Developmental cell. Jul/31/2009; 17(2): 175-186

PMID: 19686679

PMC: 2747264

Integrin-α9 is required for fibronectin matrix assemblyduring lymphatic valve morphogenesis

Summary

Dysfunction of lymphatic valves underlies human lymphedema, yet theprocess of valve morphogenesis is poorly understood. Here, we show that duringembryogenesis lymphatic valve leaflet formation is initiated by upregulation ofintegrin-α9 expression and deposition of its ligand, fibronectin-EIIIA(FN-EIIIA), in the extracellular matrix. Endothelial cell specific deletion ofItga9 (encoding integrin-α9) in mouse embryosresults in the development of rudimentary valve leaflets, characterized bydisorganized FN matrix, short cusps and retrograde lymphatic flow. Similarmorphological and functional defects are observed in mice lacking the EIIIAdomain of FN. Mechanistically, we demonstrate that in primary human lymphaticendothelial cells the integrin-α9-EIIIA interaction directly regulatesFN fibril assembly, which is essential for the formation of the extracellularmatrix core of valve leaflets. Our findings reveal an important role forintegrin-α9 signaling during lymphatic valve morphogenesis and implicateit as a candidate gene for primary lymphedema caused by valve defects.

Introduction

The lymphatic vasculature forms a network of blind-ended capillaries thatcollect protein rich fluid from the interstitial space and drain it via collectingvessels first into lymph nodes and then to larger lymphatic ducts, which connect tothe venous system (Alitalo et al., 2005; Jurisic and Detmar, 2009). The major functionsof the lymphatic vasculature are to maintain tissue fluid balance, provide immunesurveillance through transport of leukocytes and antigen-presenting dendritic cellsand participate in fat absorption (Alitalo et al.,2005; Jurisic and Detmar,2009).

Congenital malformation of the lymphatic system such as vessel hypoplasia andvalve defects cause primary lymphedema, which is usually a progressive and lifelongcondition, characterized by gross swelling of the affected limb accompanied byfibrosis and susceptibility to infections (Alitalo etal., 2005; Jurisic and Detmar,2009). The management of symptoms is based on physiotherapy andcompression garments as at present no effective treatment for lymphedema exists.Several genes, including Vegfr3, Foxc2 and Sox18,which were shown to regulate developmental lymphangiogenesis in mice (Francois et al., 2008; Karkkainen et al., 2001; Petrova et al., 2004), have been implicated in human lymphedema. Kinaseinactivating mutations in the VEGFR-3 gene lead to Milroy'sdisease (Ferrell et al., 1998), whilemutations in the transcription factors FOXC2 andSOX18 are the underlying genetic causes oflymphedema-distichiasis and hypotrichosis-lymphedema-telangiectasia, respectively(Finegold et al., 2001; Irrthum et al., 2003). Interestingly, defective luminalvalves observed as a consequence of loss of FOXC2 function (Petrova et al., 2004) highlights the criticalrole of valves in maintaining unidirectional lymphatic flow. While the mechanisms oflymphatic valve morphogenesis remain poorly characterized, it has been wellestablished that the interactions between the blood endothelial cells and theirsurrounding extracellular matrix (ECM), organized to facilitate valve function, areof key importance in regulating heart valve development and cardiac function, andconsequently, heart valve disease (Armstrong andBischoff, 2004; Lincoln et al.,2006). Similarly, ultrastructural analyses of lymphatic valves havedemonstrated a close association between ECM and lymphatic endothelial cells in thevalve leaflets (Lauweryns and Boussauw, 1973;Navas et al., 1991). These findingssuggest that the ECM has important functions in controlling endothelial cellsignalling and could also provide structural integrity during lymphatic valvemorphogenesis.

Cell-matrix adhesion receptors, such as integrins, play essential roles indevelopmental processes that involve close interactions between the cells and theirsurrounding ECM. Integrins are heterodimeric transmembrane receptors composed ofα and β subunits. Their extracellular domains bind to the ECMmolecules while the cytoplasmic domains associate with the actin cytoskeleton andaffiliated proteins, thereby providing a link between the external and internalenvironment of the cell (Geiger et al.,2001). In addition to mediating attachment to their respective ECM ligand(s),integrins have specialized signaling functions and they can regulate gene expressionas well as cell shape, migration, proliferation and survival. Furthermore, integrinbinding to ECM is not only required for transducing signals from the matrix to cellsbut this interaction also initiates responses that allow the cells to organize andremodel the matrix (Leiss et al., 2008).Despite an apparent redundancy in their ligand-binding specificities, with severalECM molecules being ligands for more than one integrin, genetic studies havedemonstrated distinct functions for individual integrins. Fibronectin (FN) receptorsintegrin-α5β1 and -α4β1, as well as components ofthe ECM, such as FN, play critical roles in the development of the blood vasculature(Hynes, 2007). However, knowledge of theexpression and function of integrins and the ECM in the lymphatic vasculature islimited (Avraamides et al., 2008).

Here we show that a member of the integrin-family, integrin-α9(encoded by Itga9), was predominantly expressed in the endothelialcells of the lymphatic valve. Interestingly, Itga9 deficiency inmice led to specific defects in the formation of luminal valves, which resulted inretrograde lymphatic flow and impaired fluid transport. We provide invivo and in vitro evidence for the requirement ofintegrin-α9 interaction with its specific ligand, fibronectin-EIIIA(FN-EIIIA, also called EDA), in regulating FN matrix assembly and thereby identifyan unexpected in vivo function for an integrin-EIIIA interaction.Collectively, our findings demonstrate an important role for integrin signaling inlymphatic valve development and provide novel insight into the previouslyundescribed morphogenetic process of lymphatic valve morphogenesis. As lymphaticvalve defects manifested in adults are likely to have origins in valve development,integrin-α9 is a candidate gene for primary human lymphedema caused bylymphatic valve defects.

Results

Integrin-α9 is expressed in mature and developing lymphaticvalves

We surveyed expression of several integrins by immunofluorescence todetermine which of these molecules might function during lymphatic valveformation. Whole-mount staining of adult skin using antibodies against specificα-subunits revealed low levels of integrin-α5 and -α6expression in lymphatic endothelia and in the valve, while no apparent stainingwas detected using antibodies against integrin-α1, -α2 or-α4 (data not shown). In contrast, staining with integrin-α9antibodies strongly highlighted the cells constituting the luminal valve (Figures 1A and 1B). In agreement withprevious studies (Huang et al., 2000), nointegrin-α9 expression was detected in the blood vessel endothelia (datanot shown).

