Anna M. Randi MD PhD. Imperial College London UK. Von Willebrand factor regulation of angiogenesis: basic mechanisms and implications for disease

Transcription

1 Anna M. Randi MD PhD Imperial College London UK Von Willebrand factor regulation of angiogenesis: basic mechanisms and implications for disease

2 Von Willebrand Disease 1926: Finnish physician Erick von Willebrand described a severe bleeding disease in a large family from the Åland archipelago in the Baltic Sea The first case was a 5 years old girl, Hjördis, one of 11 siblings of which four died from uncontrolled bleeding at an early age. Hjördis had several severe episodes of bleedings from nose and lips and following tooth extractions. At the age of 13, she bled to death from menstrual bleeding. Finland Differences with classic Haemophilia: Lack of muscle and joint bleeding Presence of mucosal bleeding Affects both male and females Sweden 2

3 VWF gene and VWD mutations The vwf gene on chromosome 12 codes for a large pro-polypeptide of 2813 aa, of which 2050 aa form the mature peptide 3

4 Von Willebrand disease (VWD) VWD : Loss or dysfunction of VWF Most common hereditary bleeding disorder due to decrease or dysfunction of VWF Type 1 and type 3 VWD: quantitative defects Type 2 VWD: qualitative defects Autosomal transmission, genetic heterogeneity Acquired VWD: associated with myeloma, aortic valve stenosis, LVAD, Treatment options: DDAVP (desmopressin) to release VWF from Endothelial cells and platelets Replacement of VWF with plasma-derived products

5 Von Willebrand Factor (VWF) VWF: large matrix and plasma glycoprotein Synthesised by megakaryocytes and endothelial cells In EC, stored within specialised organelles : Weibel-Palade Bodies (WPB) VWF mediates platelet adhesion to the subendothelium and platelet aggregation (platelet receptors GPIb & GPIIbIIIa) VWF cleavage by ADAMTS13 controls multimers size and function Control + PMA 29/04/ De Ceunynck K et al. Blood 2013;121: Sadler, PNAS 2002

6 Processing and assembly of VWF into multimers pro mature The VWF protein is synthesized as a large precursor (pro-vwf) of ~220 KD, which dimerizes in the endoplasmic reticulum (ER) through disulfide bonds at the C-terminus Dimers assemble into multimers in the Golgi after pro-peptide cleavage The higher MW multimers are the most haemostatically active Rev in Sadler, JTH

7 Defective VWF processing and increased ER retention in Type 1 VWD patient Type 1 VWD: VWF gene mutation: exon 4-5 deletion affects VWF processing : no propeptide cleavage & VWF stuck in the ER Assay VWF:Ag 15 VWF:Rco 14 VWF:CB Result nd FVIII 68 DDAVP response low Rev in Sadler, JTH 2005 Starke et al. Blood

8 The many binding partners of VWF Springer Blood 2014 Plasma proteins: FVIII ADAMTS13 Fibrin Cell surface receptors: Platelets GP Ib-IX GP IIbIIIa P-selectin Endothelial cells Integrin avb3 P-selectin VSMC Integrin avb3 Leukocytes b2 integrins PSGL-1 ECM proteins: Collagen I Collagen III Collagen IV Thrombospondin Others (plasma and/or cellular) Histones DNA NETs Angiopoietin-2 * Interleukin-8 Galectin 1&3 Osteoprotegerin Complement components Regulation of WBP storage Angiopoietin-2 * Rev in Randi et al, Blood under revision

