Role of VEGF in vasculogenesis and angiogenesis

Transcription

1 Role of VEGF in vasculogenesis and angiogenesis lecture II 14th March 2011

2 Angiogenesis and VEGF history 1787 Dr John Hunter first uses the term 'angiogenesis' to describe blood vessels growth 1983 Vascular Permeability Factor (VPF), is discovered by Dr Harold Dvorak. The molecule VPF causes leaky blood vessels associated with tumors VPF was times more potent than histamine One of the most important angiogenic factors, vascular endothelial growth factor (VEGF), is discovered by Napoleone Ferrara and by Jean Plouet. It turns out to be identical to the molecule called Vascular Permeability Factor (VPF) discovered in 1983 by Dr. Harold Dvorak.

3 Main proangiogenic factor Vascular Permeability Factor = Vascular Endothelial Growth Factor 1983, Dr H. Dvorak V P F V E G F 1989, Dr N. Ferrara Dr J. Plouet vascular permeability factor endothelial cell survival factor endothelial cell proliferation endothelial cell migration

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

5 Vasculogenesis capillaries are formed from vascular progenitor cells Vasculogenesis begins very early after the initiation of gastrulation in the mammalian embryo, with the formation of the blood islands in the yolk sac and angioblast precursors in the head mesenchyme

6 Vasculogenesis 1. First phase Initiated from the generation of hemangioblasts; 2. Second phase Angioblasts proliferate and differentiate into endothelial cells 3. Third phase Endothelial cells form primary capillary plexus In the yolk sac, these progenitors aggregate into endothelial lined blood islands that then fuse to generate a primary capillary plexus. The primary capillary plexus undergoes remodelling along with intra embryonic vessels to form a mature circulation

7 Vasculogenesis blood islands YOLK SAC MESODERM HEMANGIOBLASTS ANGIOBLASTS HEMATOPOIETIC CELLS Endothelium for vessels Developing blood cells

8 Vasculogenesis initial vessel formation MESENCHYME Endothelium ADVENTITIAL FIBROBLASTS MEDIAL SMCs Endothelium

9 ADVENTITIAL FIBROBLASTS Vasculogenesis later steps Endothelium MEDIAL SMCs VASCULOGENESIS is prominent in establishing the vascular beds of liver, lung, & heart

10 Hemangioblast Hemangioblast is a multipotent cell, common precursor to hematopoietic and endothelial cells. Hemangioblast was first hypothesized in 1900 by Wilhelm His.

11 Hematopoiesis in zebrafish (Danio rerio) Expression of the flk 1 represents the earliest marker of the developing endothelial lineage during vasculogenesis

12 SCL transcription factor is crucial for the development of blood cells and blood vessels

13 Formation of vascular network

14 Common vascular progenitor cells for endothelial cells and vascular smooth muscle cells

15 Origin of endothelial and mural cells from progenitor cells Hemangioblast Blood cells Skeletal muscles Angioblast Venous endothelium VEGF Arterial endothelium Pericytes/smooth muscles PDGF BB progenitor cells of vascular smooth muscles (various forms) VEGF PDGF BB Common vascular progenitor cells (flk 1 + )

16 Origin of endothelial and mural cells from common vascular progenitor cell Flk1+ cell differentiation in serum free culture CD31 (purple)/sma(brown) immunostaining a, Vehicle treated cells. b, PDGF BB. c, VEGF. d, Simultaneous administration of VEGF and PDGF BB.

17 Vasculogenesis in adult

18 Vasculogenesis occurs also in adult organism Previously, postnatal vascularization was thought to occur exclusively due to angiogenesis. However, recent investigations have shown that vasculogenesis is involved in blood vessel formation also during postnatal live and the cells responsible for that are EPCs. Classic Paradigm: Angiogenesis Mature ECs migrate and proliferate to form new vessels New Model: Angiogenesis + Vasculogenesis Bone Marrow derived EPCs circulate to sites of neovascularization JCI 1999;103:

19 Endothelial progenitor cells A primitive cell made in the bone marrow (or other organs) that can enter the bloodstream and go to areas of blood vessel injury to help repair the damage