Since integrin-α9 appeared to be the predominantα-subunit expressed in lymphatic endothelia and valves, we furthercharacterized its expression pattern in developing vessels and aimed our studyat determining its function in valve morphogenesis. During mouse development,the formation of valved collecting vessels occurs in late embryonic and earlypostnatal life through remodelling of a primitive lymphatic capillary network(Figures 1C-1E; (Makinen et al., 2005; Norrmen et al., 2009)). At embryonic day (E)16, mesenteric lymphaticvessels form a vascular plexus that is LYVE-1 positive and does not containvalves (Figures 1C and 1F). However,defined clusters of cells expressing high levels of Prox1 (Figure 1F) and FoxC2 (data not shown) transcriptionfactors are indicative of initiation of valve formation (Norrmen et al., 2009). At this stageintegrin-α9 expression was low in lymphatic endothelia (Figures 1F and 1G), while higher expression wasdetected in scattered cells in the surrounding tissue (data not shown) and invascular SMCs around the blood vessels (Figures 1Fand 1G, arrowheads). At E17, the lymphatic vessels formedconstrictions that showed elevated Vegfr3-lacZ reporteractivity (Figure 1D) and containedendothelial cells expressing high levels of FoxC2 (Figure 1H), Prox1 (data not shown) and integrin-α9 (Figures 1H and 1I). At E18,integrin-α9 was present in the endothelial cells of developed valveleaflets (Figures 1J and 1K). Takentogether, these expression data suggest that integrin-α9 upregulation inlymphatic endothelia correlated with the initiation of valve leafletformation.

Itga9 deficient mice display abnormal lymphaticvalves

To investigate the physiological function of integrin-α9 inlymphatic valves we analysed Itga9 deficient mice. These micewere reported to die perinatally of respiratory failure, caused by the presenceof bilateral chylothorax (Huang et al.,2000), however the potential lymphatic vascular defects remainedunknown. Analysis of chyle-filled mesenteric collecting vessels revealedcharacteristic V-shaped valves in wild-type neonates (Figures 2A and 2A′). In contrast,Itga9 mutant mice displayed fewer and morphologicallyabnormal valves (Figures 2B and2B′). Staining with an antibody against PECAM-1, which isstrongly expressed in endothelial cells forming the valve leaflets (Figure 2C), demonstrated that most of themutant valves appeared as horizontal constrictions rather than V-shapedstructures (Figures 2D, 2E and 2F). Leakageof chylous fluid from the mesenteric vessels further indicated that they weredysfunctional (arrowhead in Figure 2B). Thedefects in Itga9 deficient mice were specific to lymphaticvalves since we observed apparently normal development and gross morphology ofthe lymphatic vasculature in all tissues that were examined (Figures S1A-S2H).

Examination of longitudinal transmission electron microscopy sections ofthe valves of mesenteric lymphatic vessels revealed leaflets consisting of twoendothelial layers with a central connective tissue core (Figures 2G, 2I, 2K; (Lauweryns and Boussauw, 1973; Navaset al., 1991)). The endothelial cells of the wild-type valve leafletsformed long, overlapping cell-cell junctions, and they were tightly attached tothe matrix core (Figures 2G and 2I). In theItga9^-/- vessels the cusps of the valveswere shorter with fewer endothelial cells (Figures2H and 2K). The disorganized or absent matrix core in between thebasal sides of the endothelial cells was associated with the separation of theopposite endothelial surfaces (Figure 2J).These results suggest that integrin-α9 function is essential for theformation of the defined ECM core of a valve leaflet.

Retrograde lymph flow and impaired fluid transport inItga9^-/- mice

Luminal valves have an important role in establishing unidirectionallymphatic flow. To evaluate if lymphatic function is compromised in theItga9^-/- mice, we investigated uptake andtransport of subcutaneously injected large-molecular-weight fluorescent dextran.In wild-type mice the FITC-dextran injected into the forelimb footpad wasrapidly drained into the valved dermal collecting lymphatic vessels, which werevisualized proximal to the injection site (Figure3A). In contrast, in the Itga9 mutant skin the dyelabeled a vessel network (Figure 3B),suggesting retrograde lymphatic flow from the collecting vessels to thepre-collector vessel branches (Makinen et al.,2005; Petrova et al., 2004).In addition, the dye showed leakage into the surrounding tissue and thetransport from the injection site to the lymph nodes and the thoracic duct wasimpaired (FiguresS2A-S2D). We also analyzed the lymphatic drainage after subcutaneousinjection of FITC-conjugated Lycopersicum Esculentum lectin(LEL), which binds to the surface of lymphatic endothelial cells, in particularin the valves (Tammela et al., 2007)(Figure 3C). In theItga9 mutants valve regions were identified by strongerlectin staining, however, the valves were abnormal and often lacked obviousleaflets (Figures 3D and 3E). Together, theabove data show that Itga9 deficient mice display specificdefects in valve morphogenesis, which result in retrograde lymphatic flow andimpaired fluid transport.

Integrin-α9 is required tissue-autonomously in endothelia forlymphatic valve development

To examine if integrin-α9 has a tissue-autonomous function inlymphatic endothelia during valve development we deleted its expressionspecifically in endothelial cells by crossing the floxedItga9^lx mice (Singh et al., 2008) with Tie2-Creanimals (Koni et al., 2001). Thehomozygous Tie2-Cre;Itga9^lx/lx mice displayedchylothorax (data not shown) and reduced number of lymphatic valves (Figures S3A and S3B). Thefew valves that were detected displayed abnormal leaflets, as visualized bystaining for an ECM molecule Laminin-α5 (Figures 4A-4D).

We next asked whether integrin-α9 is also essential for themaintenance of the valves. We usedVE-cadherin-CreER^T2 mice, which allowtamoxifen-regulated activation of Cre in both blood and lymphatic endothelia (R.Adams, unpublished; FiguresS3C and S3D). To induce Itga9 gene deletion inmature valves, 4-hydroxytamoxifen (4-OHT) was injected into controlItga9^lx/⁺ (Figures 4E-4H) andVE-cadherin-CreER^T2;Itga9^lx/lx(Figures 4I-4L) mice at P1, when mostof the valves are fully developed, and the vessels were analysed at P7. In 4-OHTtreatedVE-cadherin-CreER^T2;Itga9^lx/lxvessels integrin-α9 expression was lost from lymphatic endothelia (Figure 4K). However, the valve leafletsappeared normal (Figures 4I and 4J), andintegrin-α9 negative endothelial cells remained attached to the matrixcore (Figures 4I, 4K and 4L), suggestingthat integrin-α9 is dispensable for stable adhesion of endothelial cellsin fully developed valves. Together, these results suggest thatintegrin-α9 is required tissue-autonomously in endothelia for thedevelopment of lymphatic valve leaflets, while it is not essential formaintaining valve structure and endothelial cell adhesion in mature valves.

