Angiogenesis (2015) 18:13–22 DOI 10.1007/s10456-014-9443-4

ORIGINAL PAPER

ADAM10 and ADAM17 have opposite roles during sprouting angiogenesis V. Caolo • G. Swennen • A. Chalaris • A. Wagenaar • S. Verbruggen • S. Rose-John D. G. M. Molin • M. Vooijs • M. J. Post

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Received: 28 March 2014 / Accepted: 4 September 2014 / Published online: 14 September 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract During angiogenesis, endothelial tip cells start sprouting and express delta-like 4 (DLL4) downstream of vascular endothelial growth factor (VEGF). DLL4 subsequently activates Notch in the adjacent stalk cells suppressing sprouting. VEGF also activates A disintegrin and metalloproteases (ADAMs) that induce Notch ectodomain shedding. Although two major ADAMs, i.e. ADAM10 and ADAM17, have been implicated in Notch-signalling activation, their apparent different roles in angiogenesis have not been fully understood yet. The objective of this study was to determine the roles of ADAM10 and ADAM17 activity in angiogenesis. In mouse retinas, ADAM10 or csecretase inhibition induced vascular sprouting and density in vivo, whereas attenuation of both ADAM10 and ADAM17 activity produced the opposite phenotype. Retinal blood vessel analysis in ADAM17 hypomorphic mice confirmed the requirement for ADAM17 activity in angiogenesis. However, ADAM17 inhibition did not phenocopy blood vessel increase by Notch blockage. These observations suggest that ADAM17 regulates other fundamental players during angiogenesis besides Notch,

M. Vooijs and M. J. Post have contributed equally to this work. V. Caolo  G. Swennen  A. Wagenaar  S. Verbruggen  D. G. M. Molin  M. J. Post (&) Department of Physiology, CARIM, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands e-mail: [email protected] A. Chalaris  S. Rose-John Institute of Biochemistry, Christian Albrechts University, Kiel, Germany M. Vooijs Department of Radiotherapy MAASTRO/GROW, Maastricht University, Maastricht, The Netherlands

which were not affected by ADAM10. By means of an angiogenesis proteome assay, we found that ADAM17 inhibition induced the expression of a naturally occurring inhibitor of angiogenesis Thrombospondin 1 (TSP1), whereas ADAM10 inhibition did not. Accordingly, ADAM17 overexpression downregulated TSP1 expression, and the TSP1 inhibitor LSKL rescued angiogenesis in the tube formation assay downstream of VEGF in the presence of ADAM17 inhibition. Finally, genetic and pharmacological ADAM17 blockade resulted in increased TSP1 expression in mouse retina. Altogether, our results show that ADAM10 and ADAM17 have opposite effects on sprouting angiogenesis that may be unrelated to Notch signalling and involves differentially expressed antiangiogenic proteins such as TSP1. Keywords

Angiogenesis  Tumour  Notch  ADAMs

Introduction Over the last years, different studies have highlighted the crucial function of the ADAM metalloproteinases in physiological and pathological neovascularization [1–3]. During blood vessel formation, i.e. angiogenesis, a subpopulation of ECs, called tip cells, start sprouting and elongate filopodia in response to VEGF stimulation. The proximal cells are stalk cells, which proliferate and form the endothelial lining of the newly formed vessel structure. The Notch ligand, DLL4, is expressed in tip cells and has been reported to finely regulate this process by triggering Notch1 activation in adjacent ECs and promoting their differentiation into stalk cells, which are characterized by low migration and filopodia capacity [4]. Notch signalling is initiated by two distinct proteolytic events: the ADAM-