9 The multiple roles of VWF Lenting et al, J Thromb Haemost Dec;10(12):

10 Angiodysplasia and vascular malformations in Von Willebrand Disease Angiodysplasia: degenerative lesion of blood vessels Small vascular malformations of mucosal venules and capillaries in the gastrointestinal (GI) track Fragile & dysfunctional vascular network, disrupted architecture, increased permeability In VWD, frequency varies between 5-20% Causes severe GI bleeding in VWD Angiodysplasia linked to disrupted angiogenesis Treatment with VWF/FVIII concentrates not always effective Angiodysplasia of the ileum in type 3 VWD (Franchini & Mannucci BJH 2012) GI bleeding more frequent in - type 2A VWD - type 2B VW - Type 1 VWD with very low VWF - type 3 VWD > loss of VWF HMW multimers or very low VWF

11 VWF replacement therapy and GI bleeding Retrospective study on 62 subjects from 20 centres in 10 countries: Type 3 VWD :57% Type 2A: 17% In this study, the effect of prophylaxis appeared to be most pronounced in the case of joint bleeding,..mucosal bleeding, i.e. epistaxis, GI bleeding and menorrhagia, was reduced but not to the same degree, perhaps because these haemorrhages are not only dependent on normal circulating levels of active VWF, but also on the presence of.. VWF inside the platelets and within endothelial matrices. 11

12 Vascular malformations in Von Willebrand disease Vascular malformations in nail bed of VWD patients Abnormal tortuosity, variation in calibre, and defective contractility O Brien, Proc Int Soc Hematol Blackburn, Br J Haematol Study of 100 VWD patients: positive predictive value for VWD of 99%. Koscielny, Thromb Haemost CAPILLAROSCOPY : non-invasive tool to morphologically study the microcirculation Koscielny, Thromb Haemost

13 Angiodysplasia may be linked to dysregulated angiogenesis Circulating VEGF is increased in patients with - angiodysplasia (Fujita et al, 2000) - VWD (Gritti Federici, 2011) - HHT (Junquera et al, 1999)

14 ANGIOGENESIS AND DISEASE Insufficient Baldness Vascular Malformations GI Angiodysplasia Excessive Retinal Disease MI - Ischemia Limb Fractures HHT & VWD Cerebral malformations Cancers Atherosclerosis Thrombosis Obesity AVM /CCM 14

15 How to make a blood vessel Vasculogenesis (bone marrow progenitor cell) Angiogenesis (sprouting) Arteriogenesis (collateral growth) Carmeliet Nature Medicine 6, (2000)

16 Molecular mechanisms of angiogenesis Zebrafish embryo and pruning

17 Angiogenesis live Zebrafish embryo. Endothelial cells : green (Fli-1 GFP)

18 Angiogenesis and its regulation Inhibitors: Thrombospondin-1 Angiostatin Endostatin svegf-r IL-10 IL-12 TIMPs TNF-a avb3... Maturation: Angiopoietin-1 WNT pathway AKT pathway Notch pathway ERG. Activators: VEGF family FGFs TNF-a TGF b PDGF PlGF IL-6 Factor XIII Tissue Factor avb /04/

19 R. Blanco and H. Gerhardt Vascular Endothelial Growth Factor (VEGF) and its receptors Family of 5 members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF) Three tyrosine kinase receptors: VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3; and co-receptors neuropilin (Nrp1 and Nrp2) VEGFR-2 is the major mediator of VEGF-dependent angiogenesis, activating signalling pathways that regulate endothelial cell migration, survival, proliferation.

20 Rationale: why should VWF regulate angiogenesis? Vascular malformations in VWD patients VWF directs formation of WPB which store regulators of angiogenesis VWF surrounds EC and interacts with angiogenesis regulator avb3 integrin VWF Weibel-Palade Bodies (WPB) VWF endothelium 20

21 Endothelial VWF regulates angiogenesis: in vitro evidence VWF sirna Angiogenesis (Matrigel) HUVEC VWF nuclei sictl sivwf Endothelial Migration Endothelial Proliferation Starke et al. Blood

