IntroductionAngiogenesis involves the formation of capillaries from pre-existing microvessels and therefore contributes to vascularremodeling and maturation (Carmeliet, 2000). It plays a pivotalfunction in a variety of normal and pathological conditionssuch as embryonic development, the menstrual cycle, haircycle, wound healing, arthritis, psoriasis, proliferative diabeticretinopathy, atherosclerosis, post ischemic vascularization ofthe myocardium and tumor growth and metastasis (Folkman,1995; Risau, 1997). The initiation of the physiopathologicalangiogenic response, known as the ‘angiogenic switch’,depends on the dynamic balance between exogenous orendogenous stimuli (pro-angiogenic factors) and inhibitors(anti-angiogenic factors) acting in the immediate environment

of endothelial cells (Liotta et al., 1993; Hanahan and Folkman,1996; Distler et al., 2002). Disruption of such an equilibriumcan favor the emergence of new vessel formation or lead tovessel quiescence or regression (Hanahan and Folkman, 1996).Several protein families can promote angiogenesis, amongwhich are growth factors, cytokines, integrins, proteinases,extracellular matrix components and cell adhesive proteins(Folkman, 1995; Risau, 1997; Carmeliet and Jain, 2000). Thecontribution of matrix metalloproteinases (MMPs), especiallyMMP-2, MMP-9 and membrane-type metalloproteinase-1(MT1-MMP, MMP-14) has been convincingly established bythe use of natural or synthetic MMP inhibitors, both in vitroand in vivo (Moses, 1997; Maekawa et al., 1999; Hajitou etal., 2001). Genetic studies used mice deficient in those

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Elastin-derived peptides display a wide range of biologicalactivities in a number of normal and transformed cells buttheir involvement in angiogenesis has not been reported. Inthe present study, we show that κ-elastin and VGVAPGhexapeptide elastin motif accelerated angiogenesis in thechick chorio-allantoic membrane in an in vivo model.They also stimulated pseudotube formation from humanvascular and microvascular endothelial cells in the matrigeland collagen models as well as cell migration in an in vitrowound healing assay. Confocal and scanning electronmicroscopy analyses revealed the main reorganization ofactin filaments mediated by elastin-derived peptides andchanges in cell shape that correlated with a decrease ofthe cell form factor determined by computerized imageanalysis. Such elastin-derived peptide effects wereattributed to upregulation of proMT1-MMP and proMMP-2 expression and activation at both the mRNA and proteinlevels. Batimastat, an inhibitor of furin convertase andTIMP-2, but not TIMP-1, totally abolished the influence ofelastin-derived peptides (EDPs) on cell migration andtubulogenesis, thus favoring the involvement of MT1-MMP

in such processes. To assess its contribution to EDP-mediated angiogenesis further, we used a small interferingRNA (siRNA) approach for specifically silencing MT1-MMP in human microvascular endothelial cells. Four setsof 21 bp siRNA duplexes targeting MT1-MMP mRNA weresynthesized by in vitro transcription. Two of them provedto inhibit MT1-MMP expression efficiently but did notaffect MT2-, MT3- and MT5-MMP expression. Seventy-two hours after transfection with 25 nM siRNAs EDP-induced MT1-MMP expression at the mRNA and proteinlevels was decreased fourfold. In parallel, proMMP-2activation was inhibited. A scrambled siRNA, used as anegative control, had no effect. Finally, the effect of elastinpeptides on pseudotube formation in MT1-MMP-siRNAtransfected cells was totally abolished. These dataemphasise the crucial role of MT1-MMP in the elastin-induced angiogenic phenotype of endothelial cells.

Key words: Angiogenesis, Elastin, Matrix metalloproteinase, Elastinreceptor, siRNA

Summary

Elastin-derived peptides enhance angiogenesis bypromoting endothelial cell migration andtubulogenesis through upregulation of MT1-MMPArnaud Robinet1,*, Abdel Fahem1,*, Jean-Hubert Cauchard1, Eric Huet1, Loïc Vincent2, Sandrine Lorimier3,Franck Antonicelli1, Claudine Soria2, Michel Crepin4, William Hornebeck1 and Georges Bellon1,‡

1Laboratoire de Biochimie et Biologie Moléculaire, CNRS UMR 6198, IFR 53 Biomolécules, Faculté de Médecine, Université de ReimsChampagne-Ardenne, 51 rue Cognacq Jay, 51095 Reims CEDEX, France2Groupe de Recherche MERCI, EA CNRS 2122, Faculté de Médecine et de Pharmacie, 22 Boulevard Gambetta, 78183 Rouen, France3Laboratoire Biomatériaux, INSERM EMI, IFR 53 Biomolécules, Faculté d’Odontologie, Université de Reims-Champagne-Ardenne,1 rue du Maréchal Juin, 51095 Reims CEDEX, France4Laboratoire Hémostase, Endothélium et Angiogenèse, INSERM U553, Hôpital St Louis, 1 Avenue Claude Vellefaux, 75475 Paris CEDEX 10,France*These authors contributed equally to this work‡Author for correspondence (e-mail: georges.bellon@univ-reims.fr)