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mediated S2 cleavage generates an intermediate cleavage product termed Notch extracellular truncation (NEXT), which serves as substrate for the c-secretase and facilitates the release of the signalling competent Notch intracellular domain (NICD). Although two ADAMs, i.e. ADAM10 and ADAM17, have been reported to cleave and activate Notch receptors, it is still a matter of controversy that ADAM is essential in this process. Adam17-deficient mice do not show a ‘‘Notch phenotype’’ [5], whereas conditional inactivation of ADAM10 in endothelial cells recapitulates the phenotype observed by interfering with Notch signalling: retinal vasculature exhibits increased branching and density [1]. In cell-based assays, ligand-induced Notch signalling requires ADAM10 and occurs at Val1711 S2 cleavage site [6]. Also in vivo in skin, lymphoid cells and endothelial cells, ADAM10 deficiency affects canonical NOTCH signalling [1, 7–9]. In contrast, ADAM17 seems to be involved in ligand-independent Notch signalling [10], albeit it is not sufficient in the absence of ADAM10 [11]. Moreover, conditional inactivation of ADAM17 in endothelial cells did not affect normal developmental angiogenesis, whereas it seemed to play a role during pathological neovascularization in mice in vivo [3]. In the present study, we investigated the roles of ADAM10 and ADAM17 in angiogenesis in gain of function and loss of function experiments using genetic and pharmacological approaches. Here, we show that concomitant blockage of ADAM10 and ADAM17 resulted in a marked decrease in vascular density and sprouting. In support of a positive and specific role of ADAM17 in retinal angiogenesis, we showed that homozygous hypomorphic ADAM17ex/ ex mice, characterized by dramatic reduction of ADAM17 expression in all tissues, had a less developed and organized vascular network. This phenotype strongly differs from the one observed after ADAM10 pharmacological blockage only, characterized by increased vascular density and sprouting. Altogether, these results led us to speculate that ADAM17 regulated other important players in angiogenesis than Notch and different from ADAM10. We show that ADAM17 inhibition induced the expression of the anti-angiogenic factor TSP1, whereas ADAM17 overexpression resulted in downregulation of TSP1 in ECs. Addition of TSP1 inhibitors rescued the angiogenesis in the presence of ADAM17 blockage downstream of VEGF. By contrast, no effect on TSP1 was observed following ADAM10 inhibition. In line with in vitro results, ADAM17 inhibition resulted in an increase in TSP1 when normalized to the number of blood vessels in retinas isolated from ADAM17ex/ex mice or WT pups injected with ADAM inhibitors, as compared to Wt pups and/or Wt pups injected with DSMO, respectively. Altogether, these data highlight an opposite function of the two major sheddases ADAM17 and ADAM10 during

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sprouting angiogenesis. Whereas ADAM10 appears to be the main metalloprotease to modulate Notch signalling and to suppress angiogenesis, ADAM17 positively regulates angiogenesis by regulating the crucial player TSP1.

Materials and methods Animals All animal studies were approved by the Animal Welfare Committee of the University of Maastricht and Kiel University. Wild-type C57Bl6 mouse pups were used in our experiments. In total, 100 mg/kg N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) (Sigma or Calbiochem, 565770) or 100 mg/kg of ADAM inhibitors GI254023X (ADAM-10 inhibitors) or GW413333X (ADAM10/ADAM17 inhibitor) were administered subcutaneously (injection volume 10 ll/g body weight). Control mice were injected with vehicle only (DMSO). Pups were injected daily for 48 h, starting at P5 and analysis on P7. Hypomorphic ADAM17 mice were previously described in [12]. Reagents Human cardiac microvascular endothelial cells (HCMvEC, CC-2927) and human umbilical endothelial cells (HUVECs, CC-2517) (Lonza, Vervies, Belgium) were cultured in EGM-2MV (CC-3162), with 5 % fetal bovine serum (FBS; Lonza). Recombinant VEGFA165 (300-035) was purchased from RELIATech GmbH (Brauschweig, Germany); DLL4 (rDLL4, 1506-D4-050) was from R and D System (Abingdon, Oxfordshire, UK); DAPT from SigmaAldrich (D5942); TAPI0 (CAS 143457-40-3) was from Calbiochem, Millipore (Billerica, MA, USA), LSKL (60877) was from Anaspec (Liege, Belgium). ADAM inhibitors GI254023X (ADAM10 inhibitors) and GW413333X (ADAM10/ADAM17 inhibitor) were both kindly provided by GlaxoSmithKline. Proteome Profiler Human Angiogenesis Array Kit (ARY007) was from R and D System (Abingdon, Oxfordshire, UK). Cell culture and angiogenesis proteome assay For inhibitor treatment, HCMvECs were incubated for 16 h with GI254023X or GW413333X or TAPI0 prior to VEGFA165. Cells were subcultured according to the manufacturer’s protocol (Lonza), and viable cells count was determined using trypan blue staining and the Countess Automated cell Counter (Invitrogen). Cell culture supernatant serum-free was collected and the expression levels of 55 angiogenesis-associated proteins were analysed the