Integrin-α9 ligand, fibronectin-EIIIA, is expressed in the developinglymphatic valves

To examine the function of integrin-α9 in the formation of valveleaflets we investigated the expression and localization of its ligands,including Tenascin-C (TNC), Osteopontin (OPN) and fibronectin containing theEIIIA domain (FN-EIIIA) (Liao et al.,2002; Yokosaki et al., 1998;Yokosaki et al., 1999) in thedeveloping lymphatic vessels. Confocal longitudinal cross section of a valvefrom E18.5 mesenteric vessels revealed expression of integrin-α9 and itsligand TNC on both luminal and abluminal sides of the valve endothelial cells(Figures S4A-S4C),while no staining was seen for OPN (data not shown). Interestingly, unlike FN,which was detected in all lymphatic vessel basement membrane (Figures S4D-S4F), FN-EIIIA showedlocalization restricted to the valve matrix core (Figures S4G-S4I).

To gain further insight into the role of FN-EIIIA we analysed itsexpression during different stages of lymphatic valve development so as todetermine the relationship between integrin-α9 expression and ECMdeposition. At E16, prior to upregulation of integrin-α9 expression(Figure 5A), Laminin-α5 wasfound deposited at specific sites along the vessel (Figures 5A), whereas no fibrous staining for FN-EIIIAwas detected (data not shown). Induction of integrin-α9 expressioncorrelated with appearance of continuous FN-EIIIA fibers at sites of developingvalves (Figures 5B), which were identifiedby Laminin-α5 positive matrix (Figure5C) and the presence of cells expressing high levels of Prox1 (Figure 5D). During initial steps of valvedevelopment FN-EIIIA and Laminin-α5 showed a similar localization (Figure 5C). However, during leafletelongation Laminin-α5 was found in the entire valve matrix core whileFN-EIIIA fibers were concentrated on the free edges of the leaflets (Figures 5E and 5E′). Similar to whathas been reported for other tissues (Pagani etal., 1991), FN-EIIIA expression was progressively down-regulated inpostnatal lymphatic vasculature. While prominent FN-EIIIA fibers were detectedin mesenteric lymphatic valves at P3 (data not shown), only weak staining wasobserved at P9 (FiguresS4J-S4L) and no fibers were seen in adult valves (see Figure 6D). These expression data suggest thatFN-EIIIA is primarily involved during embryogenesis and that it has a role inthe formation and extension of the valve leaflets.

Defective FN-EIIIA matrix assembly and valve leaflet formation inItga9^-/- mice

We next examined valve morphogenesis inItga9^-/- embryos. X-Gal staining of mesentericlymphatic vessels in E16Itga9^-/-;Vegfr3^lz/⁺embryos revealed normal vascular networks (Figure S5A). Consistent with thereduced number of valves observed in postnatalItga9^-/- vessels, at E17 the number ofVegfr3-lacZ positive constrictions (Figures S5B-S5E), that containedclusters of cells expressing high levels of Prox1 and FoxC2 was reduced whencompared to the wild-type (Figures S5F and S5G and data not shown). In addition, staining ofmutant valves for FN-EIIIA revealed only few short fibers (Figures 5F-5G′). Cross section view throughthe valve showed a fibrous matrix in wild-type vessels (Figure 5H). In contrast, the underdeveloped leafletsin Itga9 deficient vessels displayed predominantly a disruptedpunctuate and discontinuous pattern (Figures5I), suggesting that FN-EIIIA failed to assemble into continuousfibers. Taken together, the disorganized FN-EIIIA matrix inItga9 deficient vessels and the subsequent arrest in valvedevelopment suggest that the defect in valve formation inItga9^-/- mice is due to interaction betweenintegrin-α9 and its specific ligand (Figure 5J).

Fibronectin-EIIIA is required for normal development of lymphatic valveleaflets

To test directly if FN-EIIIA, or the other integrin-α9 bindingECM molecules, has a function in valve formation, we analyzed the lymphaticvessels in Tnc, Opn and Fn-EIIIA deficientmice (Forsberg et al., 1996; Liaw et al., 1998; Muro et al., 2003). Both Tnc andOpn deficient mice displayed apparently normal lymphaticvasculature and lymphatic valves (Figures S6A-S6F), however, micedeficient for Fn-EIIIA partially recapitulated theintegrin-α9 mutant phenotype. Quantification of Laminin-α5positive valve structures in mesenteric lymphatic vessels of newborn mice showeda 1.5-fold reduction in the number of valves inFn-EIIIA^-/- mice when compared to the wild-type(p = 0.0026 (Mann-Whitney test), Figure6A; TableS1). Notably, 76% of the valves inFn-EIIIA^-/- mice displayed abnormal matrix ringappearance or underdeveloped leaflets and disorganized Laminin-α5 matrix(Figures 6B and 6C; Table S1), similar to the valvesobserved in Itga9 mutants. In wild-type controls at thisdevelopmental stage only a minor proportion of immature valves with rudimentaryleaflets were observed, while in Itga9^-/- miceapproximately 90% of the valves were abnormal (Figure 6A; Table S1). While lymphatic valvedefects were pronounced in the Fn-EIIIA^-/- miceduring early postnatal life, they diminished during progression into adulthood,coinciding with when the expression of FN-EIIIA is down-regulated. In the earskin of three weeks old wild-type animal, punctuate staining, but no FN-EIIIAfibers, were detected in the lymphatic vessels and the valves (Figure 6D). At the same age the majority of the valvesin Fn-EIIIA mutant animals had apparently normal leaflets whencompared to the control (Figures 6E and6F). The mice also exhibited otherwise normal lymphatic vasculature,including vessel diameter and smooth muscle cell coverage (Figures 6E and 6F). However, subcutaneous injection ofFITC-dextran revealed reflux of dye from the collecting vessels, suggesting mildvalve defects, and some morphologically abnormal valves inFn-EIIIA deficient mice (Figures 6G-6I, 4/4 mice analyzed). These results suggest thatFn-EIIIA deficiency leads to lymphatic valve defects duringembryonic and early postnatal life.