Accepted 22 October 2004Journal of Cell Science 118, 343-356 Published by The Company of Biologists 2005doi:10.1242/jcs.01613

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endopeptidases and showed reduced angiogenic responses(Itoh et al., 1998; Vu et al., 1998; Zhou et al., 2000) and invitro studies used antisense oligonucleotides or antibodiesdirected against those MMPs (Fang et al., 2000; Lafleur et al.,2002). The participation of neutral proteinases on the cellinvasive program is far more complicated than originallydepicted (Hanahan and Folkman, 1996) as they can generateproteolytic fragments of the extracellular matrix (ECM), i.e.matrikines, or reveal cryptic sites within the matrix, i.e.matricryptines which behave as cytokine-like molecules,further influencing angiogenesis, either negatively or positively(Bellon et al., 2004). Overproduction of matristatins ordownregulation of stimulatory angiogenic peptides, or both,might be involved in defective wound repair, ischemia andperipheral arterial occlusive disease (Maquart et al., 2004).Conversely, the opposite imbalance could lead to excessiveangiogenesis, a hallmark of inflammatory disorders in manyorgans, and a prerequisite for tumor progression (Hanahan andFolkman, 1996; Zetter, 1998). Such matrikines, some of thembeing evidenced in the blood circulation, have been shown toexert their pro- or anti-angiogenic activity at distinct levelsof the angiogenesis process either by interfering with cellproliferation or migration or apoptosis and through differentmechanisms (Bellon et al., 2004).

Elastin occupies a privileged site in matrix biology, as it isthe major component of elastic fibers, particularly abundantin tissues such as arteries and lung, but also present in skin,breast, cartilage and certain ligaments. It is also a long-livedmacromolecule with no appreciable turn-over (Rosenbloom,1987). However, its proteolysis by elastase-type proteinasesbelonging to the metallo, serine and cysteine families islinked to the genesis of several diseases affecting elastin-richorgans (Werb et al., 1982; Visse and Nagase, 2003). Besidesaltering the rheological property of those tissues, elastolysisthrough the generation of elastin-derived peptides (EDPs)might interfere strongly with tissue homeostasis. Thus, anysign of its degradation can represent a vital signal fororganisms to initiate effective repair processes. Overall, thosepeptides were reported to display a wide range of biologicalactivities, influencing cell migration (Senior et al., 1980;Hinek et al., 1992), differentiation (Grant et al., 1989),proliferation and chemotaxis (Kamoun et al., 1995; Ghuysenet al., 1992), tumor progression (Lapis and Timar, 2002;Timar et al., 1991; Huet et al., 2002; Ntayi et al., 2004),aneurysm formation and atherogenesis (Nackman et al.,1997; Hance et al., 2002; Robert, 1996). With regard to tumorinvasion, soluble peptides from alkaline (κ-elastin) or elastasehydrolysis of insoluble elastin, as well as tropoelastin wereshown to increase MMP-2 and MMP-3 production by humanskin fibroblasts (Brassart et al., 2001; Huet et al., 2001);similarly, κ-elastin stimulated MMP-2, MT1-MMP andTIMP-2 expression in human HT-1080 fibrosarcoma celllines and, as a consequence, could promote the invasivemetastatic ability of tumor cells (Huet et al., 2002; Brassartet al., 1998). These numerous effects are mediated by thebinding of EDPs to a 67-kDa multifunctional high affinityreceptor with lectin-like properties named EBP (elastinbinding protein) (Mecham et al., 1989; Hinek, 1994). EBPwas further identified as an inactive spliced form of β-galactosidase known as S-Gal (Hinek et al., 1993; Priviteraet al., 1998), expressed in elastin-producing and non-

producing cells (Hinek, 1995; Yusa et al., 1989). S-Gal isassociated at the cell surface with two other plasmamembrane-anchored proteins, a 61-kDa moiety withneuraminidase activity and a 55-kDa protective proteincorresponding to carboxypeptidase A or cathepsin A (Hinek,1996). Cell responses to EDPs were often attributed to thebinding of a VGVAPG hexapeptide sequence, repeatedseveral times in tropoelastin, the soluble precursor form ofelastin, to a unique sequence of S-Gal, encoded by the frameshifted exon 5 of β-galactosidase. Previous investigationsindicated that the VGV tripeptide could represent the coresequence in EDPs, mediating their potent influence onvascular tone through increased intracellular calcium inendothelial cells (Faury et al., 1995; Faury et al., 1998a;Faury et al., 1998b). Conversely, a type VIII β-turnconformation adopted by elastin peptides with the GXXPGsequence was mainly involved in directing matrixmetalloproteinase expression in fibroblasts and fibrosarcomacells in culture (Brassart et al., 2001; Huet et al., 2002).

An angiogenic response has been correlated with extensivealterations and remodeling of elastic fibers and, in a rataneurysm model, administration of VGVAPG hexapeptide orelastase was shown to induce adventitial angiogenesis(Nackman et al., 1997). In addition, EDPs, at a concentrationaveraging that present in the circulation (Kucich et al., 1983;Fülöp et al., 1990), activate low specificity calcium channelsin human umbilical venous endothelial cells (HUVECs),leading to an enhancement of cytoplasmic and nuclear-freecalcium concentration (Faury et al., 1998a). Suchmodifications in calcium flux induce an endothelium-dependent vasodilatation that can be suppressed by an inhibitorof nitric oxide production (Faury et al., 1995; Faury et al.,1998a; Faury et al., 1998b).