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Human Angiogenesis Antibody Array kit (R and D Systems, Proteome ProfilerTM) as recommended by the manufacturer. Plasmids and transfection For exogenous Notch stimulation, HMvECs were stimulated for 48 h with rDLL4 coated on immobilized DLL4 in gelatin for 48 h as previously described [13] and then subjected to transfection. pcDNA-encoding ADAM17 was obtained from Addgene [14]. All transfection experiments were carried out in triplicate. Transfections were performed with lipofectamine LTX and PLUS reagent (Invitrogen, 15338-100). After 24 h of culture, mRNA was isolated and QPCR analysis performed. Western blotting Proteins were isolated using Laemmli sample buffer (Biorad) mixed with the 29 sample buffer (1:1) and 2-mercaptoethanol was added to cells. Samples were heated at 95 °C for 5 min, loaded on a 4–12 % gradient Criterion XT Bis–Tris gel and were, subsequently, subjected to electrophoresis in MOPS running buffer (BioRad) at 200 V for 55 min. Proteins were transferred to a PVDF membrane (BioRad) at 30 V overnight at 4 °C using Trisglycin transfer buffer (Biorad). Non-specific binding of the antibodies was blocked by incubating with 3 % milk powder in TBS supplemented with Tween-20 (TBS-T) for 1 h. The membranes were exposed to anti-ADAM10 and anti-ADAM17 Ab (Millipore), subsequently, incubated with appropriated horseradish peroxidase labelled secondary antibody. As loading control, bactin expression was detected using anti-bactin monoclonal antibody (Sigma). The detection was performed by using SuperSignal West Femto or Pico substrate (Pierce) and was visualized with the ChemiDoc XRS System (BioRad). RNA isolation and RT-qPCR After each experiment, RNA was isolated by using RNeasy micro kit Qiagen (Qiagen, GmbH, Hilden, Germany). A total of 100 ng RNA per sample was subjected to real-time reverse transcription polymerase chain reaction (RT-PCR) by using a BIORAD MiQ5 (Biorad, Veenendaal, The Netherlands) with Quanta reagents (VWR international BV, Amsterdam, the Netherlands) and primer concentration of 10 lM according to Van den Akker et al. [15]. PCR Primers used were: human b-ACTIN-sense: 50 -ATCCTCACCCTG AAGTACCC-30 , b-ACTIN-antisense: 50 -CACGCAGCTC ATTGTAGAAG-30 ; GADPH-sense: 50 -GCCTCAAGATC ATCAGCAAT-30 GADPH-antisense: 50 -GGACTGTGGTC ATGAGTCCT-30 ; DLL4-sense: 50 -ACAACTTGTCGGAC

