Von Willebrand Factor and Angiogenesis

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This article isprotected Allrights bycopyright. reserved. doi: 10.1111/jth.13551 thisversionandthe between lead todifferences been through the copyediting, typesetting, and proofreading which may pagination process, forpublicati accepted This articlehasbeen evidence supporting a role for VWF in blood vessel formation, the link between VWF light on thisnovel and fascinating area ofvascul these will yieldfurther evidence on the molecular pathways controlled by VWFandshed the peripheralin blood ofnormalcontrolsand patientswithnextIn the VWD. years, few pathways hasbeen greatly facilitated by the ab and extracellular, although their relative import indicate that VWF can regulate angiogenesis causemalformations can severegastrointestinal (GI)bleeding. observations that insome patients with results in enhanced, possibly dysfunctional angiogenesis is consistent with the clinical and binding partners have been identified for VWF. The finding that loss ofVWFin EC synthesized in megakaryocytes andendothelial cells (EC). In recent years, novel functions a key component of haemostasis, capturing platelets atsites of endothelial damage and opened a novel perspective onthe function of The recent discovery that von Willebrand factor (VWF) regulates blood vessel formation has ABSTRACT (250) Corresponding author email id: • • • • • Keywords (5) London, UK 1 National Heart and Lung Institute and 2 Department of Haematology, Imperial College Anna M. Randi AUTHORS Von Willebrand factor and angiogenesis: basic and applied issues TITLE Article type :ReviewArticle Date:24-Sep-2016 Accepted Revised Date :30-Apr-2016 Received Date Accepted Article Angiopoietin-2 VEGF Receptor 2 Endothelial cells Angiodysplasia Hemostasis : 05-Sep-2016 :05-Sep-2016 1 and Mike A.Laffan [email protected] 2

on and undergone full peer review buthasnot review undergone fullpeer on and Von Willebrand disease (VWD), vascular through multiple pathways, both intracellular this complex protein. VWF was discovered as ility toisolate ECfrom progenitors circulating Version of Record. PleaseVersion cite thisarticle as ar biology.Inthisarticle, we will review the ance isstill unclear.

In vitro Investigation of theseInvestigation of and in vivo studies This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Acceptedtherefore their deficiency isincompatible with li Of themany regulators ofangiogenesis, some destabilization. development starts and ends with astable blood vessel, through anintermediate phase of used [10, 11]. This may bepartly related to the circular nature ofthe process, which post- have opposite effects, namely anti- or pro-angiogenic, depending on the experimental model intracellular and extracellular fa continue to be identified, suggesting that this process is very sensitive tomodulation by regulators ofangiogenesis bothendogenous and pharmacological have beendescribed and physiological processes such as the menstrual cycle, tissue repairand wound healing. Many development. In the adult, the formation of new blood vessels is restricted to specific concert to produce astable blood vessel (rev in [9]). Angiogenesis is essential for embryonic Angiogenesis isacomplex, multistep process effective in the management of bleeding. cases where replacement therapy, the traditional choice for coagulation disorders, is not have significant implications for the treatment of understanding thepossible roles ofhaemostatic proteins inblood vessel formation may result in bleeding. Given the number of human diseases caused by defects incoagulation, establishing aspecific readout, since vascular disruption and defective haemostasis both challenge in defining the roleof haemostatic pr XIII and Factor Vcontrol the of formation bloo Articledemonstrated that several haemostatic proteins ability to investigate the two processe development or haemostasis often result in both vascular defects and bleeding [1]. The development. Indeed often co-localised in time andspace, such as during wound healing orembryonic should these areco-operate two processes that makes sense physiologically, they since the clear overlaps between the two been reco Historically, these two disciplines have been in haemostasis, i.e. the ability tocontrol bleeding by forming clots at sites ofvascular injury. angiogenesis, i.e. the formation of new bl Blood vesselscontrol many biological processes that areessential forlife. Two oftheseare INTRODUCTION bleeding refractory to VWF replacement therapy in VWD. related. Finally, we will discuss how these findin dysfunction and vascular malformations causing GI bleeding and how they may becausally 2. 1.