Integrin-α9-EIIIA interaction regulates FN assembly in lymphaticendothelial cells

The defective FN matrix observed in theItga9^-/- valves prompted us to examine thepossibility that integrin-α9-EIIIA interaction contributes to FNassembly in lymphatic endothelial cells (LECs), despite the previous findingssuggesting that the EIIIA domain is dispensable for matrix assembly infibroblasts (Tan et al., 2004). Wetherefore tested whether primary human LECs are able to assemble theirendogenously produced FN, which contains the EIIIA domain (Figure 7A). To avoid exogenous plasma FN lacking theEIIIA domain, the cells were grown in the presence of FN-depleted serum andfibril assembly was assessed by immunofluorescence for the EIIIA. After 24h inculture, a fibrillar FN-EIIIA network was detected on the surface of the LECs(Figure 7A). Inhibition ofintegrin-α9 function, or integrin-α9-EIIIA interaction, usingblocking antibodies against integrin-α9 (Y9A2) or EIIIA (IST-9) (Figures 7A and 7B), or a blocking EIIIApeptide EDGIHEL (Liao et al., 2002)(datanot shown), led to a significant decrease in FN-EIIIA fibril formation.FN-EIIIA levels were not reduced (Figures 7C), indicating that the defect was not due toimpaired mRNA synthesis. A similar effect was seen when integrin-α9expression was silenced using siRNA oligos (Figures 7A-7D). In contrast, inhibition of RGD-dependent integrininteractions, which have been considered essential for FN assembly viaα5β1 and αv integrins, had no effect (Figure 7B), while siRNA-mediated knock-down ofintegrin-α5 (Figure 7D) partiallyblocked LEC mediated FN-EIIIA assembly (Figure 7Aand 7B). Deoxycholate (DOC) differential solubilization assay andWestern blot analysis were further used to analyse the conversion of DOC-solublefibrils into insoluble stable matrix containing high molecular mass FNmultimers. No DOC-insoluble material was associated with the LECs in whichintegrin-α9 expression or the integrin-α9-EIIIA interaction wasblocked (Figure 7E, upper panel). Instead,similar quantities of DOC-soluble FN matrices were found in all cells (Figure 7E, lower panel). Consistent with theimmunofluorescence data, silencing of integrin-α5 expression partiallyblocked the formation of DOC-insoluble FN-EIIIA matrix (Figure 7E). These results suggest that althoughintegrin-α5 cannot functionally compensate for the loss ofintegrin-α9, it contributes to FN fibrillogenesis in the LECs. However,the low levels of integrin-α5 detected in the developing lymphaticvessels (Figure 7F) suggest that it doesnot play a major role during valve morphogenesis. The above results demonstratethat integrin-α9-EIIIA interaction can directly regulate FN matrixassembly, suggesting a functionally indispensable, integrin-specific mechanismfor FN fibrillogenesis in the LECs.

Discussion

The present study establishes integrin-α9 as an essential regulatorof the morphogenetic process controlling the formation of lymphatic valve leaflets,which allow opening and closing of the valve in response to pressure changes. Thisaction of the valve is critical for the maintenance of unidirectional lymphatic flowand the functionality of the entire lymphatic vascular system, highlighted by thelack or insufficient function of lymphatic valves as an underlying cause of humanlymphedema (Alitalo et al., 2005).

The leaflets of a mature valve consist of a well-defined matrix in betweentwo sheets of lymphatic endothelial cells, which forms a strong but elasticconnective tissue core (Figure 1). Duringembryogenesis, the formation of the lymphatic valve is initiated by upregulation ofFoxC2 and Prox1 transcription factors in distinct clusters of endothelial cells,which define the positions of future valves ((Norrmen et al., 2009), see Figure5J). We found that the development of the valve leaflet is subsequentlyinitiated by upregulation of integrin-α9 expression and deposition of ECMcontaining its ligand, the EIIIA splice isoform of FN. The localization of FN-EIIIAat the distal tip of the developing valve suggested its function in regulating valveleaflet elongation. However, from previous studies the precise function of FN-EIIIAhas remained elusive. Despite strong expression in the angiogenic blood vessels, novascular phenotypes were reported in Fn-EIIIA deficient mice (Astrof et al., 2004). In vitrostudies demonstrated that inclusion of the EIIIA segment enhanced the ability of FNto incorporate into existing matrix (Guan et al.,1990). This suggests that FN-EIIIA may participate in FN fibrillogenesis,which is a highly regulated, multistep process initiated by binding of integrins tospecific sites in the FN molecule (Leiss et al.,2008; Wierzbicka-Patynowski andSchwarzbauer, 2003). Studies in cultured cells have revealed thatgeneration of cytoskeletal tension through integrin-mediated cell attachment andanchoring via focal adhesions causes a change in integrin-bound FN conformation thatexposes cryptic self-association sites to allow FN fiber assembly (Leiss et al., 2008; Wierzbicka-Patynowski and Schwarzbauer, 2003). The small GTPases,including Rho and Rac, play critical roles in cytoskeletal tension generation viaregulation of actin polymerization and myosin phosphorylation (Dzamba et al., 2009; Zhong et al., 1998). In vivo, tissue tension and FNfibrillogenesis can also be generated by cell-cell adhesion and cohesivity (Dzamba et al., 2009). The observation that FNassembly proceeded normally in Fn-EIIIA deficient fibroblasts(Tan et al., 2004) led to the conclusionthat EIIIA domain is not required for matrix assembly (Leiss et al., 2008). Instead, fibril formation in thesecells was most likely mediated through binding of the major FN receptor,integrin-α5β1, to the Arg-Gly-Asp (RGD) motif located in the typeIII-10 module of FN (Fogerty et al., 1990),which has been considered the most important mechanism of FN assembly (Leiss et al., 2008).

Surprisingly, we found that in lymphatic endothelial cells theintegrin-α9-EIIIA interaction directly regulated FN fibril assembly, whileRGD-dependent interactions appeared dispensable for matrix formation. Although theEIIIA domain has not been previously implicated in FN fibrillogenesis, otherRGD-independent mechanisms for FN assembly have been observed. In fact, therequirement of the RGD motif for the formation of a functional fibrillar FN networkin vivo was recently challenged as it was found that miceexpressing FN with a non-functional RGD motif assembled an apparently normal FNmatrix (Takahashi et al., 2007). The assemblyof the RGD-deficient FN was mediated via integrin-αvβ3 binding toNGR sequence in the fifth N-terminal type I module of FN, which is converted to ahigh affinity binding site through deamidation (Takahashi et al., 2007). In addition, binding of Mn²⁺activated integrin-α4β1 to the CS-1 site of the alternativelyspliced V region was shown to promote FN assembly in vitro (Sechler et al., 2000).

