Endothelial dysfunction in von Willebrand disease: angiogenesis and angiodysplasia

Thrombosis Research 141S2 (2016) S55–S58

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Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r . c o m / l o c a t e / t h r o m r e s

Endothelial dysfunction in von Willebrand disease: angiogenesis and angiodysplasia Anna M. Randi* National Heart and Lung Institute (NHLI) Vascular Sciences, Hammersmith Hospital, Imperial College London, London, W12 0NN, UK

K E Y W O R D S

A B S T R A C T

von willebrand factor endothelial cells weibel palade bodies vascular malformations extracellular matrix

I n recent years, new functions for the haemostatic protein von Willebrand Factor (VWF) have emerged. Amongst these is the ability to modulate the development of new blood vessels, a process called angiogenesis. The subtle effects that VWF exerts on blood vessel formation and stability may be relevant for the small but significant fraction of patients with von Willebrand disease (VWD) who also present with vascular malformations (angiodysplasia) in the gastrointestinal tract, often responsible for intractable bleeding. This review will briefly summarise the evidence and discuss the molecular pathways involved. © 2016 Elsevier Ltd. All rights reserved.

von Willebrand disease and acquired von Willebrand syndrome von Willebrand disease (VWD), the most common inherited bleeding disorder in humans (reviewed in [1]), is caused by congenital decrease or dysfunction of von Willebrand factor (VWF), a large glycoprotein best known for its role in haemostasis. The classification of VWD identifies 3 main types and a number of subtypes. VWD can also be acquired (acquired von Willebrand syndrome or AVWS), due to dysfunction or degradation of VWF, often in association with malignant disorders, aortic valve stenosis or left ventricular assist devices (reviewed in [2,3]). Vascular abnormalities in patients with VWD or AVWS Up to 20% of patients with VWD present with gastrointestinal (GI) bleeding [4] which can be severe and not responsive to VWF replacement therapies [5]. GI bleeding has been linked to the presence of angiodysplasia [6,7]. The true prevalence of angiodysplasia in VWD or AVWS is not known, partly because of the risk of complications from the invasive endoscopic techniques required for diagnosis. Angiodysplastic lesions are thought to develop due to dysregulated angiogenesis, leading to the production of fragile vessels prone to bleeding [8]. Angiogenesis is a complex, multistep process which involves numerous pathways acting in concert to produce a stable blood vessel (reviewed in [9]). Loss of balance between proliferation and stabilization may result in excessive, unstable and dysfunctional new vessels, such as those found in angiodysplastic lesions. Interestingly, vascular

  * Correspondence to: Anna M. Randi, Professor of Cardiovascular Medicine, NHLI Vascular Sciences, Hammersmith Hospital, Imperial College London, Du Cane Road, London, W12 0NN, United Kingdom. Tel.: +44 20 7594 2721.

E-mail address: [email protected]

malformations outside the GI track have also been reported in patients with VWD [10,11]. Until recently, the pathological mechanism underlying angiodysplasia in patients with VWD was unexplained. The discovery that VWF regulates angiogenesis [12] has provided a novel prospective on this syndrome and opened the way to new strategies for the treatment of angiodysplasia. von Willebrand factor, angiogenesis and angiodysplasia: molecular mechanisms VWF is a multifunctional glycoprotein best known for its essential roles in haemostasis, as a mediator of platelets adhesion and as carrier for coagulation Factor VIII. VWF is present in three pools: cells (endothelial cells [EC] and megakaryocytes); plasma (mostly from EC release) and subendothelium (through release from EC). In EC, VWF is stored in organelles called Weibel Palade Bodies (WPB) [13,14], as multimeric molecules which reach a molecular weight (MW) up to 20,000 kDa. Both the multimeric size and the conformation of VWF determine its platelet binding activity (reviewed in [15]). Besides its well-characterised role in haemostasis, VWF is increasingly been implicated in other biological processes, from inflammation to permeability and cell survival (reviewed in [16]). We recently identified a role for VWF in the control of angiogenesis [12]. In vitro studies on EC showed that inhibition of VWF expression using siRNA resulted in increased proliferation, migration and in vitro angiogenesis [12]. A similar overall pattern is found in blood outgrowth endothelial cells (BOEC) from patients with VWD [12,17], although differences in the cellular phenotypes have been observed depending on different molecular defect. In VWF deficient mice, angiogenesis and vascular density were increased in several in vivo models [12], whilst recruitment of VSMC, a sign of arterial maturation, was delayed in the developing retinal vasculature [18]. Because of the numerous molecular interactions and functions of VWF in the vasculature, multiple molecular pathways