Angiogenesis: basic concepts in health and disease disease and health in concepts basic Angiogenesis: haemostasis and Angiogenesis in vivo studies show that mutation ofgenes essential for vascular ctors. To add to the complexity, the same factors or drugs may ordrugs same the factors complexity, to the ctors. Toadd s through ever moresophisticated vestigated separately, and only recently have d d Howevervessels [2-8]. in humans a major ood vessels from pre-existing ones, and ones, pre-existing from vessels ood are essential for allormost blood vesselsand fe. Examples ofthese are growth factors ofthe gspoint tonoveltherapeutic approaches to which involves numerous pathways acting in such as tissue factor, prothrombin, Factor gnised. Fromafunctional perspective, it oteins inangiogenesis patients. This is particularly relevant in

in vivo is the difficulty in models has This article is protected by copyright. All rights reserved. copyright. This articleis protectedby sequence involved in binding to AcceptedADAMTS13, which controls VWF’smultimeric (A1), heparin(A1) andcollagen (A1 and3). The A2 domaincontains the cleavage site for A central domain toFVIII; the bind domains D'D3 monomers areassembled into largemultimers of up to 20 000kDa. At the N terminus, the of VWF have beenThe VWFmonomer is 220 KDa,and contains multiple domains towhich the many functions mapped [20, 21]. By a process of C- and then N- terminal linking, the three pools: endothelial in VIII.VWFissynthesised Factor secondary haemostasis, as amediator of platelet adhesion and as carrier for coagulation VWF isamultifunctional glycoprotein best vessels [19]. stabilization during angiogenesis may result in excessive, unstable and dysfunctional new angiodysplastic lesions are poorly understood, but aloss of balance between proliferation and of anemia inadults [18].The molecular mechanisms underlying the formation of the most commoncause of gastrointestinal obscure Articlegast the in [17]. malformations Vascular bleeding trivial to life-threatening or disabling problems as aresult of location, pain, tissue damage and lesions ofblood vesselsthat occur because of engineering approach. A lessinvestigated area is goal in diseases characterized by tissue isch efficacy in a number of diseases. Conversely, the ability to promote neo-vascularization is the abnormal angiogenesis[16]. Thus anti-angiogenic chronic inflammatory diseases, ocular diseases, diabetes and obesity involve enhanced, often Numerous diseasesare associatedwith incr healing defects [15]. orcoagulatio [14], vitreoretinopathy exudative network. Examples of these are mutations in avascular of absence complete than rather malformations in results defect or deficiency appear to be partly dispensable and possibly act in a tissue-restricted manner, so that their [13]. Many other proteins however, which modulate the process of new vessel formation, VEGF family [12] and transcriptional master re • • • 3.

surface receptors. of the extracellular matrix (ECM) and endothelial and vascular smooth muscle cell Subendothelial VWF:arising fromabluminal release fromECand bound to molecules EC release. Plasma VWF:circulating as alarge globular protein andderived almost entirely from and in alpha granules respectively and released upon stimulation. Cellular VWF:present in vascular EC andinplatelets, stored inWeibel Palade bodies Von Willebrand factor in haemostasis β 3 integrins. Both the multimeric size and the conformation cells (EC) and megakaryocytes and is present in ispresent and megakaryocytes cells (EC)and known foritsessential roles inprimaryand emia, and isa major component of any tissue eased angiogenesis: aswelltumors,many the WNT receptor FZD4 which cause familial n factor XIII deficiency which can cause wound wound cause can which XIIIdeficiency n factor defects during angiogenesis. Theserange from gulators of endothelial lineage differentiation size; theC4 domaincontains theRGD s are involved in binding to platelet GP 1b GP platelet to in s areinvolved binding that of vascular malformations, i.e. localized agents have shown variouslevels of clinical rointestinal tract, called angiodysplasia, are bleeding, which is the most frequent cause cause frequent most the is which bleeding,