Our findings suggest that the defective FN matrix organization inItga9 mutant valves is a direct consequence of the lack of aspecific integrin-matrix interaction and contributes to the observed defects invalve leaflet formation. In agreement, we found that a large proportion of lymphaticvalves in neonatal Fn-EIIIA deficient mice displayed defectiveleaflets similar to those observed in the Itga9 mutants. However,the absence of chylothorax and only partial recapitulation of theItga9^-/- phenotype suggest that the defect inlymphatic development in mice deficient of Itga9 cannot be solelyexplained by a defect in EIIIA-containing FN matrix assembly. In particular, theobservation that the valve defect in Fn-EIIIA^-/- micediminished in adulthood suggests that the animals can eventually overcome therequirement of FN-EIIIA, coinciding with when its expression is down-regulated. Thismay imply that during postnatal development other integrin-α9 ligand(s)become involved, or that FN fibrillogenesis can be mediated via alternativeintegrin-FN interactions. In agreement, we found that acute deletion ofintegrin-α9 postnatally in mature valves did not lead to degeneration of theleaflets within the one-week period investigated, suggesting that once the stablematrix fibrils are assembled, integrin-α9-EIIIA interaction is not requiredto maintain ECM organization and valve structure. However, our results do notexclude that integrin-α9-EIIIA is required for long-term maintenance ofvalves. Notably, although not normally present in adult tissues, FN-EIIIA isupregulated in various pathological situations, such as cancer, atherosclerosis andthrombosis (Muro et al., 2003; Tan et al., 2004; Villa et al., 2008). It is therefore likely that it isreactivated and plays a role also in adult lymphatic vessels under specificcircumstances, for example during repair processes or when the vasculature ischallenged by inflammation.

Why is integrin-α9 important specifically in valves? Unlike otherparts of the lymphatic vasculature, which have thin or absent basement membranes,the discrete architecture of the connective tissue of a valve leaflet suggestsspecific requirement for highly regulated mechanism of matrix organization. Indeed,the development of a related structure, the heart valve, is known to rely on theformation of a highly organized ECM, and consequently, dysregulation of ECM, oftencaused by genetic defects in matrix protein structure or expression, is linked toheart valve disease (Armstrong and Bischoff,2004; Lincoln et al., 2006). FNassembly is likely to play a key role in coordinating the formation of complexmatrices as it has been shown to initiate the organization of other ECM proteins,such as collagens, and control the stability of matrix fibers (Kadler et al., 2008; Sottile and Hocking, 2002). Our observations demonstrate thatintegrin-α9 is the predominant alpha subunit in lymphatic endothelia.Although the major FN receptor, integrin-α5, appeared to contribute to FNfibrillogenesis in the LECs in vitro, it was not able tofunctionally compensate for the loss of integrin-α9 either in cultured cellsor in vivo. Low expression levels of integrin-α5 in thedeveloping valves may provide one explanation for the dependency of these cells onalternative FN assembly pathways.

FN matrix assembly, as well as integrin-α9 signaling, promoteadhesion-dependent cell growth and migration (Leisset al., 2008; Liao et al., 2002;Singh et al., 2008; Sottile et al., 1998) and defects in these processes maytherefore underlie the disrupted valve leaflet formation inItga9^-/- andFn-EIIIA^-/- mice. Although we cannot exclude acontribution of integrin-α9-mediated adhesion during the early events ofvalve formation, the observation that acute deletion of integrin-α9 inpostnatal valves did not lead to cell detachment and degeneration of the valvesargue against a role of integrin-α9 in mediating stable adhesion of valveendothelial cells in vivo. Furthermore, an in vivoBrdU incorporation assay showed that the valve endothelial cells expressing highlevels of FoxC2, Prox1 and integrin-α9 do not proliferate during leafletformation (E.B. and T.M., unpublished). These results suggest that endothelial celladhesion and proliferation are unlikely to be controlled by integrin-α9 orinvolved in valve leaflet formation, respectively. Finally, the observed defect invalve leaflet elongation may be caused by failure in morphogenetic cell movements,which were shown to rely on proper organization of FN matrices in otherdevelopmental processes (Dzamba et al., 2009;Rozario et al., 2009). Therefore, wepropose that the key function of the integrin-α9-EIIIA interaction is toprovide an integrin-specific mechanism of FN matrix assembly and thereby coordinateECM organization to allow formation of a leaflet of necessary length and strength,capable of supporting valve function.

In summary, we demonstrate that integrin-α9 and its ligand,FN-EIIIA, play specific roles in lymphatic valve development and remodeling intofunctional leaflets. To our knowledge, the only other mouse mutants identifiedto-date, which show a failure of valve formation, show additional lymphatic defects;mice lacking the C-terminal PDZ binding domain of ephrinB2 display vesselhyperplasia (Makinen et al., 2005) whileFoxC2 deficient mice have patterning defects (Norrmen et al., 2009; Petrova et al., 2004). The specific valve defects observed in theItga9 mutant mice therefore reveal a previously undescribedmorphogenetic process and provide potential insights into the development of valvesin other parts of the vascular system, such as in the veins and the heart, which arelikely to be regulated by similar molecular mechanisms. Lack of integrin-α9signaling in mice results ultimately in chylothorax and postnatal death (Huang et al., 2000). Recent identification ofmutations in the ITGA9 gene in fetuses with congenital chylothorax(Ma et al., 2008) suggests conservationof the signaling pathway from mice to human, making integrin-α9 a candidategene for primary lymphedema caused by valve defects.

Experimental Procedures

Antibodies

The antibodies were rat antibodies to mouse PECAM-1 (MEC3.1, BDBiosciences), Tenascin-C (MTn-12, Abcam) and FoxC2 (Petrova et al., 2004); rabbit antibodies to mouseFibronectin (Millipore), Laminin-α5 (Ringelmann et al., 1999) and LYVE-1 (Reliatch); rabbit antibodies tohuman Prox1 (Reliatech) and Fibronectin (Abcam); mouse antibodies to humancellular Fibronectin recognizing the EIIIA domain ((Liao et al., 2002), FN-3E2, Sigma and IST-9, Abcam),hamster antibody to mouse PECAM-1, goat antibodies to mouse integrin-α9and Osteopontin (R&D Systems) and Cy3-conjugated mouse antibody againstα-smooth muscle actin (Sigma). The monoclonal hamster antibody to mousePodoplanin (8.1.1) developed by Andrew Farr was obtained from the DevelopmentalStudies Hybridoma Bank. Secondary antibodies conjugated to Cy2, Cy3 or Cy5 wereobtained from Jackson ImmunoResearch.