In the present study, we have shown that occupancy ofEBP by VGVAPG motif-containing peptides on endothelialcells from vascular and microvascular origins triggeredneoangiogenesis by promoting cell migration andtubulogenesis. The effects induced by EDPs were linked toupregulation of proMT1-MMP and proMMP-2 expression andactivation. The contribution of MT1-MMP in EDP-mediatedangiogenesis was demonstrated by the use of specific inhibitorsof MT1-MMP and a siRNA approach for specifically silencingMT1-MMP expression in human microvascular endothelialcells (HMECs). We have demonstrated that two 21-bp siRNAduplexes targeting MT1-MMP mRNA at position 107-127 and228-248 relative to the start codon, respectively, proved todecrease MT1-MMP expression fourfold after a 72-hourtransfection. In parallel, siRNA107-127 totally suppressed theeffect of elastin peptides on pseudotubes formation byHMECs.

Materials and MethodsReagentsInsoluble elastin and soluble κ-elastin peptides (KE) were obtained aspreviously described (Jacob and Hornebeck, 1985). VGVAPG andVVGSPSAQDEASPL (V14) peptides were synthesized using 9-fluoromethoxycarbonyl (Fmoc) chemistry using a Fmoc-Val resin(0.22 meq/g). Couplings were performed with Fmoc-amino acid-pentafluorophenyl esters (Pfb) (4 molar excess). Each Fmocdeprotection step involved treatment with 20% (v/v) piperidine/dimethylformamide for 10 minutes. Cleavage of the peptide from the

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resin was achieved by a six-hour treatment with trifluoroaceticacid/water (95/5; v/v), followed by successive washings of the resinwith ether. Purity of peptides was confirmed by HPLC and by fastatom bombardment mass spectrometry.

In vivo angiogenesis assay in the chick chorio-allantoicmembrane (CAM) modelIn vivo angiogenesis was performed according to an established shell-less culture technique, exposing the CAM to direct access forexperimental handling (Averbar et al., 1974). At day six of embryonicdevelopment, angiogenic areas were delimited with a silicon ring(Weber Métaux, France) and PBS (as control) or κ-elastin (50 ng) orVGVAPG peptide (200 ng) in a final volume of 20 µl were placed insidethe rings. The embryos were then placed in an incubator to inducespontaneous angiogenesis and were treated daily. Treated areas werephotographed daily from day 6 to day 10 of embryonic development.

Cell culturesHuman microvascular endothelial cell-1 line (HMEC) was providedby E. W. Ades (Center for Disease Control and Prevention, Atlanta,GA). HMECs are representative of the microvasculature and haveproperties similar to those of the original primary culture (Ades et al.,1992). HMECs were cultured in endothelial cell growth medium(ECGM) MV (PromoCell, Heidelberg, Germany) supplemented with0.4% (w/v) ECGS/H, 2% (v/v) fetal calf serum (FCS), 10 ng/mlepidermal growth factor (EGF), 1 µg/ml hydrocortisone, 50 ng/mlamphotericin B and 50 µg/ml gentamicin. Human umbilical veinendothelial cells (HUVECs) were purchased from PromoCell(Heidelberg, Germany) and were used between the second and eighthpassages. HUVECs were cultured in ECGM supplemented with 0.4%(w/v) ECGS/H, 2% (v/v) FCS, 0.1 ng/ml EGF, 1 ng/ml basic fibroblastgrowth factor (bFGF), 1 µg/ml hydrocortisone, 50 ng/ml amphotericinB and 50 µg/ml gentamicin.

Cell proliferation and survivalHMECs (104 cells) were seeded in 24-well culture plates and culturedfor various periods in FCS-free ECGM MV in the absence (control)or presence of varying concentrations of κ-elastin (KE) or VEGF (20ng/ml) or bFGF (20 ng/ml). At the end of each period of incubation,the cell number was determined using the violet crystal assay (Kuenget al., 1989). A standard absorbance curve was established.

Capillary tubes formation on matrigelMatrigel matrix (Sigma, France) was kept on ice for 24 hours. Then,200 µl matrigel per well was added to a 24-well culture plate. Afterincubation at 37°C for 30 minutes, the gels were overlaid with 500 µlECGM for HUVECs or ECGM MV for HMECs containing 4×104