This article is protected by copyright. All rights reserved. copyright. This articleis protectedby raising thepossibility that VWF Acceptedlarge number of vasoactive molecules. Many of these have been shown to bind to VWF, on the synthesis of VWF, butanalysis ofWPB contents has shown that they also contain a other biological processes (rev in[35]). The itsBesides well-characterised role in haemostasi also been reported in patients with VWD [33, 34]. a leftventricular device [32]. from angiodysplasia has been reported to present as soon as 11 days following placement of angiogenesis, leading to the production of fr VWD [29, 31]. Asnoted above, these are angiodysplastic lesions in the GI tract and this has been reported in 2-4% of patients with 2A VWD [27] and in AVWS [30]. GI bleeding has also been linked to the presence of particularly common when there is a deficiency severe andrequire repeated blood transfusions. Bleeding from this site seems to be Up to20% ofpatients with VWD present with gastrointestinal (GI) bleeding [29]; this can be involved. in gastrointestinal (GI)bleed VWF-containing concentrates, but a number of reports have shown this to beless effective in[26]). In hereditary VWD, bleeding is generally controlled by replacement therapy with (rev (LVAD) devices assist ventricular left with patients in and valvestenosis aortic disorders, clinical conditions such monoclonal gammopathy, myeloproliferative andmalignant Articlesyndrome or AVWS), due to dysfunction or degradation of VWF, seen in association with described. Deficiency of VW based on differences and degree of quantitative and qualitative defects has been thoroughly common inherited bleeding disorder in humans (rev in [24, 25]). The classification of VWD, Congenital decreaseordysfunction ofVWFca and becoming susceptible to ADAMTS13 cleavage [23]. process while it remains tethered to the EC, mediating platelet adhesion to the endothelium vascular injury,VWFisreleasedfrom the e ADAMTS13, which determines the size range of Met1606 bond inthe A2domainfacilitatin GPIb for site binding to surfaces such as collagen and the extracellular matrix exposes the buried binding conformation that does not bind to its main platelet receptor GPIb of VWFdetermine itsplatelet binding activity 5. 4.

The multiple roles of von Willebrand Factor in the vasculature Willebrand syndrome Vascular abnormalities in patients von Willebrand disease & Acquired von α , localized in theVWFA1 domain. Unfo

ing ([27, 28]), suggesting that Interestingly, vascular malformations outside theGI tract have F function can also be acquired (acquired von Willebrand plays a role in directing andregulatingplays arolein theiractionsafter ndothelium andcan undergo asimilar unraveling formation of WPBin ECisabsolutely dependent uses VonWillebrand disease (VWD),themost agilevessels prone tobleeding[19].Bleeding . Inplasma,VWFadopts afoldedglobular thought to develop due to dysregulated g itscleavagebythe metalloproteinase circulating VWF multimers [22]. Following s, VWFhas increasingly been implicated in of the larger VWF multimers, asin type lding of VWF also exposes the Tyr1605-lding of VWFalsoexposesthe

additional mechanisms may be α . Fluid shear stress or This article is protected by copyright. All rights reserved. copyright. This articleis protectedby activation of downstream signaling Acceptedand Figure 2). VEGFR2– complex, reciprocal relationship exists between VEGFR-2 and and cruciallyto vascular endothelial growth Multiple pathways downstream of interaction with other receptors (revin [48]). possibly depending on phases of angiogenesis, different ECM ligands, crosstalk and/or bimodal effect on angiogenesis, both as activator and inhibitor, playing different roles genetic Pharmacological inhibition of a criticalbutcomplex roleinangiogen integrin angiogenesis [41], indicating the existence of In vitro pathways extracellular angiogenesis: of VWF control 6.1 VWF mayregulate angiogenesis isshown in Figure 1. extracellular and intracellular pathways. Amo to far, theevidencepoints So VWF. multiple molecular pathways arelikely tobein Because of the numerous molecular interactions and functions of VWF in the vasculature, deficient mouse, in several physiological and pathological models [41, 43]. [41, 42]. Articlethe cellular phenotypes have been observed depending on the individual molecular defect blood outgrowth endothelial cells (BOEC) frompatients with VWD although differences in proliferation, migration and expression in human umbilical vein EC(H vascular malformation inpatients with decrea angiogenesis [41], which has provided the first plausible explanation for the presence of Our contribution to this fieldhas been the identification of VWF as aregulator of haemostasis. of control the beyond emerging, indicating that VWFmaybeinvolvedin being identified, acomplex network ofinteract reviewed elsewhere [35]. Thus as novel binding partners and novel functions of VWF are inflammatory pathologies andstroke [38-40]. Themultiple functions of VWFhave been regulation of vascular permeability inthe brain,with possible relevance forbrain control of vascular inflammation andleukocyte recruitment [36], metastasis [37]and in the release with consequent effects onangiogenesis. 6.