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Figure 1. von Willebrand factor (VWF) controls angiogenesis and vascular maturation through multiple pathways: model. VWF modulates endothelial proliferation, migration and angiogenesis through intracellular and extracellular endothelial pathways, which converge to control vascular endothelial growth factor receptor (VEGFR)-2 signalling. Intracellular pathway: within endothelial cells (EC), VWF is essential for the formation of WPB, organelles which store the growth factor Angiopoietin (Ang)-2. Decrease in intracellular VWF results in increased release of Ang-2 from the EC. Upon release, Ang-2 can bind to the tyrosine kinase receptor Tie-2 and trigger signals which synergize with VEGFR-2 signaling to destabilize blood vessels and promote angiogenesis. Extracellular pathway: VWF, either in plasma or released into the subendothelial space, can interact with integrin αvβ3, a heterodimeric adhesion receptor with multiple ligands, involved in angiogenesis and vascular homeostasis. In EC, αvβ3 integrin modulates VEGFR-2 activity and downstream signaling. On the EC surface, VWF stabilizes integrin αvβ3 expression. Loss of VWF in EC results in decreased αvβ3 expression, which may causes over-sensitivity to VEGF/ VEGFR-2 signalling, leading to formation of disrupted and immature blood vessels, similar to those described in angiodysplasia. Finally, during vascular development, expression of αvβ3 can also be upregulated in vascular smooth muscle cells (VSMC). VWF binding to αvβ3 on VSMC is required for their recruitment, thus promoting arterial maturation during vascular development. Thus lack of VWF may result in defective vascular maturation also because of reduced VSMC recruitment.

are likely to be involved in the regulation of angiogenesis by VWF. So far, the evidence points to VWF modulating angiogenesis through extracellular and intracellular pathways, schematically summarized in Figure 1. VWF control of angiogenesis: extracellular pathways In vitro, plasma-derived VWF inhibits endothelial tube formation in a basic model of angiogenesis [12], indicating the existence of an extracellular pathway. On endothelial cells, VWF binds to integrin αvβ3 [19], a heterodimeric adhesion receptor with multiple ligands, which plays a critical but complex role in angiogenesis and vascular homeostasis [20,21]. Pharmacological inhibition of αvβ3 inhibits angiogenesis in experimental models; however, genetic β3 deficiency results in enhanced angiogenesis in vivo. αvβ3 appears to exert a bimodal effect on angiogenesis, both as activator and inhibitor, playing different roles possibly depending on phases of angiogenesis, different ECM ligands, crosstalk and/or interaction with other receptors (reviewed in [21]). Multiple pathways downstream of αvβ3 link this receptor to regulation of gene expression and crucially to vascular endothelial growth factor receptor-2 (VEGFR)-2 signaling. A complex, reciprocal relationship exists between VEGFR-2 and αvβ3

integrin (reviewed in [22]). VEGFR2–αvβ3 integrin association is important for full VEGFR-2 activity and activation of downstream signalling [23]; however, lack of endothelial β3 causes over-sensitivity to VEGF leading to immature blood vessels [24]. As well as binding to αvβ3, VWF controls its expression by stabilizing its surface levels [12]. The effect of VWF on the pathways above is unknown and requires further investigation. Interestingly, VWF has been shown to interact with αvβ3 also on vascular smooth muscle cells (VSMC), in a pathway which involves Notch signaling [18]. This process has been proposed to promote arterial maturation during vascular development, thus providing a further mechanism through which VWF may modulate blood vessel formation. The observation that vascular malformations are most frequent in patients with AVWS and with type 2A VWD [5] suggests that VWF high MW multimers, which are crucial for haemostasis, may also be important for the control of angiogenesis, perhaps by enhancing VWF binding to EC. VWF control of angiogenesis: intracellular pathways VWF drives the formation of WPB, the endothelial storage organelles which contain multiple proteins, including the angiogenesis regulator as Angiopoietin-2 (Ang-2) [13,25]. In vitro