20 Plasticity of adult stem cells the ability to form specialized cell types of other tissues (also called transdifferentiation)

21 Differentiation pathways for pluripotent bone marrow stromal cells

22 Adult progenitor cells

23 Endothelial Progenitor Cells 1997 Asahara et al. reported putative EPCs or angioblasts Cells display progenitor phenotype Differentiate into mature ECs in vitro Participate in neovascularization when injected into ischemic animal model Science 1997;275:

24 Endothelial Progenitor Cells

25 DiI AcLDL uptake EPC 1:200 2h Anna Grochot Przeczek

26 BODIPY AcLDL uptake Anna Grochot Przeczek

27 Isolectin binding by EPC Lectin from Bandeira simplicifolia control isolectin Anna Grochot Przeczek

28 Immunohistochemical characterisation of culture expanded expanded EPCs 2 weeks culture Cytoplasmic factor VIII (von Willebrand factor) Uptake of acetylated low density lipoproteins tube formation on Matrigel Dzau et al., Hypertension 2005,

29 Markers of endothelial progenitor cells CD34 VEGFR2 CD133 (prominin, AC133) CD133 is absent on mature endothelial cells and monocytic cells

30 Mechanisms of EPCs homing and differentiation Urbich & Dimmeler, Circ Res 2004

31 Mobilization of vascular progenitors Tissue ischemia results in expression of cytokines like VEGF Recruites progenitor cells Steady state 0.01% MNCs in blood are CEPs Amount of circulating progenitors are increased after trauma, infectious injuries or tumour growth 24 h after injury: 12%

32 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

33 VEGF belongs to VEGF family Endogenously expressed in mammals Encoded by the double stranded DNA virus, orf VEGF-A

34 Ferrara et al. 2004

35 VEGF VEGF (vascular endothelial growth factor, vascular permeability factor, vasculotropin) homodimeric protein, kda produced by many types of cells (e.g. macrophages, VSMC, fibroblasts, and cancer cells) expression is induced in response to hypoxia and proinflammatory cytokines receptors (VEGF R1 and VEGF R2) are present mostly on endothelial cells, therefore VEGF acts specifically on endothelium (but also on neurons and Schwann cells). VEGF R1 is expressed also on monocytes and vascular smooth muscle cells their activation upregulates expression of metalloproteinases and increases cell migration.

36 VEGF It protects endothelial cells from apoptosis and induces their proliferation, migration, and formation of capillaries VEGF acts protectively on neurons VEGF is required for the normal development of embryonic vasculature, the cyclic growth of blood vessels in the female reproductive tract, and the formation of capillaries during wound repair however, VEGF is also involved in abnormal angiogenesis, as seen in proliferative retinopathies, rheumatoid arthritis, psoriasis, and malignancies

37 VEGF is highly conserved between species VEGF has been found in all vertebrate species : fish(thezebrafishdanio 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).

38 Presence of VEGF like proteins in different animals In the nematode Caenorhabditis elegans four possible homologs of PDGF/VEGF receptors (VER 1 to VER 4) and one ligand (PVF 1) are known PVF 1 has the ability to bind to human receptors VEGFR 1 and VEGFR 2 and to induce angiogenesis in two model systems derived from vertebrates Control HUVEC HUVEC + VEGF HUVEC + PVF-1 Jorgensen & Mango, Nat Rev Gen 2002; Tarsitano et al. FASEB J 2006.

39 Drosophila melanogaster respiratory (tracheal( tracheal) ) system Branching tubular system of trachea delivers oxygen to the tissues of insects. Its development shows parallels to the angiogenesis Branchless (a homolog of mammalian FGF), PVF1, PVF2, PVF3 (homologs of mammalian VEGF/PDGF) and PVR receptor regulate the migration of early hemocytes and are necessary for formation of tracheal system. Tracheal tree of Drosophila embryo DB dorsal branch; DT dorsal trunk; GB ganglionic branch; VB visceral branch O Farrell, J Clin Invest 2001

40 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

41 VEGF isoforms 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

42 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 Heparan Sulfate Proteoglycan After Robinson and Stringer 2001, J Cell Science 114:853-65

43 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

44 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 is produced in big amount by different tumor 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. VEGF165 is most abundantly expressed, but VEGF189 is a major isoform in lungs, and both VEGF165 and VEGF189 predominate in heart. Furthermore, the relative levels of VEGF isoforms may vary during development or in response to cytokine stimulation.