Von Willebrand Factor regulates angiogenesis. angiogenesis. regulates Factor Willebrand Von , plasma-derived VWFinhibits endothelial tube formation inabasicmodelof β α In vivo 3 deficiency results in enhanced angiogenesis v β 3 [44, 45], a heterodimeric adhesion receptor with multiple ligands, which plays , angiogenesis and vascular density were found to be increased in the VWF ανβ 3 integrin association isimport in vitro α v β 3 inhibits angiogenesis inexperimental models; however, angiogenesis [41]. Asimilar overall pattern was found in α v [50]. However, lack ofendothelial β 3 link this receptor to regulation of gene expression VWF modulating angiogenesis through both an extracellularpathway. del ofthe molecular pathways through which esis andvascular homeostasis [46, 47]. se ordysfunction ofVWF. Inhibition of VWF UVEC) usingsiRNA resu factor receptor-2 (VEGFR)-2 signaling. A volved inthe regulation ofangiogenesis by thepathogenesis of vascular diseaseswell In vivo ions with multiple vascular pathways is in vivo

studies haveimplicated VWF in the ant for full VEGFR-2 activity and ανβ . Thus . Thus 3 integrin inEC(rev[49] α v β VWFbinds to EC via 3 appears to exert a lted inincreased β 3 causes over- This article is protected by copyright. All rights reserved. copyright. This articleis protectedby enhanced Ang-2 mRNA levels. Moreover, BOECAccepted froma patient with type 3 VWD and (Smith and Randi, unpublished) and in heart tissue from VWF-deficient mice [60] results in Randi, unpublished). VWF also controls Ang-2 synthesis: loss ofVWF effect on allWPB proteins, asIL-8release is increased release of Ang-2 in the cellsupernatan storage and release of Ang-2: inhibition of promote angiogenesis [59]. angiogenesis [58]. Ang-2 can act to destabilize blood vessels and synergise with VEGF to Angiopoietins/Tie-2 the system acrucial of pathway, regulating vascular and homeostasis proteins, including the angiogenesis regulator Angiopoietin-2 (Ang-2) [56, 57]. Ang-2 is part VWF drives the formation ofWPB, the endothelial storage organelles which contain multiple pathways intracellular angiogenesis: of VWF control 6.2 physiological blood vessel growth and stabilization. VWF multimers, maycontribute angiogenes molecules to their receptors. [55] It is also conceivable that platelets, recruited by HMW angiogenesis; itis possible that the larger receptors. Moreover, VWFcaninteract example by clustering of between VWF and its endothelial receptors, thus potentiating the signaling effect– for this are presently unknown. It is possible that HMW multimers mayenhance the interaction crucial for haemostasis, may alsobecritical for the control of angiogenesis. Thereasons for with type 2AVWD suggests that VWFhigh molecular weight multimers (HMW), which are The observation that vascular malformations are most frequent in patients with AVWS and Article ofstages bloodvessel development. evidence that VWF is required for physiological interact with sub-endothelial VWFleading tovessel maturation [54]. These dataadd to the smooth muscle cells(VSMC)leads deficient mice. In anelegant study, they showed that Notch signaling from EC tovascular development, Scheppke etal reported that arterial maturation was delayed in VWF- development, namely arterial maturation. Using a mouse model of retina vascular Interestingly, the interaction of VWF with on the pathways above is unknown and requires further investigation. surface expression by preventing its internalization [41]. Theconsequent neteffect ofVWF VWF normally controls angiogenesis by inhibiting VEGFR2 signaling. VWF also controls enhanced VEGFR2-dependent endothelial migration and proliferation [41], suggesting that in proposed angiodysplasia been has signaling vessels [47,51],similar toangiodysplastic sensitivity to VEGF and increased VEGFR2si α v β In vitro 3 and/or mediating crosslinki to expression of integrin studiesshow that VWFregulates theendothelial α not increased in VWF-deficient cells (Starke and v multimers mayenhance the affinity for these β3 VWF expression results in VWF in WPBwith results loss of expression lesions: indeed, arole for increasedVEGF gnalingimmature,leading to fragileblood with circulating molecules which affect may alsoaffect another aspect of vascular [52,53]. VWFdeficiency alsoresultsin angiogenesis, possibly acting atmultiple is regulatorswhich are essential for t [41]. Interestingly, this is not ageneral