cells. Endothelial cells were then incubated in the absence or presenceof KE, VGVAPG peptide or various agents at concentrations indicatedin the text. Aiming to verify the contribution of S-Gal on EDP-mediated angiogenesis, we used lactose and V14 peptide asantagonists. Lactose was used to inhibit the binding of EDPs on EBPin keeping with the galactolectin-like property of this receptor (Hineket al., 1998). V14 in turn, a 14mer peptide corresponding to part ofthe elastin binding sequence of EBP was used to compete with thebinding of EDPs to EBP. The involvement of MMPs wasdemonstrated through the use of specific inhibitors: batimastat, apotent broad-spectrum MMP inhibitor, TIMP-2 known to inhibitMT1-MMP and MMP-2 without distinction, TIMP-1 that inhibitsMMP-2 but not MT1-MMP and Dec-RVLR-cmk, a chloromethylketone peptide known to specifically impede proMT1-MMPactivation by furin convertase, its intracellular activator. Forexperiments with siRNA (see below), 250 µl per well of matrigel was

added to a 24-well culture plate. After incubation at 37°C for 15minutes, the gels were overlaid with 500 µl ECGM MV containing3×104 HMECs pre-transfected with 25 nM siRNA107 or scrambledsiRNA107. HMECs were then incubated in the absence or presence of1 µg/ml KE in ECGM MV containing 2% (v/v) FCS. Capillary tubeformation was observed at different times during a culture period of24 hours with a phase-contrast microscope (Axiovert 25, Zeiss)equipped with a digital camera (Sony). Images were taken with aphase-contrast microscope with a 10× lens and semi-quantitativeevaluation of pseudotube formation was performed in ten randomlyselected fields (after black and white pixelization) by determining thenumber of black pixels relative to the total pixels. Experiments wereperformed in quadruplicate.

Capillary tubes formation in type I collagen latticeCapillary tube formation in three-dimensional type I collagen gel wasperformed as previously reported (Trochon et al., 1996). Briefly,HMECs (10,000 cells/well) were seeded on 2% (w/v) agarose gel ina 96-well plate. After an 18-hour incubation in complete ECGMMV, aggregate-forming cells were then incorporated into a three-dimensional type I collagen gel and incubated for 3 days at 37°C inFCS-free ECGM MV in the absence or presence of 100 ng/ml KE.Tube formation was observed by phase-contrast microscopy.

Wound healing assayEndothelial cell migration assay was performed as previouslydescribed (Vincent et al., 2001). Briefly, HMECs (1×105 cells/well)were seeded in 24-well plates and grown to confluence in ECGM MV.The cell monolayer was disrupted with a 1 mm cell scraper, and afterwashing with PBS, endothelial cells were incubated in basal ECGMMV supplemented with 2% (v/v) FCS (a concentration of FCS thatallows cell survival but not cell proliferation) and stimulated in theabsence or presence of KE (10, 100 and 200 ng/ml), VGVAPG peptide(10, 100, 200 ng/ml), bFGF (20 ng/ml) or VEGF (20 ng/ml). In someexperiments, bFGF or VEGF were simultaneously added with KE orVGVAPG peptide. The influence of TIMP-2 or batimastat on elastinpeptides-induced cell migration was evaluated at concentrationsindicated in the text. After 24 hours of incubation, images were takenusing a phase-contrast microscope with a 5× lens and cell migrationwas determined by measuring the number of cells invading a 0.5 mm2

wounding area. Experiments were done in triplicate and ten fieldsfrom each well were randomly selected for cell counting. Thepercentage of migrating cells was determined relative to the controlin the absence of effector.

Confocal microscopy analysisConfocal microscopy analysis of actin filaments was performed onHUVECs and HMECs cultured on matrigel for 14 and 24 hours,respectively, in the absence or presence of KE (50 µg/ml). Actinfilaments were visualized by tetramethyl-rhodamine isothiocyanate-labeled phalloidin as described (Manelli-Oliveira and Machado-Santelli, 2001). Confocal microscopy analysis of siRNAs was thenperformed. siRNAs were fluorescently labelled with Cy3 using thesilencer™ siRNA labeling kit (Ambion, Huntingdon, UK) accordingto the manufacturer’s instructions. HMECs were cultured oncoverslips (LabTek, Nunc) in 24-well plates (Nunc, Roskilde,Denmark) in ECGM MV at 60% subconfluency and then transfectedwith 25 nM labeled siRNAs using 2 µl/well oligofectamine(Invitrogen, Cergy Pontoise, France) as transfection reagent in FCS-free medium for 72 hours at 37°C. Cells were observed by confocallaser-scanning microscopy using an MRC-1024 imaging system(Bio-Rad, Microscience, Hemel Hempstead, UK) coupled to anepifluorescence microscope (Olympus, Tokyo, Japan), equipped witha ×60 water immersion lens.

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Scanning electron microscopyHUVECs were cultured on matrigel in the absence or presence of KE(50 µg/ml) for 14 hours and scanning electron microscopy analysiswas performed as previously described (Anderson, 1966). Theobservations were carried out with a JEOL JSM 5400 LV scanningelectron microscope. Changes in HUVEC shape were determinedby semi-automatic and automatic quantitative image analysis aspreviously described (Godeau et al., 1986). Cell form factor wasdetermined on 60 cells. For this purpose, the perimeter (P) and thesurface (S) of a cell were determined from the outline of individualendothelial cells which were manually delimited with the pointer ofthe computer using the Imagenia 3000 Program (Biocom 200,Courtaboeuf, France). Evaluation of the form factor was determinedby the formula 4πS/P2, which is used as an index of the circularity ofcells (Baak and Ort, 1983; Flook, 1987; Lorimier et al., 1998). Asmentioned by Flook, the value of the circularity can decrease byelongation of the shape or by the addition of highly textured orserrated edge to an otherwise circular feature as can be observed whena cell progresses from an adhering state with a rounded aspect to aspreading or migrating state.