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TTCCAG-30 , DLL4-antisense: 50 -CAGCTCCTTCTTCTG GTTTG-30 ; HES-1-sense: 50 -CCAAAGACAGCATCTGA GCA-30 , HES-1-antisense: 50 -GCCGCGAGCTATCTTTCT T-30 ; TSP1-sense: 50 -CTGACTCAGGACCCATCTAT-3; TSP1-antisense: 50 -CCAGTCTTTGTCTCCCTTTC-30 . Primers were designed with oligoperfectTM Designer (Invitrogen), Primer3 and Mfold (http://www.idtdna.com/scitools/Applica tion/mfold/) and were synthesized by Eurogentec (Seraing, Belgium). Matrigel assay For Matrigel network assay, 15 l Slide Angiogenesis (Ibidi, 81506) were filled with 10 ll Reducing Growth Factor Matrigel (BD Bioscience) were incubated in appropriate growth medium with or without 10 ng/ml of VEGFA, LSKL and 5 lM GI, GW or TAPI0. After 30 min, 10,000 HUVECs were seeded. The following day cells images were taken with EVOS fluorescent microscope and analysed by ImageJ (Version 1.48b NIH public domain software. The number of junctions was quantified by ‘‘Angiogenesis Analyzer’’ by Gilles Carpentier (http://image.bio.methods.free.fr/ImageJ/ ?Angiogenesis-Analyzer-for-ImageJ). Retina isolation and immunohistochemistry Retinas were isolated and stained as previously described in [16]. Isolectin GS-IB4 (20 lg/ml, I21411, Invitrogen) was used to stain blood vessels. TSP1 was stained with a rabbit polyclonal antibody (Abcam ab85762, 5 lg/ml) and secondary antibody Alexa Donkey Antibody 594 (R37119, Invitrogen). Photos were taken using a Leica DFC350 FX digital camera or Leica TCS SPE confocal. Quantifications vessel branch points: To assess the density of the vascular plexus, we quantified the number of branch points per area 200 9 200 lM fields, micrographs taken with 20 9 0.75 NA lens, 24–32 fields, 6–8 retinas for Ctrl-, DAPT-, GIand GW-treated groups or Wt, Wt/ex and ex/ex groups. To define vascular density, percentage of Isolectin B4-positive vessels areas was measured and calculated using ImageJ. Relative TSP1 immunofluorescent intensity was measured on four micrographs taken with 40 9 1.25 NA lens per retina. A total of four retinas per group were analysed. TSP1 intensity values were normalized to vascular density. Statistical analysis Statistical significance was determined by applying oneway ANOVA/Tukey’s or Student’s t (SigmaStat Software, San Jose, USA) tests. Values are expressed as mean ± SD of 3 in vitro experiments or 4 or 6–8 retinas per group. P values of \0.05 were considered significant.

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Fig. 1 ADAM10 and ADAM17 differentially affect angiogenesis. a IsolectinB4 staining of retinas from 7 day-old pups injected and treated with DAPT, GI and GW and DMSO (control; Ctrl) for 48 h (magnification 95 (I–IV), 910 (V–VIII) and 940 (IX–XII),

respectively). b, c Quantification shows that DAPT and GI treatment both resulted in a significant increase (*), whereas GW resulted in a significant decrease (**), of vascular density and branching points per field compared to Ctrl

Results

Fig. 1b, c), essentially phenocopying pharmacological inhibition of Notch activation with the c-secretase inhibitor DAPT (Fig. 1a II, VI, X; Fig. 1b, c). Conversely, combined ADAM10 and ADAM17 inhibition with GW resulted in reduced vascular density and sprouting angiogenesis (Fig. 1a IV, VIII, XII; Fig. 1b, c) (n = 6, P \ 0.001). These results support recent observations that ADAM10 mediate ligand-induced Notch processing in vitro and in vivo [6, 7, 10], recapitulating the phenotype observed following Notch inhibition with DAPT. ADAM17 on the other hand seems to regulate other fundamental function of endothelial cells independent of Notch signalling [17], which are essential for angiogenesis.

ADAM17 and ADAM10 inhibition produces an opposite phenotypes in mouse retinal vasculature In order to assess the effect of ADAM10 and ADAM17 inhibition on sprouting angiogenesis, we injected early postnatal (P5) wild-type mice with GI a potent ADAM10 inhibitors, GW a dual ADAM 10/17 inhibitor or the csecretase inhibitor DAPT for 48 h. Whole-mount analysis of the retinal vasculature after isolectin B4 staining revealed that the inhibition of ADAM10 alone with GI (Fig. 1a III, VII, XI; Fig. 1b, c) increased vessel sprouting and density compared with controls (Fig. 1a I, V, IX;