22 Endothelial VWF regulates blood vessel formation : in vivo evidence Increased angiogenesis in VWF KO mice (Matrigel plug) Control Increased vascularization in VWF KO mouse ear VWF is required for VSMC coverage in mouse retina vasculature Starke et al. Blood 2011 VWF KO Also: increased neovascularization in the ischemic brain of VWF KO mice Xu et al. Blood 2017;130:11-22 Vascular Density Fractal Dimension Total Ear Area From Lea Scheppke et al. Blood 2012;119:

23 Regulation of VEGFR-2 signalling by integrin avb3 VWF binds to integrin avb3 on endothelial cells (Cheresh PNAS 1987) avb3 expression upregulated in angiogenic vessels avb3 involved in regulation of angiogenesis: pro- and anti-angiogenic role (Hodivala-Dilke et al. Cell Tissue Res. 2003; Brooks et al Cell 1994, Reynolds et al. Nat. Med ) avb3 regulates VEGF Receptor 2 (VEGFR2) function and levels VWF regulates levels of integrin avb3 Starke et al. Blood Modified from Smyth and Patterson, Cell Biol 2002;158:17-21

24 Storage of angiogenesis regulators in Weibel Palade Bodies Angiopoietin-Tie2 ligand-receptor system VWF Ang-2 merged HUVEC McKinnon, unpublished Ang-2 VWF merged From P. Kümpers Ang-1 and Ang-2 are antagonistic ligands of the Tie2 receptor Angiopoietin-2 (Ang-2) antagonises Ang-1 signalling through the Tie2 receptor, and promotes vascular instability and VEGF-dependent angiogenesis Reviewed in Thurston and Daly, Cold Spring Harb Perspect Med From Fiedler Nature Medicine - 12, (2006) Human colon 24

25 VWF regulates Ang-2 storage and synthesis in EC VWF Ang-2 merged Ang-2 release 25

26 VWF control of Ang-2 expression is tissue-specific Ang-2 levels increased in heart and brain from VWF KO mouse Yuan et al 2016; Nat Comms Brain microvessels Xu et al, Blood

27 VWF regulation of angiogenesis: model Vascular maturation VSMC avb3 Tie-2 VEGFR2 avb3 VWF Ang-2 VWF WPB WPB From Randi, Thromb Res Angiogenesis Endothelial Cell

28 Excess VEGF signalling results in dysfunctional, leaky blood vessel Unstable (VEGF + control) Stable (VEGF + ERG) - In vivo Matrigel angiogenesis assay with FITC-dextran (2x10 6 MW, green) to visualize blood vessel and TRITC-dextran (4.4x10 4 MW, red) to measure permeability - ERG: transcription factor which promotes vascular stability (Birdsey et al, Dev Cell 2015) 28

29 VWF, angiodysplasia and blood vessel formation: open questions Multiple pathways involved in VWF regulation of blood vessel formation and maturation EC: VEGFR-2 signalling; avb3; Ang-2 (tissue-specific) VSMC recruitment Role for VWF high molecular weight multimers Possible role for platelets and leukocytes? Are angiogenic pathways differently affected depending on the VWD type? Clinical implications: Angiodysplasia and GI bleeding Does molecular dysfunction in BOEC correlate with angiodysplasia? vascular imaging Angiogenesis-related diseases in VWD Tissue-specific vascular dysfunction? Therapeutic implications for angiodysplasia: Systemic targeting of relevant angiogenic pathways? Localised treatment for angiodysplasia? 29

30 Study of endothelial cells in patients using Blood Outgrowth Endothelial Cells (BOEC) 30 Endothelial progenitors of unknown origin (also known as Endothelial Colony Forming Cells) (Lin et al, JCI 2000; Ingram et al, Blood 2004) BOEC phenotypically resemble HUVEC (Toshner, PLoS1 2014) BOEC studies in patients with endothelial dysfunction HHT (Fernandez-L et al 2005) COPD (Paschalaki et al Stem Cells 2013) Premature neonates (Vassallo et al Blood 2014) blood ml PBMC Blood outgrowth EC (BOEC) Collagen Type I EBM2 + 10% FCS HUVEC Green: VWF BOEC HUVEC BOEC Red: VE-Cadherin 3-4 weeks culture