α v β ng tomultiple cellsurface 3 inVSMC. Thiswould then in vitro inHUVEC ανβ 3 This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted themselves. lesions ineffective and so attention has focused onways toinduce regression ofthe angiodysplastic treatments. Moreover, for those patients with dependent on frequent redcell transfusions despite conventional local andsystemic important forhemostasis intheGI tract. Thus asignificant proportion of patients remain [27]. It is also possible that the lack ofplatelet, EC andsubendothelial VWF is particularly effective than for bleeding atother sites and that higher doses ofconcentrate are required therefore, published reports indicate that are alsoreduced bycleavage within a few minutes ofinfusion [67]. has not been previously exposed to ADAMTS13 contains ultra-large multimers, but these intermittent infusions due to the persistent action of ADAMTS13. Recombinant VWF, which haemostasis atthis site andclearly it is difficult to sustain high levels ofHMW using excess ofsurgical approaches of limited efficacy but also due to the GI nature of the lesions. The relative bleedingwidespread and recurrent nature of the angiodysplastic lesions which makes local or in typerecognized as adifficult and sometimes intractable problem. This is inpart due to the often 2A comparedIntestinal blood loss from angiodysplasia in patients with VWD has been frequently to 2M points to an importancemalformations in platelet dysfunction would be important. syndrome[65]. of HMW in been reported inpatients with Glanzmann thrombasthenia [66] and Bernard-Soulier Articlemajor platelet function defects orofthrombocytopenia [65] VWD may contribute to angiodysplasia. Angiodysplastic lesions are not afeature of the protective effect occurs in humans [64]. It is also possible that failure of this mechanism in atherosclerosis was noted in the VWF-null mouse [63], but it is not clear that any such and healing and also tobeone ofthe first steps inatherogenesis. Areduction in numerous vasoactivemediators. Theseare thought to be important in vesselwall repair of capture and subsequent binding interactions results in platelet activation with release of be bound onto the surface of endothelial cells or to the subendothelial matrix. The process and inflammation, especially under conditions of high shear stress. The platelets may then VWF has awell-established role inmediating platelet capture at sites of vascular damage 6.3 angiodysplasia has been identified, with rais patients, remains to be determined. Recently, an association between Ang-2 and sporadic VWF inexperimental models,and cruciallyin [61]. Whether Ang-2 plays aroleintheenhanced, disrupted angiogenesis caused by lack of complete lack of endothelial VWF synthesis al

7. VWF-dependent platelet adhesion: potential role inangiogenesis

Treatment of angiodysplasia in VWD in angiodysplasia of Treatment

Detailed reviews of the clinical data ed Ang-2 levels in plasma and tissues [62]. replacement therapy for GI bleeding isless so Ang-2 release synthesis and enhanced show the pathogenesis ofangiodysplasia inVWD AVWS,replacement therapy isinevitably possibly associated with vascular associated with possibly