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Fig. 2 Reduced angiogenesis in ADAM17 ex/ex hypomorphic mice. a Isolectin-B4 staining of retinas from 7 day-old Wt, Wt/ADAM17 ex and ADAM17 ex/ex hypomorphic mice (magnification 5X (I–III), respectively). Retinas isolated from hypomorphic ADAM17 (ex/ex) animals showed less vascular density and more disorganized vascular

network compared to Wt and heterozygous Wt/ex corresponding animals. b, c Quantification analysis shows that ex/ex mice retinas had higher vascular density and branching points (*) per field compared to Ctrl

Genetic inactivation of ADAM17 in hypomorphic mice (ADAM17ex/ex) affects retinal vasculature

In order to identify the specific ADAM17 targets, we performed an angiogenic proteome assay following stimulation of ECs with VEGF and ADAM10 (GI) or ADAM10/ADAM17 (GW) or ADAM17 inhibitor (TAPI0). By screening 55 angiogenesis proteins, we found that ADAM17 inhibition upregulated a well-known natural inhibitor of angiogenesis, thrombospondin-1, TSP1 (Fig. 3a), whereas ADAM10 inhibition did not display any effect on the expression of TSP1 (Fig. 3a). In line with these results, ectopic ADAM17 expression, in HMvECs downregulated TSP1 expression (Fig. 3b), whereas transfection of ADAM10 did not show any effect (Fig. 3b).

To confirm the non-redundant role of ADAM17 in blood vessel formation, we assessed angiogenesis in retinas obtained from P7 ADAM17ex/ex hypomorphic mice witch display a dramatically reduced ADAM17 expression in all tissues [12]. As shown in Fig. 2a–c vascular density and sprouting were markedly decreased in ADAM17ex/ex homozygous mice compared with the wild-type (Wt) and heterozygous (Wt/ex) animals used as control groups (n = 6, P \ 0.001. Also in ADAM17ex/ex homozygous mice, retinal vasculature appeared highly disorganized as compared to Wt and Wt/ex mice (Fig. 2a). ADAM17 regulates TSP1 expression in ECs

TSP1 inhibitors rescued angiogenesis inhibition by knockdown of ADAM17 downstream of VEGF

The reduction in blood vessel density in ADAM17ex/ex and following combined ADAM10 and ADAM17 inhibition compared to ADAM10 inhibition only, suggested an opposite role for ADAM10 and ADAM17 during angiogenesis. Whereas ADAM10 blockage recapitulated the typical Notch phenotype in endothelium, ADAM17 seemed to modulate angiogenesis in a Notch signalling-independent manner.

To further determine the inhibitory potential of TSP1 on angiogenesis downstream of ADAM17, we stimulated HUVECs with VEGF and ADAM10 or ADAM17 inhibitors in the presence or absence of TSP1 inhibitor LSKL. In line with the in vivo data, ADAM10/ADAM17 or ADAM17 inhibition reduced, whereas ADAM10 alone augmented, VEGF-induced ECs tube formation on matrigel (Fig. 4a, b).

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Fig. 3 ADAM17 modulates TSP1 expression. a Human angiogenesis array was screened using conditioned media from HMvECs treated with or without VEGFA ? GI, GW or TAPI0 and were compared with control (Ctrl, set at 1). Inhibition of ADAM17 with GW or TAPI0 increased TSP1 protein level. b Western blot analysis of protein extracts from HMvECs transiently transfected with ADAM10

or Mock pcDNA (left panel) and ADAM17 or Mock pcDNA (right panel). Results are from one representative experiment. Overexpression of ADAM17 in HMvECs resulted in significant TSP1 mRNA downregulation, whereas ADAM10 overexpression did not show any effect

TSP1 inhibitor LSKL administration neutralized the antiangiogenic effect of ADAM17 blockage by GW or TAPI0 administration, downstream of VEGF. Altogether, these data indicate that ADAM17 positively regulates angiogenesis by reducing TSP1 expression.