MMP and TIMP quantificationPreparation of cell extracts

Gelatinolytic activities of MT1-MMP and EBP associated withplasma membranes were identified by gelatin zymography andwestern blot analysis as previously described (Brassart et al., 1998;Ntayi et al., 2004). Quantitative determination of total MT1-MMPwas performed using the Biotrak MT1-MMP activity assay system(Amersham Biosciences Europe, Orsay, France) according to themanufacturer’s instructions.

Zymography analysisMMPs and TIMPs were analyzed by gelatin zymography and reversezymography, respectively, as previously described (Huet et al., 2002).Molecular weight markers (Bio-Rad Laboratories) and recombinantMMP-2 (VWR International, Strasbourg, France) were added to eachgel analyzed. Recombinant TIMP-1 and TIMP-2 (Calbiochem, MerckEurolab, Fontenay-sous-Bois, France) were used as markers. Areas ofgelatinolytic or TIMP activity were measured by automated imageanalysis, as described previously (Huet et al., 2002).

Western blot analysisProtein samples were separated in 0.1% (w/v) SDS, 10% (w/v)polyacrylamide gel (Laemmli, 1970). After electrophoresis, proteinswere transferred to Immobilon-P membranes (Millipore, Saint-Quentinen Yvelines, France) as previously described (Towbin et al., 1979). Then,the membranes were saturated with 5% (v/v) FCS, in 50 mM Tris-HCl,150 mM NaCl, pH 7.5 (TBS) for 2 hours, and probed with either a mouseanti-human MT1-MMP monoclonal antibody (clone 114-1F2 fromCalbiochem) (1:1000) or rabbit anti-human MT1-MMP (hinge region)polyclonal antibodies (1:5000) (Euromedex, Soufflewehersheim,France) or rabbit anti-human EBP polyclonal antibodies (Ntayi et al.,2004) directed against the EBP binding sequence of elastin (V14peptide), overnight at 4°C. Membranes were then extensively washed inTBS containing 01% (v/v) Tween-20 (TBS/Tween), then probed withhorseradish peroxidase (HRP)-conjugated sheep anti-rabbit or anti-mouse IgG antibodies (1:10,000) for 1 hour at 20°C. Membranes werewashed in TBS/Tween and immunocomplexes were visualized bychemiluminescence using the ECL+ system according to themanufacturer’s instructions (Amersham, France).

RT-PCR analysesTotal RNA from HUVECs or HMECs cultured as monolayers was

harvested by using RNAzol B (Biogenesis LTD, Poole, UK), andextracted by the acid guanidinium/phenol/chloroform extractionmethod (Chomczynski and Sacchi, 1987). RNA concentration wasdetermined by absorbance measurement at 260 nm and its integritychecked by 1.5% (w/v) agarose gel electrophoresis. RT-PCR wasperformed with 1 µg total RNA using the Hybaid Omnigenthermocycler (Teddington, Middx, UK) and two pairs ofoligonucleotides (Invitrogen, France) as already reported(Giambernardi et al., 1997). Forward and reverse primers for humanMT1-MMP and GAPDH were designed. MT1-MMP primers: forward,5′-CGCTACGCCATCCAGGGTCTCAAA-3′; reverse, 5′-CGGTCAT-CATCGGGCAGCACAAAA-3′; and GAPDH primers: forward, 5′-ACCACAGTCCATGCCATCA-3′; reverse, 5′-TCCACCACCCTGT-TGCTGT-3′.

Forward and reverse primers for human MT2-MMP, MT3-MMPand MT5-MMP were made as previously described (Ueda et al.,2003). cDNA products were amplified for 26, 28 and 32 cycles for allassays. PCR products were separated on 1.5% (v/v) agarose gelcontaining 1 µl/ml ethidium bromide and fragments of 497 bp, 578bp, 461 bp, 565 bp and 452 bp lengths were obtained for MT1-MMP,MT2-MMP, MT3-MMP, MT5-MMP and GAPDH, respectively. Thefluorescence of the bands was evaluated by scanning the gel at 312nm and computerized using the Bio-Profile software (VilbertLourmat, Marne la Vallée, France).

