 VEGF165 VEGF-C VEGF-D VEGF-E VEGF-C VEGF-D VEGF145 VEGF165 VEGF189 VEGF206 VEGF-B167 VEGF-E PlGF-2 Sema-III Sema-E Sema-IV VEGF165 PlGF-2 VEGF-B VEGF-E Sema-E Sema-IV VEGF165 Heparan-Sulfate Proteoglycan Neuropilin-1 Neuropilin-2 VEGF-R1 VEGF-R2 VEGF-R3 After Neufeld et al , FASEB J 13:9-22

26 Expression of VEGF receptors - endothelial cells: VEGFR-1, VEGFR-2, co-receptors - other cells: monocytes vascular smooth muscle cells? tumor cells? hematopoietic stem cells neuronal cells

27 Expression of VEGF receptors is not restricted to endothelial cells Zachary et al., 2003

28 Significance of VEGF and VEGF receptors has been recognized by targeting disruption of those genes in mice

29 Semaphorin receptors Np-1 and Np-2 - form complexes with type A plexins - complexes serves as signaling receptors for class-3 semaphorins - involved in axonal guidance Np-1 and Np-2 in angiogenesis - binds VEGF165, VEGF-B, PlGF-2 - knockout of Np-1 lethal at E overexpression of Np1- excessive capillary formation, dilated blood vessels extensive hemorrhage - no discernible abnormalities in Np.-2 knockout mice, but Np-2-/-Np1+/- are lethal - double knockouts Np-1-/-Np-2-/- - died in utero at E8.5, completely avascular yolk sacs

30 Transgenic mice technology

31 The Nobel Prize in Physiology or Medicine, 2007 Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their discoveries of "principles for introducing specific gene modifications in mice by the use of embryonic stem cells" M. Capecchi Univ. of Utah Sir M. Evans Cardiff Univ., UK O. Smithies UNC Chapel Hill First KO mice using ES cell technology were produced in 1989

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33 Vector with a transgene tk1 & tk2 - Herpes Simplex Virus thymidine kinase genes (make cells susceptible to gancyclovir) Neo - neomycin resistance gene Homologous regions - homologous to the chromosomal target Transgene - foreign gene

34 The Problem: DNA Integration Can occur by homologous (H) or nonhomologous (N-H) recombination Frequency of N-H >> H (by at least 5000-fold) in mammalian cells If you want H integrants, which you need for knock-outs, you must have a selection scheme for those.

35 Example of what happens with N-H recombination Nonhomologous recombination homologous homologous tk1 sequence neo transgene sequence tk2 chromosome tk1 homologous homologous sequence neo transgene sequence tk2 chromosome Transformed cells are neo-resistant, but gancyclovir sensitive.

36 What happens with HR Homologous recombinants homologous homologous tk1 sequence neo transgene sequence tk2 chromosome homologous sequence neo transgene homologous sequence chromosome If DNA goes in by HR, transformed cells are both neo-resistant and gancyclovir-resistant! Use double-selection to get only those cells with a homologous integration event.

37 Gene knockout technology

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39 How to make a transgenic mouse? Transfection Blastocyst (mouse) Embryonic Stem cells (ES cells) with DNA Grow in culture. Select for those that carry the transgene. Neomycin and gancyclovir resistant! Inject into a blastocyst

40 Inject into a blastocyst Implant int o pseudopregnant mouse pseudopregnant mouse (by mating a female mouse with a vasectomized male). Chimeric mouse Ident if y of f spring which carry t he t ransgene in t heir germline.

41 Gene knockout technology

42 Lethal effects of VEGF gene knockout

43 Knockout of VEGF is lethal in heterozygous form Ferrara &Alitalo, Nature Med. 2000

44 Effect of knockouts of VEGF receptors VEGFR-1 Flt1-/- mice die in utero between days 8.5 and EC develop but do not organize into vascular chanels - excessive proliferation of angioblasts Thus, at least during early development, VEGFR-1 is a negative regulator of VEGF action

45 Effect of knockouts of VEGF receptors VEGFR-2 Flk1-null mice die between day 8.5 and 9.5 Lack of vasculogenesis and failure to develop blood islands and organized blood vessels VEGFR-2 is the key receptor for VEGF-A-induced angiogenesis. It signals mitogenic, chemotactic and prosurvival effects

46 Why effect of knockouts of VEGF receptors is different?

47 VEGF receptors role in angiogenesis Affinity of VEGF to VEGFR pm Affinity of VEGF to VEGFR nm

48 Functions of VEGF receptors Ferrara N et al., Nature Med., June 2003

49 Effects of knockouts of VEGF genes and VEGF receptors genes

50 Functions of the VEGF receptors family VEGFR-1 Crucial to embryonic angiogenesis Does not appear to be critical in pathogenic angiogenesis VEGFR-2 VEGFR-3 Mediates the majority of VEGF angiogenic effects Found only in lymphatic endothelial cells Associated with lymph node metastasis