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studies on VWF-deficient EC (siRNA-treated or BOEC from type 3 VWD) show that VWF regulates the endothelial storage and release of Ang-2 [12,26]. Interestingly this is not a generalised effect on all WPB proteins, as IL-8 release is not regulated by VWF (Starke and Randi, unpublished). VWF also controls Ang-2 synthesis: mRNA levels of Ang-2 are increased in EC treated with VWF siRNA and in BOEC from a type 3 VWD patient [26] and recently, Yuan et al have shown an increase in Ang-2 mRNA and protein levels in hearts of VWF–deficient mice [27]. Interestingly, this pathway appears to be tissue-specific, at least in mouse, since Ang-2 levels in lung or liver of VWF-deficient mice were unchanged [27]. Ang-2 is part of the Angiopoietins/Tie-2 pathway, a crucial system regulating vascular homeostasis and angiogenesis [28]. Ang-2 has been shown to destabilize blood vessels and synergize with VEGF to promote angiogenesis [29,30]. Recently, an association between Ang-2 and sporadic angiodysplasia has been identified, with raised Ang-2 levels in plasma and tissues [31]. Thus it is possible that significant disruption of WPB storage in endothelial cells, as seen in BOEC from type 3 or severe type 1 VWD [26], may result in local release of Ang-2 which could act to destabilize small blood vessels, in synergy with pro-angiogenic and/or pro-inflammatory signals. This model is supported by the recent report by the Aird group [27], who found Ang-2 levels increased in hotspots in the heart of VWF-deficient mice, accompanied by significant microvascular damage of capillaries in the heart. These findings were associated with abnormal cardiac function, apparently unrelated to any focal microhemorrhaging or enhanced plasma extravasation. These data suggest that VWF’s control of Ang-2 storage and expression, possibly in a tissue specific manner, affects other endothelial pathways besides angiogenesis, and is essential for endothelial homeostasis and vascular functionality. VWF control of angiogenesis via VEGFR-2 signalling Crucially, both extracellular and intracellular pathways described above converge on one of the major regulators of blood vessels growth and stability, namely VEGFR-2 signaling. Indeed, VWF deficiency results in enhanced VEGFR-2-dependent endothelial migration and proliferation [12], suggesting that VWF controls angiogenesis by inhibiting signalling through VEGFR-2. Excessive VEGF signalling is known to cause formation of unstable, fragile and leaky vessels [32], similar to angiodysplastic lesions; a role for increased VEGF signalling has in fact been proposed in angiodysplasia [33]. Multiple pathways act downstream of VEGFR-2 to control angiogenesis [34]; future studies will show which of these are modulated by VWF and the functional relevance of these networks in VWF’s control of angiogenesis. Importantly, VEGF signaling controls many vascular functions besides angiogenesis, including EC and cardiomiocyte survival, myocardial blood flow and hemodynamics [35], indicating another possible pathway underlying the abnormal cardiac function described by Yuan et al in VWF-deficient mice [27]. Angiogenic pathways in VWD From this brief summary, it is clear that VWF has the potential of influencing multiple important pathways involved in angiogenesis and blood vessel stability. In which circumstances VWF plays a role and how these pathways are affected are clearly important questions. According to the intracellular vs extracellular models, it is possible to speculate that in VWD patients with disrupted endothelial WPB, such as seen in BOEC from type 3 and severe type 1 patients, the Ang-2 pathway may be predominant [17,36]. However in patients with dysfunctional VWF, such as type 2 VWD, WPB appear mostly normal [17,36]; in these patients therefore the interaction of VWF with cell surface receptors and/or other extracellular protein may be more relevant.