Mouse lines

Itga9^-/- (Huang et al., 2000), Itga9^lx (Singh et al., 2008),Vegfr3^lz/⁺ (Dumont et al., 1998),Fn-EIIIA^-/- (Muro et al., 2003), Tnc^-/- (Forsberg et al., 1996),Opn^-/- (Liawet al., 1998), Tie2-Cre (Koni et al., 2001) and Rosa26R(Soriano, 1999) mice have beendescribed previously. VE-cadherin-CreER^T2 mice willbe described elsewhere (R. Adams, unpublished). For the induction of Cremediated recombination in newbornVE-cadherin-CreER^T2 mice, 4-hydroxytamoxifen(4-OHT; 2 μl of 10 mg/ml dissolved in ethanol) was injected i.p. at P1and P2 and the vessels were analyzed at P7-8. All animal experiments wereperformed in accordance with UK Home Office and institutional guidelines.

Immunostaining and X-Gal staining

For whole-mount staining, the tissue was fixed in 4%paraformaldehyde (PFA), permeabilized in 0.3% Triton-X100 in PBS (PBSTx)and blocked in 5% milk or serum. Primary antibodies were added to theblocking buffer and incubated with the tissue overnight at 4°C. Afterwashes in PBSTx, the tissue was incubated with fluorochrome-conjugated secondaryantibodies in the blocking buffer for 2h at RT, followed by washing in PBSTx andmounting in Mowiol. The samples were analyzed using Zeiss LSM 510 laser scanningconfocal microscope. All confocal images, except Figures 5H, 5I and S4A-S4I, represent 2D projections of Z-stacks. Alternatively,biotin-conjugated secondary antibodies and ABC staining kit (VectorLaboratories) were used and the bound antibodies were visualized usingdiaminobenzidine (DAB) as a substrate. For histological analyses, skin biopsieswere fixed in 4% PFA overnight, dehydrated and embedded in paraffin.Sections (5μm) were stained after heat-induced epitope retrieval usingTyramide Signal Amplification kit (TSA, NEN Life Sciences) and3-Amino-9-Ethylcarbazole (AEC) as a substrate. For the visualization oflymphatic vessels in Vegfr3^lz/⁺reporter mice, the tissues were fixed with 0.2% glutaraldehyde andstained by the β-galactosidase substrate X-gal (Promega).

Visualization of lymphatic vessel function

FITC-dextran (Sigma, 8 mg/mL in PBS) or FITC-conjugatedLycopersicum Esculentum lectin (Vector Laboratories, 1mg/ml in PBS) was injected subcutaneously into an anesthetized mouse and thelymphatic vessels were analyzed by fluorescence microscopy.

Electron microscopy

The intestines were dissected from P5 mice and immediately fixed in4% PFA/2.5% glutaraldehyde in 0.1M phosphate buffer pH 7.4. Thesamples were post fixed in reduced osmium tetroxide for 1 hour followed by1% tannic acid in 0.05M sodium cacodylate for 45 minutes. Samples werethen dehydrated through a graded series of ethanol and embedded in Araldite(Agar Scientific). Semi-thin sections were cut on a UCT ultramicrotome (LeicaMicrosystems UK), stained with 1% Toluidine Blue in 1% Borax andviewed under a III RS light microscope (Carl Zeiss UK) to locate the area ofinterest. Ultra-thin sections were cut and stained with lead citrate beforebeing examined in a JEOL 1010 microscope and imaged with a Bioscan CCD (GatanUK).

Cell culture

Human lymphatic endothelial cells (LEC) were isolated from primarydermal microvascular endothelial cell cultures (PromoCell) using rat antibody tohuman Podoplanin (NZ-1, Angiobio) and Mini/MidiMACS magnetic separation system(Miltenyi Biotech), as previously described (Makinen et al., 2001). The cells were cultured on fibronectin(Sigma) coated plates in the presence of 10 ng/ml VEGF-C (R&D Systems)and used at passages 4-6. For siRNA mediated knock-down, LECs were transfectedtwice during 48h using calcium phosphate (Dharmacon) in DMEM, supplemented with20% FBS, followed by recovery in Endothelial Cell Medium (PromoCell) for24h before the cells were used for experiments. ON-TARGETplus siRNAs wereobtained from Dharmacon. The following targeting sequences were used:ITGA9_1: GAAGAAAGUCGUACUAUAG, ITGA9_2GUGCAGAGAUGUUUCAUGU and ITGA5: UCACAUCGCUCUCAACUUC. Controltransfections were carried out using ON-TARGETplus siControl Non-targeting siRNAfrom Dharmacon.

Analysis of FN fibril assembly

LECs in 5% FN-depleted serum were plated on glass coverslips.Similar results were obtained when the coverslips were coated with 0.2%gelatin (no exogenous FN) or with extracellular matrices extracted fromFN-EIIIA⁺/⁺(Muro et al., 2003) embryonicfibroblasts (containing small quantities of exogenous FN, all of which containsthe EIIIA domain). The function-blocking antibodies (mouse anti-humanintegrin-α9 (Y9A2; Millipore) and EIIIA (IST-9; Abcam)) and the blockingpeptides were used at a concentration of 10 μg/ml. 24 hours afterplating the cells were fixed in 4% PFA and FN fibril assembly wasexamined using immunofluorescence. Images from five randomly chosen view fieldsfrom two independent experiments were acquired. Image processing and analysiswas performed using MetaMorph Imaging software (Molecular Devices). Scaledimages were thresholded and filtered so that only FN-EIIIA-specific fibersgreater than 4 pixels (1.8 μm) were recorded. The total fiber length percell was calculated by dividing the total value recorded with the number ofcells present in each image. Isolation of DOC-soluble and – insolublematrix was done as previously described (Wierzbicka-Patynowski et al., 2004). Equal amounts of total proteinwere separated by non-reducing 5% SDS-PAGE, transferred tonitrocellulose and probed with anti-EIIIA antibody (FN-3E2).