pharmacological and genetic inhibition of ADAM17 resulted in the opposite phenotype in mouse retinal blood vessels (Fig. 1). The data, while supporting the general importance of ADAM10-induced Notch signalling [7], highlight a new role for ADAM17 during retinal sprouting angiogenesis. In support of a specific role of ADAM17 in retinal angiogenesis, we showed that homozygous hypomorphic ADAM17ex/ex mice had a less developed and organized vascular network. These results differ from a recent study, which provided evidence that ADAM17 from endothelial cells is not involved in normal developmental angiogenesis or vascular homeostasis [3]. Conditional ADAM17 deletion on endothelial cells did in fact not result in a particular retinal vasculature phenotype. For our experiments, we used hypomorphic mice that show a 95 % decrease in ADAM17 expression in all tissues. It is thus possible that other sources of ADAM17 besides endothelial cells are responsible for its activity during developmental angiogenesis or may rescue lack of endothelial ADAM17 activity. In accordance with our data in hypomorphic mice, ADAM17 inhibition prevented VEGF-induced ECs proliferation and tube-like formation. Also in line with our results, Lucitti et al. [17], by the use of conditional and global knockouts, demonstrated that ADAM10 and ADAM17 display opposite roles during pial collateral formation based on mechanisms associated with sprouting/branching. ADAM17 promoted collateral formation, whereas ADAM10 suppressed excessive tip cell formation. Similar to EC-specific ADAM10 knockdown [1], pharmacological inhibition of c-secretase activity resulted in increased collaterogenesis.

ADAM17 blockage resulted in increase TSP1 in mouse retinas In order to assess the effect of ADAM17 on TSP1 expression in vivo, retinas from ADAM17 inhibitor GWinjected mice and ADAM17ex/ex hypomorphic were isolated and subjected to immunofluorescent staining for TSP1. We found that TSP1, besides being expressed in retinal vasculature, was also localized in the proximity of endothelial cells in the vascular regions. These observations are consistent with previous studies showing that TSP1 acts as a secreted angiocrine suppressor, which is promptly degraded [18, 19]. Here, we show that following pharmacological and/or genetic inactivation of ADAM17, TSP1 expression is increased when normalized to the vascular area, as compared to Wt pups and/or Wt pups injected with DSMO, respectively (Fig. 5).

Discussion In the present study, we report on opposite angiogenic phenotypes observed following ADAM10 inhibition and loss of ADAM17 function. While inhibition of ADAM10 activity mimicked loss of Notch signalling using c-secretase inhibitors and induced vessel sprouting and density,

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Fig. 4 LSKL counteracts the negative effect of ADAM17 inhibition on tube formation. a HUVECs tube formation (n = 3) was significantly increased following VEGFA or VEGFA ? GI treatment (II, III) as compared to control (I). By contrast, ADAM17 inhibitor GW or TAPI0 (IV, V) reduced tube formation despite VEGFA administration. Addition of 10 ng/ml TSP1 inhibitor LSKL neutralized the anti-angiogenic effect of ADAM17 blockage (VI, VII). Each photograph shows a representative well of triplicate wells set-up for each group. b Tube formation was quantified by assessing the number of junctions counted for each group (*) significantly different from Ctrl

It appeared from these results that ADAM17 differs from ADAM10 by mainly regulating angiogenesis in a Notchindependent way. Therefore, we investigated what other molecular mechanisms are affected by ADAM17 during the process of angiogenesis. By means of an angiogenic proteome assay, we demonstrated that ADAM17 inhibition was able to upregulate a well-known natural inhibitor of angiogenesis, TSP1. In support of this observation, overexpression of ADAM17 resulted in downregulation of

TSP1 expression in ECs. Consistently, pharmacological and genetic inhibition of ADAM17, resulted in a relative upregulation of TSP1 expression when normalized to vascular area. Recently, Ghajar et al. [19] have demonstrated that TSP1 is highly expressed in stable vasculature in dormant tumour niches. On the other hand, the tumour-suppressive nature of microvascular endothelium is lost at the sprouting endothelial cells, which are characterized by reduced TSP1 expression. Altogether, these observations