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More studies on VWD BOEC will help to shed some light on these questions, which could in the future help to optimize the treatment of patients with angiodysplasia and intractable GI bleeding. Of note, this brief review does not take into account the role that platelets and leukocytes, recruited by VWF, may also play in the process; their relevance is still to be investigated. Conclusions and future directions The identification of a role for VWF in the control of blood vessel formation adds to the increasing web of functions and interactions that this large and fascinating protein can engage in. The recent findings of abnormal cardiac function and microvascular damage in VWF-deficient mice raises the possibility of further vascular roles, controlled by pathways which may crossover with angiogenesis. Although clinical evidence from nearly a century and innumerable studies in patients and models clearly show that haemostasis is the key role of VWF, novel functions being identified, including the control of angiogenesis, may provide a new prospective in the understanding and ultimately care of patients with VWD. In the near future, the characterization of new molecular pathways controlled by VWF and their link to angiodysplasia in VWD could provide novel candidate targets for the treatment of this unresolved clinical issue. Conflicts of interest None. References [1] Lillicrap D. von Willebrand disease: advances in pathogenetic understanding, diagnosis, and therapy. Blood 2013;122:3735–40. [2] Shetty S, Kasatkar P, Ghosh K. Pathophysiology of acquired von Willebrand disease: a concise review. Eur J Haematol 2011;87:99–106. [3] Uriel N, Pak SW, Jorde UP, Jude B, Susen S, Vincentelli A, et al. Acquired von Willebrand syndrome after continuous-flow mechanical device support contributes to a high prevalence of bleeding during long-term support and at the time of transplantation. J Am Coll Cardiol 2010;56:1207–13. [4] Abshire TC, Federici AB, Alvarez MT, Bowen J, Carcao MD, Cox GJ, et al. Prophylaxis in severe forms of von Willebrand’s disease: results from the von Willebrand Disease Prophylaxis Network (VWD PN). Haemophilia 2013;19:76–81. [5] Castaman G, Federici AB, Tosetto A, La MS, Stufano F, Mannucci PM, et al. Different bleeding risk in type 2A and 2M von Willebrand disease: a 2-year prospective study in 107 patients. J Thromb Haemost 2012;10:632–8. [6] Makris M. Gastrointestinal bleeding in von Willebrand disease. Thromb Res 2006;118 Suppl 1:S13–S17. [7] Warkentin TE, Moore JC, Anand SS, Lonn EM, Morgan DG. Gastrointestinal bleeding, angiodysplasia, cardiovascular disease, and acquired von Willebrand syndrome. Transfus Med Rev 2003;17:272–86. [8] Bauditz J, Lochs H. Angiogenesis and vascular malformations: antiangiogenic drugs for treatment of gastrointestinal bleeding. World J Gastroenterol 2007;13:5979–84. [9] Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of angiogenesis. Cell 2011;146:873–87. [10] Quick AJ. Telangiectasia: its relationship to the Minot-von Willebrand syndrome. Am J Med Sci 1967;254:585–601. [11] Koscielny JK, Latza R, Mursdorf S, Mrowietz C, Kiesewetter H, Wenzel E, et al. Capillary microscopic and rheological dimensions for the diagnosis of von Willebrand disease in comparison to other haemorrhagic diatheses. Thromb Haemost 2000;84:981–8. [12] Starke RD, Ferraro F, Paschalaki KE, Dryden NH, McKinnon TA, Sutton RE, et al. Endothelial von Willebrand factor regulates angiogenesis. Blood 2011;117:1071–80. [13] Metcalf DJ, Nightingale TD, Zenner HL, Lui-Roberts WW, Cutler DF. Formation and function of Weibel-Palade bodies. J Cell Sci 2008;121:19–27. [14] Wang JW, Groeneveld DJ, Cosemans G, Dirven RJ, Valentijn KM, Voorberg J, et al. Biogenesis of Weibel-Palade bodies in von Willebrand’s disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation. Haematologica 2012;97:859–66. [15] Stockschlaeder M, Schneppenheim R, Budde U. Update on von Willebrand factor multimers: focus on high-molecular-weight multimers and their role in hemostasis. Blood Coagul Fibrinolysis 2014;25:206–16. [16] Lenting PJ, Casari C, Christophe OD, Denis CV. von Willebrand factor: the old, the new and the unknown. J Thromb Haemost 2012;10:2428–37.