Western blot analysis

The cells were lysed in Triton-X100 lysis buffer (50 mM Tris pH 7.5, 120mM NaCl, 10% glycerol, 1% TritonX-100) supplemented withComplete Protease Inhibitors (Roche). Lysates were clarified by centrifugationand subjected to immunoprecipitation and/or Western blot analysis using standardprotocols. The antibodies were mouse antibody to human integrin-α5(GBS5; Millipore) and human integrin-α9 (Y9A2; Millipore, for IP),chicken antibody to human integrin-α9 (GenWay, for WB) and mouseantibody to human α-tubulin (TAT-1). The bound antibodies were detectedusing HRP-conjugated secondary antibodies (Jackson ImmunoResearch) and ECLchemiluminescence.

Relative quantitative PCR

RNA from LECs was reverse transcribed using random hexamers and an avianmyeloblastosis virus reverse transcriptase (Promega). cDNA was amplified byquantitative real-time PCR (ABI 7900HT) using SYBR Green PCR master mix reagent(Qiagen). Each primer was used at a concentration of 0.5 μM. Cyclingconditions were as follows: step 1, 15 min at 95°C; step 2, 20 s at94°C; step 3, 20 s at 60°C; step 4, 20 s at 72°C, withrepeat from step 2 to step 4 35 times. Data from the reaction were collected andanalyzed by the complementary computer software. Relative quantitations of geneexpression were normalized to the endogenous control (GAPDH).The primers were ITGA9:5′-CGGAATCATGTCTCCAACCT-3′ and5′-TCTCTGCACCACCAGATGAG-3′, EIIIA:5′-TTGATCGCCCTAAAGGACTG-3′ and5′-ACCATCAGGTGCAGGGAATA-3′ and GAPDH:5′-GAAGATGGTGATGGGATTTC-3′ and5′-GAAGGTGAAGGTCGGAGT-3′.

Statistical analysis

P values were calculated using the non-parametric Mann-Whitney test,unpaired two-tailed Student T-test or χ² test asindicated.

Supplementary Material

NIHMS141065-supplement-01.doc

Figure 1

Expression of integrin-α9 in mature and developing lymphaticvalves

(A, B) Immunofluorescence staining of adult ear skin with antibodies againstintegrin-α9 (green), FoxC2 (red) and α-smooth muscle actin(α-SMA, blue). Arrow in (B) points to a luminal valve. (C-E)Development of mesenteric lymphatic vessels. Whole-mount X-Gal staining ofmesenteric lymphatic vessels fromVegfr3^lz/⁺ embryos. Thetissues were taken from embryos at the indicated ages (E16.5-E18.5).

(F-K) Immunofluorescence staining of developing mesenteric lymphatic vesselsof E16.5 (F, G), E17.5 (H, I) and E18.5 (J, K) with antibodies againstintegrin-α9 (green), Prox1 (F, red) or FoxC2 (H, J, red) and LYVE-1(F, H, J, blue). Arrowhead in (F, G) points to a blood vessel, the smoothmuscle coverage of which is positive for integrin-α9 staining.Arrows point to clusters of cells expressing high levels of Prox1 andFoxC2.

Scale bars; A, B, H-K: 50 μm, C-G: 1 mm.

Figure 2

Abnormal valves in Itga9 deficient mice

(A-B′) Luminal valves in chyle-filled mesenteric lymphatic vessels ofwild-type (A, A′) and Itga9 mutant mice (B,B′). Note the difference in the shape of a wild-type in comparisonto a mutant valve (A′, B′, arrows in A, B) and leakage ofchyle from the mutant vessels (arrowhead in B). BF = brightfield.

(C, D) PECAM-1 immunohistochemistry of P5 mesenteric vessels and luminalvalves (arrow) in wild-type (C) and Itga9^-/- (D)mice.

(E) Quantification of the number of luminal valves in P5 wild-type andItga9^-/- mesenteric lymphatic vessels(mean ± s.d., n = 4 animals per genotype, 3 vessels each).Black bar = normal V-shaped valves; white bar = abnormalvalves with ring appearance. *** p < 0.0001(Mann-Whitney test).

(F) Schematic representation of luminal valves (arrows) in the collectinglymphatic vessels ofItga9⁺/⁺and Itga9^-/- mice.

(G-J) Transmission electron micrographs of wild-type (G, I) andItga9^-/- (H, J) valves in mesentericlymphatic vessels of P6 mice. Arrows in (G, H) point to the matrix core(red) anchored into the vessels wall, arrowheads mark the free edges of thevalve leaflets. (I) shows the valve leaflet with a connective tissue core(red). Note the rudimentary (arrows in H) or absent (J) matrix core in themutant valves and the gaps in between the two endothelial sheets (redasterisks in J).

(K) Schematic representation of luminal valves in theItga9⁺/⁺and Itga9^-/- mice. Matrix core is indicated inred.

Scale bars; A, B: 100 μm, C, D: 50 μm, G, H: 10 μm,I, J: 1 μm.

Figure 3

Defective lymphatic drainage in Itga9 deficientmice

(A, B) Visualisation of dermal collecting vessels following injection ofFITC-dextran into the footpads of P6 wild-type (A) andItga9^-/- mice (B). Note the presence ofan abnormal vessel network (arrowheads in B) and a valve in a vessel branchpoint in the Itga9 mutants (arrow in B).

(C, D) FITC-lectin (LEL) staining of the valves in dermal lymphatic vesselsfollowing footpad injection. No valve leaflets are seen in theItga9 mutant (D). Arrows in (C) point to the two valveleaflets seen from the 90° angle when compared to Figure 2A, D.

(E) Schematic representation of a side view of luminal valves as visualizedby FITC-LEL staining in the collecting lymphatic vessels ofItga9⁺/⁺(left) and Itga9^-/- (right) mice. Arrowsindicate the direction of the flow.

Scale bars; A, B: 100 μm, C, D: 50 μm.

Figure 4

Endothelial cell specific deletion of Itga9 duringdevelopment and in mature valves

Lymphatic vessels of Tie2-Cre;Itga9^lx/lx mouse(A-D), and of 4-OHT treatedItga9^lx/⁺ (E-H) andVEcad-CreER^T2; Itga9^lx/lx(I-L) mice stained with antibodies against Laminin-α5 (red),integrin-α9 (green) and PECAM-1 (blue). Expression ofintegrin-α9 is detected in the vascular SMC (arrowhead in A, C, E,G, I, K) but is lost from the endothelial cells inTie2-Cre;Itga9^lx/lx (C) and from mostendothelial cells of the valves of VEcad-CreER^T2;Itga9^lx/lx mutant animals (I, K, open arrowheadpoints to a single integrin-α9 expressing cell). Note the abnormalvalve in Tie2-Cre;Itga9^lx/lx mouse (arrow in B),which has undergone embryonic deletion of Itga9 allele, butintact valve leaflets (arrow in J) and the attachment of LECs on theleaflets in the VEcad-CreER^T2;Itga9^lx/lx mutant (arrow in L), which has undergonepostnatal deletion of Itga9 allele, as compared to acontrol (arrows in F, G).