45 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

46 Receptors for VEGF A Main receptors: VEGFR 1 (Flt 1) VEGFR 2 (Flk1; KDR) Accessory receptors Neuropilin 1 (NRP1) Neuropilin 2 (NRP2) Storage heparan sulfate proteoglycans

47 VEGF A belongs to VEGF family

48 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.

49 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 TK TK Heparan-Sulfate Proteoglycan Neuropilin-1 Neuropilin-2 VEGF-R1 VEGF-R2 VEGF-R3 After Neufeld et al , FASEB J 13:9-22

50 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

51 Expression of VEGF receptors is not restricted to ECs Zachary et al., 2003

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

53 Knockout of VEGF is lethal in heterozygous form Yolk sac of E10.5 VEGF+/+ and VEGF +/ mouse embryos Ferrara & Alitalo, Nature Med. 2000

54 Effect of knockout of VEGF receptors VEGFR 1 Flt1 / mice die in utero between days 8.5 and 9.5 EC develop but do not organize into vascular channels excessive proliferation of angioblasts 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

55 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 E12.5 overexpression of Np1 excessive capillary formation, dilated blood vessels extensive hemorrhage no visible 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

56 Why effect of knockouts of VEGF receptors is different?

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

58 Effects of knockouts of VEGF genes and VEGF receptors genes

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

60 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

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

62 Angiogenic and vasculoprotective functions of VEGF

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

64 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

65 Mechanisms of chemotactic VEGF signaling Zachary, Cardiovasc Res 2001

66 VEGF level has to be tightly regulated during development and postnatal life

67 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

68 Too high and unbalanced expression of VEGF after gene delivery using adenoviral vectors A and B. Note prominent tissue edema and new blood vessel formation. C. Note also a prominent leakage of plasma protein complexes from locally hyperpermeable ear vessels.

69 The effect of different isoforms of VEGF on angiogenesis

70 Vessel formation and sprouting angiogenesis in embronyic bodies in response to the different VEGF ligands Formation of peripheral vascular plexus in two dimensional EB cultures, visualized by anti CD31 immunostaining (red), was induced by 1 nmol/l VEGF A165, but not by VEGFA165b,VEGF A121, VEGF A145, or vehicle. Kawamura et al. Cancer Res. 2008

71 Vessel formation and sprouting angiogenesis in embronyic bodies in response to the different VEGF ligands Vascularization of Matrigel plugs in nude mice. Plugs were fixed and stained to detect CD31 on endothelial cell (red) and α SMA (ASMA) on pericytes (green) by immunofluorescent detection. Kawamura et al. Cancer Res. 2008

72 VEGF A165b inclusion of VEGF A165b led to invasion of endothelial cells into the Matrigel, but the cells failed to organize into vessels and also failed to attract pericytes endothelial cells pericytes Kawamura et al. Cancer Res. 2008

73 VEGF A121 In the VEGF A121 containing Matrigel plugs, occasional vessel structures were seen, which lacked branch points and a pericyte coat endothelial cells pericytes Kawamura et al. Cancer Res. 2008

74 VEGF A145 In VEGF A145 containing Matrigel plugs, branching, pericyte clad vessels were seen but to a much lesser extent than in the VEGF A165 containing Matrigel plugs endothelial cells pericytes Kawamura et al. Cancer Res. 2008

75 VEGF A165 Inclusion of VEGF A165 induced abundant vascularization of the Matrigel with richly branched, pericyte covered vessels endothelial cells pericytes!vegf165 is the crucial isoform! Kawamura et al. Cancer Res. 2008

76 How to assess the role of different VEGF isoforms, if the knockout of the gene is lethal?

77 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.

78 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

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

80 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

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

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

83 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

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85 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

86 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 werenormal VEGF 188/188 halfofembryosdiedinutero surviving gain less weigth, were less fertile

87 Take home messages 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 coreceptors: 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