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Fig. 5 TSP1 expression was increased in mouse retina blood vessels following ADAM17 blockage. a IsolectinB4 (green) and TSP1 (red) of retinas from 7 day-old pups injected GW and DMSO (control; Ctrl) for 48 h (magnification 940). b Quantification shows that GW treatment resulted in a significant increase (*P \ 0.05) of TSP1 expression when normalized to vascular area compared with Ctrl.

c IsolectinB4 (green), TSP1 (red) of retinas from 7 day-old pups from Wt and ADAM17 ex/ex hypomorphic mice (magnification 940). d Quantification shows that ADAM17 ex/ex hypomorphic mice have a significantly increased (*P\0.05) TSP1 expression when normalized to vascular area compared to Wt mice. (Color figure online)

are consistent with our observations that ADAM17 negatively regulates TSP1 and that the decrease in blood vessel density and sprouting following ADAM17 inhibition is likely due to an upregulation of TSP1. Accordingly, TSP1 inhibition rescued angiogenesis in the tube formation assay in the presence of ADAM17 blockage downstream of VEGFA. Due to its crucial role in angiogenesis and its high expression level in tumour stroma and vasculature, DLL4 Notch signalling has been considered a suitable therapeutic target to block angiogenesis in cancer [20–22]. Paradoxically, despite an increased vessel number by DLL4 blockade, such vessels were not functional and poorly perfused, resulting in tumour size decrease [23, 24]. For

this reason, anti-DLL4 antibodies have recently entered clinical trials [23]; however, the toxic side effects after Notch inhibition are still poorly understood. DLL4-blocking therapies present a number of serious limitations. Chronic DLL4 blockade has been reported to abnormally activate ECs, disrupt normal organ homeostasis and produce significant pathology in multiple organs, inducing vascular neoplasms [25]. Intriguingly, Notch blockade results in an increase in tip cell number. Recently, it has been shown that tumour growth accelerates around tip cells, which release growth factors activating dormant tumour cells [19]. Moreover, following Notch blockade blood vessels become leaky, creating a hostile tumour environment. Long-term vessel disruption causes hypoxia

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in the tumour, which may trigger a pro-angiogenic response by the release of vascular endothelial growth factors and activation of metalloproteases. Therefore, research is now focusing on the identification of new molecular targets, besides Notch, in order to find an effective strategy to prevent blood vessel formation in solid cancer. Recent studies have highlighted the positive role of ADAM17 in pathological angiogenesis [3, 26], suggesting it as a potential therapeutic target. Specific ADAM17 inhibition could provide a new tool to achieve blood vessel inhibition in tumours. In this study, we showed that ADAM17 and ADAM10 display opposite effects on angiogenesis. Whereas ADAM10 negatively regulates angiogenesis, ADAM17 has a positive role in this process by inhibiting the expression of the antiangiogenic factor TSP1. Noteworthy, inhibition of both ADAM10 and ADAM17 prevented a dramatic increase in blood vessel density and fusion observed after administration of the Notch inhibitor DAPT or an ADAM10 inhibitor alone. Further studies are needed to investigate whether inhibition of concomitant Notch and ADAM17 could provide a new potentially applicable clinical tool to prevent excessive non-functional angiogenesis in solid tumours characterized by highly active Notch signalling. Acknowledgments We thank Drs. Lorraine Bray from GlaxoSmithKline for the inhibitors GI254023X and GW413333X. We are grateful to Arjan. J. Groot for technical support. The authors gratefully acknowledge the support of the PENT and the CTMM Eminence Programs of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science. M.Vooijs is supported by ERC-Starting grant 208259. A. Chalaris and S. RoseJohn are supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB877, project A1) and the cluster of excellence ‘‘inflammation and interfaces’’. D.G.M. Molin is supported by European grants Interreg IVa Euregio Meuse Rhine BioMIMedics and Interreg IVa Flanders— The Netherlands VaRiA. Conflict of interest

The authors declare no conflicts of interest.

Ethical standard All of the reported work has been carried out according to applicable laws and regulations.

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