Scale bars; A-L: 20 μm.

Figure 5

Development of lymphatic valve leaflets in wild-type andItga9^-/- mice

(A-C) Immunofluorescence staining of developing mesenteric lymphatic vesselsof E16 (A) and E17 (B, C) wild-type embryo using antibodies againstLaminin-α5, integrin-α9 and FN-EIIIA (colors as indicated).The dotted lines outline the vessels.

(D-G) Immunolabeling of lymphatic valves in wild-type (D-E′) andItga9^-/- (F-G′) mesentericvessels for Prox1 (green) and FN-EIIIA (red; E, F; at E17) or forLaminin-α5 (red), FN-EIIIA (green) and the endothelial markerPECAM-1 (blue; E, G; at P2). The arrows in (D, E′, F, G′)point to FN-EIIIA fibers.

(H, I) View through the opening of the valve in P0 wild-type (H) andItga9^-/- (I) vessels, labelled for FN(red) and FN-EIIIA (green). Note the punctuate localization of FN-EIIIA inthe Itga9^-/- valve (arrow in I) compared to thefibrous staining in the wild-type (arrow in H).

(J) Schematic model of lymphatic valve formation. Upregulation of Prox1 andFoxC2 transcription factors (blue nuclei) in lymphatic vessels define thepositions of future valves. Deposition of extracellular matrix (red)containing Laminin-α5 and FN-EIIIA and re-orientation of cellsexpressing high levels of Prox1 and FoxC2 perpendicular to the vessel wallis followed by upregulation of integrin-α9 (green) on the outflowside of the future valve. Itga9^-/- mice (below)display defective organization of the extracellular matrix and failure ofleaflet formation.

Scale bars; A-G: 50 μm, H, I: 10 μm.

Figure 6

Abnormal lymphatic valves in mice lacking the integrin-α9 ligand,FN-EIIIA

(A) Luminal valve numbers in newborn wild-type,Fn-EIIIA^-/- andItga9^-/- mesenteric lymphatic vessels(mean ± s.d., n ≥ 4 animals per genotype, ≥ 2vessels each, see Suppl.Table 1). The percentage of abnormal valves is indicated:*** p < 0.0001 (χ²test).

(B, C) Visualization of lymphatic valves in P1 wild-type (B) andFn-EIIIA^-/- (C) mesenteric vessels usingantibodies against Laminin-α5. Note the incomplete development ofthe valve as evident by lack of leaflets (arrow in C) in theFn-EIIIA^-/- vessels.

(D) Immunofluorescence staining of three weeks old ear skin forintegrin-α9 (green) and EIIIA (red).

(E, F) Dermal lymphatic vessels in the ears of three weeks old wild-type (E)and Fn-EIIIA^-/- (F) mice labeled forLaminin-α5 (green), podoplanin (blue) and α-SMA (red).

(G-I) FITC-dextran assay in three weeks old wild-type (G) andFn-EIIIA^-/- mice (H, I). Note the refluxof dye (arrows in H) and an abnormal valve (arrow in I) in the mutantskin.

Scale bars; B, C: 20 μm, D:50μm, E, F, I: 100 μm, G,H: 400 μm.

Figure 7

Integrin-α9-EIIIA interaction regulates FN fibril assembly inprimary human lymphatic endothelial cells

(A) FN fibrils in primary human lymphatic endothelial cells (LECs).Integrin-α9-EIIIA interaction was blocked using antibodies againstEIIIA (IST-9) or integrin-α9β1 (Y9A2), or siRNA againstintegrin-α9 or -α5, and stained with EIIIA antibodies.

(B) Quantification of FN fibrillogenesis in the LECs, in whichintegrin-α9-EIIIA interaction (IST-9, Y9A2, α9 siRNA) orintegrin-α5/RGD-dependent integrin interactions (RGDSP peptide,α5 siRNA) were inhibited, in comparison to the control cells(untreated, ctrl siRNA or RGESP peptide). Data represent mean FN-EIIIA fiberlength per cell (± s.d) from five randomly chosen view fields in twoindependent experiments. *** p< 0.003,n.s.= non-significant, p = 0.881 (Student T-test).

(D) siRNA mediated knock-down of integrin expression in primary human LECs.Western blot analysis of immunoprecipitated (IP) cell lysates usingintegrin-α9 or -α5 antibodies (upper panels). For theloading control, the total cell lysates (TCL) were blotted againstα-tubulin and EIIIA.

(E) Conversion of DOC-soluble FN fibrils into insoluble stable matrix.DOC-insoluble (upper panel) and -soluble matrix (lower panel) isolated fromthe LECs were separated in non-reducing SDS-PAGE and probed for EIIIA.

(F) Immunofluorescent staining of wild type E18 mesenteric vessels usingantibodies against integrin-α9 (left panel) and integrin-α5(right panel). Note low levels of integrin-α5 expression in thevalve (arrows) in comparison to strong staining in the blood vesselendothelia (arrowhead).

Scale bar = 20 μm.

Footnotes

Author contributions: E.B. and T.M. designed research; E.B., S.X., C.C. and A.W.performed research; N.M., L.S., R.A., A.M. and D.S. contributed reagents; E.B.,S.X., C.C., A.W. and T.M. analysed data; and E.B. and T.M. wrote the paper.

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Acknowledgments

We are grateful to K. Alitalo for theVegfr3^lz/⁺ mice and R.Fässler, A. Faissner and T. Czopka for the Tnc mice andtissues. We thank I. Rosewell for help with the establishment of mouse colonies; H.Chapman, A. Horwood, E. Murray, S. Lighterness and C. Watkins for animal husbandry;and E. Nye for assistance with the histological analyses. We would like to thank M.Way, N. Hogg, M. Graupera and I. Ferby for helpful discussions and critical commentson the manuscript. This work has been supported by Cancer Research UK (E.B., S.X.,A.W., R.A., T.M.) and by grant HL64353 from the National Heart Lung and BloodInstitute (USA) (D.S.). The authors declare no financial conflict of interest.

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