SiRNA preparationThe 21-nucleotide RNAs were synthesized by in vitro transcriptionusing the silencer™ siRNA construction kit (Ambion). Four sets ofspecific siRNAs targeting human MT1-MMP mRNA (GenBankaccession number Z48481) corresponding to the coding regions 107-127, 228-248, 949-969 and 1462-1482 relative to the start codon weretested but only the results obtained with siRNA107-227 were shown. Aleader sequence (CCTGTCTC) complementary to the T7 promoterprimer was incorporated at the 3′ end of each sense and antisenseoligonucleotide. In vitro transcription was carried out according to themanufacturer’s instructions except for annealing of each siRNAoligotemplate to the T7 promoter primer, which was performed for 1minute at 90°C followed by 1 hour at 37°C. The filling DNA step withKlenow DNA polymerase was performed for 45 minutes insteadof 30 minutes. Antisense and sense siRNA107-127 oligonucleotidetemplates were as follows: antisense, 5′-AAGCCTGGCTACAGCA-ATATGCCTGTCTC-3′; sense, 5′-AACATATTGCTGTAGCCAGG-CCCTGTCTC-3′. Scrambled antisense and sense oligonucleotidetemplates were obtained by replacing four nucleotides (underlined initalics): GCTA was replaced by TCAG and TAGC by CTGA.Scrambled siRNAs were used as a negative control. SiRNAs werecharacterized by gel electrophoresis on 2% (w/v) agarose gel as wellas by a nondenaturing 12% (w/v) polyacrylamide gel electrophoresis.The molar concentration (M) of the siRNAs was equal to theconcentration of siRNA/14 in µg assuming that 1 nmol of an average21-mer double strand (ds)RNA contained 14 µg RNA. HMECs weretransfected with siRNAs at various concentrations and for variousperiods as indicated in the text. In some experiments, HMECs wereincubated in the absence or presence of 1 µg/ml KE.

Statistical evaluationStatistical significance of the results was determined by using theStudent’s t-test. A value of P<0.05 was considered to be significant.Mean±s.d. values of three to four experiments are presented.

ResultsElastin-derived peptides stimulate the angiogenicphenotype of endothelial cells in vivo and in vitroAs EDPs were previously reported to influence the growth rate,

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the invasive potential or apoptosis of both normal andtransformed cells, we suspected that such matrikines mightsimilarly modify the angiogenic phenotype of endothelial cells.To validate those premises we initially evaluated the influenceof EDP in vivo using the chick CAM model. From day 6 ofembryonic development, membranes were treated daily withPBS (control), κ-elastin (KE) (50 ng) or VGVAPG (200 ng).As shown in Fig. 1A, in contrast to controls whereangiogenesis was detected at day 8 of embryonic developmentand reached a maximum at day 9 (not shown), KE as well asVGVAPG peptide led to an enhanced rate of angiogenesis,occurring as early as 24 hours following peptidesupplementation of embryos; it was also associated with anincreased level of microvessel density reaching a maximum atday 8. In parallel to these in vivo experiments, the influence ofKE on the ability of HMECs to reorganize in capillary tubes,when embedded in type I collagen lattice, was evaluated.KE considerably enhanced pseudotube formation (Fig. 1B).Similarly, when HUVECs or HMECs were cultured onmatrigel, the surface area occupied by pseudotubes wasincreased by KE in a time- and concentration-dependentmanner (Fig. 1C). Such an effect occurred within 6 hours ofincubation and with peptide concentrations as low as 10 ng/ml,but was significant only after 14 hours of culture, withformation of numerous cellular extensionslinking cellular aggregates at branch points ofthe array (Fig. 1C,D). Such effects of KE aresimilar to those obtained with VEGF.

Consistent with the intricate relationshipbetween membrane calcium channel activationand cytoskeleton actin microfilaments, confocalmicroscopy analysis of HUVECs and HMECSrevealed a main reorganization of actin bundleswhen endothelial cells were cultured onmatrigel in the presence of EDPs or VEGF (Fig.2A). In contrast to their spindle-shaped andsometimes rounded aspect in the control,endothelial cells adopted a more spread outshape in the presence of KE. Similarobservations were made by scanning electron

microscopy analysis of HUVECs cultured on matrigel (Fig.2B). In the absence of KE, HUVECs reorganized into a sparsehoneycomb network consisting of strings of rounded cells (Fig.2Ba,c). By contrast, in the presence of KE, they formed a densearray of pseudotubes with numerous tight cell contacts andcellular protrusions within matrigel (Fig. 2Bb,d). Such changesin cell shape were then quantified on an image analysis basis.As shown in Fig. 2C, KE significantly decreased the cell formfactor of HUVECs following just 4 hours of incubation, aphenomenon that was accentuated following 14 hours ofincubation. As elongation and/or spreading of a cell isinversely proportional to the magnitude of such a factor, thisresult supported our proposal that cells adopted a more spreadshape in the presence of KE.

The influence of EDPs on endothelial cell tubulogenesismight originate from either modulation of cell growth orincrease of cell migration, or both. The kinetics of cellproliferation in the absence or presence of growth factor areshown in Fig. 3A,B. In the absence of growth factor, KE (200ng/ml) did not significantly influence endothelial cellproliferation. However, in the absence of KE and growth factor,the number of cells drastically decreased with the time ofculture. The kinetics of cell proliferation in the presence of KEand growth factor were similar to that obtained with growth

Fig. 1. Elastin-derived peptides enhanced in vivoand ex vivo angiogenesis. (A) Representativephotomicrographs of CAM from chick embryos atday 7 (D7) and 8 (D8) of embryonic developmenttreated daily from day 6 of embryonic developmentwith PBS (control), 50 ng of κ-elastin (KE) or 200ng VGVAPG peptide. (B) Representativephotomicrographs of capillary tube formation inthree-dimensional type I collagen gel by HMECsincubated in the absence or presence of 100 ng/mlKE. (C) Representative photomicrographs ofcapillary-like structures from HUVECs cultured onmatrigel for 14 hours in the absence (control) orpresence of KE (10 and 100 ng/ml). As a positivecontrol, VEGF (20 ng/ml)-induced tubulogenesis isshown in comparison to KE (100 ng/ml). (D) Semi-quantitative evaluation of pseudotube formation wasperformed by determining the number of blackpixels per field. Results, each based on ten randomlyselected fields, are expressed as mean±s.d. of fourexperiments. ***P<0.001 compared to the controllevel.