51 The VEGFRs differ in their downstream signaling effects Receptor VEGFR-1 VEGFR-2 Effects Possible decoy receptor effect Induction of other factors Proliferation Migration Survival Angiogenesis VEGFR-3 Effects mainly in lymphatic cells

52 Angiogenic and vasculoprotective functions of VEGF - vascular permeability functions - endothelial cells survival factor - endothelial cell proliferation - endothelial cell migration - inhibition of thrombosis

53 VEGF level has to be tightly regulated during development

54 Embryonic development is disrupted by modest increases in VEGF gene expression Miquerol L, Langille BL, Nagy A. Development, 2000: 127: fold overexpression is deletorious to embryonic development Enlarged hearts Embryos died between E12.5 and E14.5

55 Enhanced hair growth and follicle size in VEGF transgenic mice Detmar et al., JCI - hair re-growth after depilation accelerated, - hair more dense

56 Hair growth is dependent on angiogenesis Hair growth and angiogenesis VEGF Hair follicle involution TSP-1 (an inhibitor of angiogenesis)

57 Conditional knockouts of genes This strategy is based on a tissue-specific inactivation of the gene of interest. This can be achieved by means of a Cre recombinase, that catalyzes site-specific recombination of DNA between loxp sites. A Cre recombinase is an enzyme that deletes the DNA fragment located between the two recombinase-specific (LoxP) sites. A mouse bearing the recombinase-specific sites (introduced by homologous recombination in Embryonic Stem cells) is bred with a mouse expressing the recombinase (generated by homologous recombination or transgenesis). The tissue-specific expression of the recombinase allows the inactivation of the gene of interest only in the tissue where the recombinase is expressed.

58 Use of Cre recombinase for conditional knockouts DNA recognition site for recombinase enzymes. The DNA recombinases have a similar basic recognition site, as shown here for the Cre enzyme. Two palindromic sequences (loxp sites for the enzyme Cre) are separated by a DNA core. The core sequence can vary, whereas the palindromic sequences must contain a subset of the nucleotides shown to support integration. Gorman, Curr Opinion Biotech 2000

59 Cre recombinase mediated deletion ADS Ryding et al., J Endocrinol 2001

60 Cre-driven conditional expression of genes Transgenic animals, in which the target gene is flanked by Lox sequences, must also express Cre recombinase Thus, they have to be cross-bred with mice expressing Cre. The expression of Cre can be: 1. Tissue specific Cre gene is driven by the tissue specific promoter, eg. heart, liver etc. 2.Conditionally induced Cre gene is driven by the inducible promoter, eg. tetracycline-induced or IFN α induced

61 VEGF is required for growth and survival in neonatal mice One allele of VEGF deleted in exon 3 Gerber et al., 1999

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63 VEGF is required for growth and survival in neonatal mice 1. 38% mortality at day 7 in mice without VEGF (its synthesis was blocked from day 3); 2. Liver changes - smaller hepatocytes, immature sinusoids, increased extramedullary hematopoiesis and almost complete absence of Flk-1 positive endothelial cells; 3. Similar effects as after targeted knockouting of VEGF were obtained when mice were treated with anti-vegf antibodies

64 Splice variants of human VEGF a.a. 115 a.a. 6a 24 a.a. 6b 17 a.a a.a. 8 6 a.a. VEGF121 VEGF145 VEGF165 NRP-1 VEGF183 VEGF189 VEGF206 signal peptide VEGF-R1 VEGF-R2 HSPGs After Robinson and Stringer 2001, J Cell Science 114:853-65

65 Differential role of VEGF isoforms VEGF165 is the crucial isoform How to assess the role of different VEGF isoforms, if the knockout of the gene is lethal?

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67 Targeting of VEGF isoform-specific specific alleles Stalmans et al., JCI 2002

68 Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms The present study investigates the distinct role of the different VEGF isoforms in retinal vascular development. Retinal vascular development was normal in VEGF164/164 mice. In contrast, VEGF120/120 mice exhibited severe vascular defects, with impaired venous and severely defective arterial vascular development in the retina. VEGF188/188 mice had normal venous development, but aborted arterial outgrowth. Stalmans et al., JCI 2002