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[17] Starke RD, Paschalaki KE, Dyer CE, Harrison-Lavoie KJ, Cutler JA, McKinnon TA, et al. Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells. Blood 2013;121:2773–84. [18] Scheppke L, Murphy EA, Zarpellon A, Hofmann JJ, Merkulova A, Shields DJ, et al. Notch promotes vascular maturation by inducing integrin-mediated smooth muscle cell adhesion to the endothelial basement membrane. Blood 2012;119:2149–58. [19] Huang J, Roth R, Heuser JE, Sadler JE. Integrin alpha(v)beta(3) on human endothelial cells binds von Willebrand factor strings under fluid shear stress. Blood 2009;113:1589–97. [20] Brooks PC, Montgomery AM, Rosenfeld M, Reisfeld RA, Hu T, Klier G, et al. Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994;79:1157–64. [21] Hodivala-Dilke K. alphavbeta3 integrin and angiogenesis: a moody integrin in a changing environment. Curr Opin Cell Biol 2008;20:514–9. [22] Somanath PR, Malinin NL, Byzova TV. Cooperation between integrin alphavbeta3 and VEGFR2 in angiogenesis. Angiogenesis 2009;12:177–85. [23] Mahabeleshwar GH, Chen J, Feng W, Somanath PR, Razorenova OV, Byzova TV. Integrin affinity modulation in angiogenesis. Cell Cycle 2008;7:335–47. [24] Weis SM, Lindquist JN, Barnes LA, Lutu-Fuga KM, Cui J, Wood MR, et al. Cooperation between VEGF and beta3 integrin during cardiac vascular development. Blood 2007;109:1962–70. [25] Fiedler U, Scharpfenecker M, Koidl S, Hegen A, Grunow V, Schmidt JM, et al. The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies. Blood 2004;103:4150–6. [26] Starke RD, Paschalaki KE, Dyer CE, Harrison-Lavoie KJ, Cutler DF, McKinnon TA, et al. Defective angiopoietin-2 release from von Willebrand disease patients’ blood outgrowth endothelial cells. J.Thromb.Haemost. 11. 2013.

[27] Yuan L, Chan GC, Beeler D, Janes L, Spokes KC, Dharaneeswaran H, et al. A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity. Nat Commun 2016;7:10160. [28] Thomas M, Augustin HG. The role of the Angiopoietins in vascular morphogenesis. Angiogenesis 2009;12:125–37. [29] Daly C, Eichten A, Castanaro C, Pasnikowski E, Adler A, Lalani AS, et al. Angiopoietin-2 functions as a Tie2 agonist in tumor models, where it limits the effects of VEGF inhibition. Cancer Res 2013;73:108–18. [30] Felcht M, Luck R, Schering A, Seidel P, Srivastava K, Hu J, et al. Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling. J Clin Invest 2012;122:1991–2005. [31] Holleran G, Hall B, O’Regan M, Smith S, McNamara D. Expression of Angiogenic Factors in Patients With Sporadic Small Bowel Angiodysplasia. J Clin Gastroenterol 2015;49:831–6. [32] Reginato S, Gianni-Barrera R, Banfi A. Taming of the wild vessel: promoting vessel stabilization for safe therapeutic angiogenesis. Biochem Soc Trans 2011;39:1654–8. [33] Junquera F, Saperas E, de T, I, Vidal MT, Malagelada JR. Increased expression of angiogenic factors in human colonic angiodysplasia. Am J Gastroenterol 1999;94:1070–6. [34] Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling in control of vascular function. Nat Rev Mol Cell Biol 2006;7:359–71. [35] Taimeh Z, Loughran J, Birks EJ, Bolli R. Vascular endothelial growth factor in heart failure. Nat Rev Cardiol 2013;10:519–30. [36] Wang JW, Bouwens EA, Pintao MC, Voorberg J, Safdar H, Valentijn KM, et al. Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease. Blood 2013;121:2762–72.