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factor alone (Fig. 3Ab,c). As shown in Fig. 3B, in the absenceof growth factor, survival of endothelial cells after 5 days ofculture was improved following treatment of cells with KEat concentrations ranging from 10 to 200 ng/ml, whereas nodifference was observed in the presence of growth factor (Fig.3Bb,c). By contrast, KE induced a concentration-dependentincrease of endothelial cell migration in an in vitro woundhealing assay (Fig. 3C). A 2.5- and 1.6-fold increase of cellmigration was obtained with 200 ng/ml KE (2.67 nM) and200 ng/ml VGVAPG peptide (400 nM), respectively. Suchincrements were higher than those obtained with 20 ng/mlbFGF (1.2 nM) or VEGF (0.6 nM). EDPs and growth factorsacted independently of each other in terms of endothelial cellmigration (Fig. 3Cb,c). These data suggested that EDPstriggered an increased rate of tubulogenesis through enhancinginfluence on endothelial cells migration.

Influence of EDP on the angiogenic phenotype is due toS-Gal occupancyAs already mentioned, most effects of EDPs on cell phenotypeare consecutive to the binding of those peptides to a cell-associated elastin-binding protein (EBP) with lectin-likeproperty, which proved to be identical to a spliced form of β-galactosidase (S-Gal). To assess whether EDP-mediated

influence on endothelial cell tubulogenesiscould be attributed to the binding of suchpeptides to their cognate receptor, we firstattempted to reproduce endothelial cellmorphotype using VGVAPG peptide, theknown repeated motif in tropoelastininteracting with EBP. VGVAPG peptidereproduced the effect of KE on pseudotubeformation (Fig. 4A,B). Such effects were

also reproduced by several peptides displaying an (X)GXXPGconsensus sequence with type VIII β-turn conformation thatfavors S-Gal binding (data not shown). As a second set ofexperiments, aiming to verify the contribution of S-Gal on EDP-mediated angiogenesis, we used lactose or V14 peptide asantagonists. Both compounds attenuated the effect of EDPson pseudotube formation (Fig. 4). Interestingly, polyclonalantibodies directed against the V14 sequence of EBP involvedin the binding of elastin peptides reproduced the ligand effecton tubulogenesis (data not shown). As shown in Fig. 4B, EBPwas expressed in HUVECs and HMECs as a 67-kDa protein asin fibroblasts.

EDPs selectively modulate the expression and activationof proMT1-MMP and proMMP-2 in endothelial cellsAs EDPs were previously shown to enhance MMP expression,acting as key players in angiogenesis and the invasive propertyof several cell types, we evaluated the influence of thosematrikines on MMPs expression by HUVECs and HMECs.EDPs had no influence on MMP-1, MMP-3 and MMP-9expression by HUVECs and HMECs (data not shown).Considering that MTI-MMP has recently been shown to playa major role in endothelial cell migration and tubulogenesis inseveral endothelial cell culture models, we next examined the

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Fig. 2. Morphological changes induced byelastin-derived peptides. (A) Confocalmicroscopy analysis of actin filaments stainedwith tetra methyl rhodamine isothiocyanate-labeled phalloidin from HUVECs and HMECscultured on matrigel for 24 hours in the absence(control) or presence of KE (50 µg/ml) orpresence of VEGF (20 ng/ml). (B) Scanningelectron micrographs of HUVECs cultured for14 hours on matrigel in the absence (a and c) orpresence (b and d) of 50 µg/ml KE. Sampleswere fixed, dehydrated and desiccated asdescribed in the methods section. KE induces adense honeycomb network (arrowheads in a andb) where several cell-cell interactions with closecontacts are frequently observed (arrows in d),whereas it is sparse in the control with anumber of rounded cells (arrow in c).(C) Quantitative evaluation of the cell formfactor after 4 and 14 hours’ incubation ofHUVECs in the absence or presence of KE wasperformed by computerized image analysis.Cell form factor was calculated according to theformula 4πS/P2 where S is the surface of celland P its perimeter. Results represent themean±s.d. obtained from 60 cells. **P<0.01;***P<0.001 when compared to levels in therelevant controls.