; however angiodysplasia has

Not surprisinglyNot This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted novel candidate targets for the treatment of this unresolved clinical issue. of VWF and angiodysplasia should contribute very valuable tools in the development of generation of animal models to investigatethe understanding and ultimately the care of patients with VWD. In the near future, ththe other functions, including themodulation of angiogenesis, may provide new prospects for patients and models clearly show that the key role of plasma VWFis to control haemostasis, can engagein. Although clinical evidence from increasing web ofroles andfunctional interactions that this large and fascinating protein The identification ofarole forVWFinthe control ofblood vesselformation adds tothe Conclusions and future directions sensitive to growth factor imbalance, as described in tissue samples from patients. found. However, cellular models ofangiogenesis could bemodified to make themmore complexity, including the mechanical stress, of the environment where these lesions are be used as surrogate.possible to develop amouse model where analysis ofvascular malformations in the skin can patients arenot uniquely localised in thegut but arealso present inthe nail bed,it It may be is difficult GI on the models relevant of absence the to envisageappropriate treatments. However, nospecific an The development of models ofangiodysplasia would undoubtedly helpthe development of useful in patients with haemostatic defects. Articlebut GI and nasal bleeding was afrequent complication, indicating that they may not be (angiostatin, endostatin). These agents were developed for use in patients with malignancy, including anti-VEGF and anti-VEGFR antibodies, VEGF-trap andsmall molecule inhibitors subjected to arandomised trial. Anumber ofanti-VEGF, strategies have been developed, but regression of angiodysplasia has not been directly demonstrated and it has not been angiogenic effects athigher doses [72, 73]. The reduction in bleeding has beensignificant, recently, success has been reported with atorvastatin which is also known to have anti- bleeding, but its use is frequently limited by the development of neuropathy. Most angiodysplastic VWD-associated in reported alsobeen have VEGF[71].plasma Responses in areduction wasassociated arate of compared 4%to with therapy. Response iron from a response rate of71% inpatients with refractory bleeding fromvascular malformations properties, interestingly mediated by suppression VWD and angiodysplasia [70]. patients with HHT [69]. It has subsequently been reported to also benefit patients with shown inadouble-blind randomised and placebo-controlled trial toreduce epistaxis in not reproduced incontrolled trials [19, 68]. However, Tamoxifen (an anti-oestrogen) was Early reports of success in treating angiodysplasia with oestrogens and progesterones were

Thalidomide is an agent with

system, since vascular malformationssystem, since in VWD vascular e molecular links between lack or dysfunction in vivo nearly acentury and innumerable studies in ofVEGF,and inarandomised trialyielded model isdescribed in the literature. In in vitro

modelthat could capture the establishedanti-angiogenic This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Ref [49]. in found of the effects of VWF on this signalling path capable of inhibit VEGFR2 activity, possibly based onits ligand occupancy. This might be one over-sensitivity to VEGF and increased VEGFR2 signaling, suggesting that outside-in further and signalling triggers This in turn results inconfor of domain cytoplasmic the phosphorylate which c-src, VEGFR2 by VEGF results inVEGFR2 phosphorylation and recruitment of adaptors including important forfull VEGFR-2 activity andactivation ofdownstream signaling. Activation of between VEGFR2 and of The schematic model co showsasimplified the endothelium. the Figure 2:Functional cross-talk between VEGFReceptor-2 (VEGFR2) and integrin multiple pathways, involving both ECand VSMC. development. Therefore, loss ofVWFresults in disrupted blood vessel formation through is required for their recruitment, thus prom can be upregulated in vascular smooth muscle cells (VSMC). VWFbinding to signaling. downstream and EC, In ECsurface. expression the its on stabilizes ligands, involved in angiogenesis and VWF caninteract with integrin Articlewith VEGFR-2 signaling to destabilize blood its storage and inhibiting its synthesis. Ang-2 is released upon EC activation and synergizes which store the growth factor Angiopoietin (Ang factor receptor (VEGFR)-2 signalling.VWF is and angiogenesis through pathways which converge tocontrol vascular endothelial growth panel: Left through multiple pathways: model Figure 1:von Willebrand factor (VWF)controls angiogenesis and vascular maturation Figure legends A. Randi reports grants from LFB. personal fees from Octapharma, outside the submitted work. M. A.Laffan reports personal fees from LFB, Disclosure within endothelial cells (EC),VWFregulates endothelial proliferation, migration α v β 3. As discussed in the text, VEGFR2– text, the in 3. Asdiscussed Right panel: Right mational changes and increase inligand binding affinity, which α v β 3, aheterodimeric adhesion receptor with multiple vascular homeostasis. VWF binding to to VWF binding homeostasis. vascular during vascular development, expression of activation. However, lack ofendothelial grants and personal fees from CSLBehring, and essentialorganelles fortheformationWPB, of vessels and promote angiogenesis. Moreover, ways. Adetailedreview on this topic can be

)-2. VWFcontrols Ang-2 levels bypromoting oting arterial maturation during vascular ανβ mplex cross-talk and functional interaction 3 integrin canrepress VEGFR-2activity α v

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