31 Von Willebrand disease (VWD) VWD : Loss or dysfunction of VWF Most common hereditary bleeding disorder due to decrease or dysfunction of VWF Type 1 and type 3 VWD: quantitative defects Type 2 VWD: qualitative defects Autosomal transmission, genetic heterogeneity Acquired VWD: associated with myeloma, aortic valve stenosis, LVAD, Treatment options: DDAVP (desmopressin) to release VWF from Endothelial cells and platelets Replacement of VWF with plasma-derived products

32 Type 1 but not type 2 VWD patients show reduced VWF mrna and protein in BOEC VWF mrna Intracellular VWF protein Type 1 VWD Quantitative defect Type 2 VWD Qualitative defect 2A 2M 2A 2M Starke et al. Blood

33 Type 1 but not type 2 VWD patients show reduced VWF mrna and protein in BOEC VWF mrna Intracellular VWF protein Healthy Type 1 VWD Quantitative defect Type 1 Type 2 VWD Qualitative defect VWD 10 VWD 8 2A 2M 2A 2M Starke et al. Blood 2013 VWD 2 Type 2M 33

34 Defective VWF processing and increased ER retention in Type 1 VWD patient Pt 8 VWF gene mutation: exon 4-5 deletion Assay VWF:Ag 15 VWF:Rco 14 VWF:CB Result nd FVIII 68 DDAVP response low Rev in Sadler, JTH 2005 Starke et al. Blood /04/

35 BOEC from Type 2M patient shows normal VWF expression and release Patient ID VWF release Mutation VW D VWF:Ag (NR ) VWF:R Co VWF:Ag Post DDAVP VWD-2 I1416T 2M PMA +PMA Starke et al. Blood

36 VWF, angiodysplasia and blood vessel formation: open questions Multiple pathways involved in VWF regulation of blood vessel formation and maturation EC: VEGFR-2 signalling; avb3; Ang-2 (tissue-specific) VSMC recruitment Role for HMW multimers Possible role for platelets and leukocytes? Are angiogenic pathways differently affected depending on the VWD type? Clinical implications: Angiodysplasia and GI bleeding Does BOEC dysfunction correlate with angiodysplasia & vascular imaging? Angiogenesis-related diseases in VWD? Therapeutic implications for angiodysplasia: Systemic targeting of relevant angiogenic pathways? Localised treatment for angiodysplasia? 36

37 VWF, angiodysplasia and blood vessel formation: open questions Multiple pathways involved in VWF regulation of blood vessel formation and maturation EC: VEGFR-2 signalling; avb3; Ang-2 (tissue-specific) VSMC recruitment Role for HMW multimers Possible role for platelets and leukocytes? Are angiogenic pathways differently affected depending on the VWD type? Clinical implications: Angiodysplasia and GI bleeding Does BOEC dysfunction correlate with angiodysplasia & vascular imaging? Angiogenesis-related diseases in VWD Therapeutic implications for angiodysplasia: Systemic targeting of relevant angiogenic pathways? Localised treatment for angiodysplasia? 37

38 Acknowledgements ENDOTHELIAL HOMEOSTASIS GROUP COLLABORATORS Koval Smith Koralia Paschalaki Neil Dufton Claire Peghaire Viktoria Kalna Josefin Janssen Oisin King Linda Inuabasi Claudio Raimondi Previous lab members Richard Starke Beth Payne Flavia Ribezzo Carolina Bianchi Imperial College Graeme Birdsey Justin Mason Mike Laffan Tom McKinnon Carolyn Millar Mervin Yoder (Indianapolis, USA) Augusto Federici (Milan, Italy) Giancarlo Castaman (Florence, Italy) Ralf Adams (Munster, Germany) Dan Cutler (UCL, London) 38