69 Impaired retinal vascular development in VEGF 120/120 and VEGF 188/188 mice Stalmans et al., JCI 2002

70 They all died before postnatal day 14

71 Effect of conditional knockout of VEGF164 on myocardial angiogenesis Capillary density increases 300% in VEGF+/+ hearts (filled bars) but not in VEGF120/120 hearts (stippled bars). There were fewer α-actin stained coronary vessels per section in VEGF120/120 hearts (stippled bars) than in VEGF+/+ hearts (filled bars). Carmeliet et al., 1999

72 Viability of VEGF-isoform mice VEGF 120/120 half neonates died shortly after births because of cardiorespiratory distress; the other died within 2 weeks after birth, in part due to impaired myocardial angiogenesis resulting in cardiac failure VEGF 164/164 were normal VEGF 188/188 half of embryos died in utero - surviving gain less weigth, were less fertile and had smaller litter size

73 Role of VEGF in arteriogenesis Stalmans et al., JCI 2002 VEGF120 not sufficient for venular and even less so for arteriolar VEGF188 allows venular development only VEGF164 sufficient for both

74 Vascularization of tumours grown from cells expressing only a single isoform of VEGF Day 6 Day 7 Day 8 Tozer GM VEGF 188 VEGF 120 The different splice variants of VEGF-A influence vascular maturation in a number of ways. Fibrosarcoma cell lines expressing only single VEGF isoforms were developed from corresponding knock-out mice, in order to investigate these processes and the consequent vascular characteristics of the arising solid tumours. Fragments of VEGF 120 tumours expanded rapidly in the chambers despite poor internal vascularization. Within 4 days of implantation, haemorrhage of the surrounding blood vessels was observed. In contrast, the rate of VEGF 188 fragment growth was slower until the tumours were well vascularized, with narrower vessels and no haemorrhage. The different VEGF isoforms clearly have markedly different effects on the vascularization process of tumours, influencing both the morphology and function of tumour blood vessels

75 Angiogenic and vasculoprotective functions of VEGF - vascular permeability functions - endothelial cells survival factor - endothelial cell proliferation - endothelial cell migration - inhibition of thrombosis

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77 Mechanisms of anti-apoptotic apoptotic VEGF signaling Focal adhesion kinase Phosphotydyloinositol 3 kinase AKT = PKB Zachary, Cardiovasc Res 2001

78 Mechanisms of mitogenic VEGF signaling phosphatydylinositol 4,5 biphosphate Phospholipase C Proteins with src homology (SH) 2 domain Diacyloglicerol+ Inositol 1,4,5 -trisphosphate Extraxellular signal-regulated kinases Zachary, Cardiovasc Res 2001

79 Mechanisms of chemotactic VEGF signaling Zachary, Cardiovasc Res 2001

80 Why different VEGF isoforms have different angiogenic potentials? Is there a role of some downstream mediators in those differences? Nitric oxide as a mediator of VEGF signaling

81 Sources of NO in the organism

82 Nitric oxide synthases enos - endothelial (constitutive) NOS (NOS III) nnos - neuronal (constitutive) NOS (NOS I) inos - inducible (NOS II) L-arginine O 2 NOS cofactors. NO L-citrulline

83 Nitric oxide is produced from L-arginine by nitric oxide synthases

84 VEGF-induced signaling in endothelial cells Ca 2+ independent Zachary, Cardiovasc Res 2001

85 Involvement of nitric oxide in angiogenic activities of VEGF isoforms NO concentration [nmol/l] VEGF VEGF121 VEGF165 Release of NO by VEGF-stimulated endothelial cells is stronger in case of VEGF 121 isoform Time [sec] * * Synthesis of cgmp by VEGF-stimulated endothelial cells is higher in case of VEGF 121 isoform cgmp [fmol/ml] # # 2 Józkowicz, Dulak et al., Growth Factor, control L-NAME D-NAME L-NAME 165 D-NAME

86 Involvement of nitric oxide in angiogenic activities of VEGF isoforms No difference in angiogenic potentials of various VEGF isoforms VEGF121 VEGF121 control control VEGF165 VEGF165 Matrigel assay Spheroid assay

87 Properties of VEGF 121 and VEGF 165 isoforms A. VEGF 121 enos activity Migration Assembly Capillary sprouting Proliferation B. VEGF 165 enos activity enos expression Migration Assembly Proliferation Capillary sprouting

88 Take-home messagess VEGF (VEGF-A) is a key mediator of vasculogenesis, angiogenesis and arteriogenesis VEGF is generated in the form of several isforms, being the results of alternative splicing The most common and the most active and crucial isoform is VEGF 165 VEGF exerts its activity by binding to its receptors: VEGFR1, VEGFR2 and co-receptors: neuropilin 1 & 2. VEGFR2 is the key receptor, mediating the majority of actions of VEGF. VEGFR1 is a decoy receptor, playing an important role in modulating VEGF activity during development