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effect of EDPs on the proMT1-MMP/proMMP-2system in HMECs. Gelatin zymography analysiswas used to evaluate gelatinase A secreted into theculture medium and associated with cell extracts inHMECs cultured as monolayer, following a 24-hourcell exposure to different concentrations of KE. Asillustrated in Fig. 5A, HMECs were found toconstitutively release latent proMMP-2 into theconditioned medium (Fig. 5Aa). KE also induced anincrease of both pro and active MMP-2 forms in cellextracts (Fig. 5Ab). Such effects were concentration-dependent and were observed with 100 ng/ml KE, aconcentration shown above to influence cellmorphogenesis and migration. At the highest KEconcentration (100 µg/ml), secreted and membrane-associated MMP-2 levels were increased two- and19-fold, respectively, and active MMP-2 represented31.8% of total cell-bound enzyme. It suggested thatelastin peptides could trigger major quantitative alterations inpartners involved in proMMP-2 activation process i.e. MT1-MMP and TIMP-2. However, levels of TIMP-2, TIMP-1 (Fig.5Ac) as well as αVβ3 integrin (data not shown) were notsubstantially modified by supplementing HMEC culturemedium with KE. However, those peptides enhanced proMT1-MMP expression and activation, similar to MMP-2, at theprotein (Fig. 5Ad) and mRNA levels (Fig. 5B). Similar data wereobtained with HUVECs instead of HMECs and could also bereproduced by VGVAPG peptide (data not shown).

MT1-MMP involvement in EDP-mediated angiogenicphenotypeTo investigate the contribution of the MT1-MMP/MMP-2system in the elastin peptide-mediated influence on HMECtubulogenesis and migration, experiments were performed inthe presence of agents known to interfere with the activity ofthose enzymes. As shown in Table 1, batimastat nearly totallyabolished the effect of KE on pseudotube formation by HMECscultured on matrigel. A similar level of inhibition was obtainedwith TIMP-2 whereas TIMP-1 displayed only a partialrepressive effect on tubulogenesis. In addition, Dec-RVLR-cmksuppressed the effect of KE on tubulogenesis, in a similarmanner to batimastat. Overall, these data argue for the main

Fig. 3. Influence of elastin-derived peptides on cellproliferation, survival and migration. (A,B) Cellproliferation and survival: HMECs (104 cells) wereseeded in 24-well culture plates and cultured in FCS-free ECGM MV for various periods (A) or for five days(B) in the absence of KE (control, C) and growth factor(none) or in the presence of KE and VEGF (20 ng/ml)(b) or bFGF (20 ng/ml) (c). Cell number was determinedat the end of each period of incubation. (C) To examinecell migration, HMEC monolayers were disrupted witha 1mm cell scraper and incubated in the absence orpresence of KE or VGVAPG peptide and in the absence(a, none) or presence of VEGF (20 ng/ml) (b) or bFGF(20 ng/ml) (c) for 24 hours. Cell migration wasdetermined in triplicate experiments as percentage ofmigrating cells relative to control value in the absence ofgrowth factors and expressed as mean±s.d. NS, notsignificant; *P<0.05; **P<0.01; ***P<0.001.

Table 1. Involvement of MT1-MMP in EDP-mediatedHMEC migration and pseudotube formation

A Pseudotube formation* Number of black pixels per field �2·10–2

κ-elastin 100±8.7+ Batimastat (10–7 M) 11.0±5.7 (89)†

+ TIMP-2 (1 µg/ml) 20.2±8.7 (79.8)+ TIMP-1 (1 µg/ml) 77.1±5.7 (22.9)+ Dec-RVLR-cmk (0.75 mg/ml) 16.8±5.7 (83.2)

B Cell migration (%)‡ Control TIMP-2 Batimastat

None 100 82±7 (18)† 62±6 (38)KE (200 ng/ml) 253±11 103±6 (59.3) 61±12 (75.9)VGVAPG (200 ng/ml) 161±4 91±11 (43.5) 62±4 (61.5)

*HMECs were cultured on matrigel in the presence of κ-elastin (100ng/ml) and various inhibitors of proteinases for 24 hours. Semi-quantitativeevaluation of pseudotube formation was performed after pixelization ofphotomicrographs from ten selected fields. Results are expressed as mean?s.d.of four experiments.

†Values in brackets represent the % inhibition determined relative to therespective controls.

‡HMEC monolayers were disrupted and incubated for 24 hours in theabsence (none) or presence of κ-elastin (KE) or VGVAPG peptide. TIMP-2(1 µg/ml) or batimastat (10–7 M) was added to the culture medium. Results,each based on triplicate experiments, are expressed in percentage relative tocontrol (none) and as mean±s.d.

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involvement of MT1-MMP in the EDP-mediated influence onendothelial cell behavior. Indeed, TIMP-2, as well as batimastat,also inhibited the effect of KE on cell migration as determinedby the wound healing assay, thus further emphasising thecentral role of MT1-MMP in such phenomena (Table 1).

To address this issue in a less ambiguous way, we useda siRNA approach for silencing proMT1-MMP mRNAexpression. Four independent sets of 21-bp siRNA duplexeswere chosen. In preliminary experiments, we showed that onlysiRNA107 and siRNA228 proved to be efficient in suppressing

proMT1-MMP expression in endothelial cells (data notshown). Further experiments were performed with siRNA107.Confocal microscopy analysis was first performed to determinethe subcellular distribution of siRNA107 within HMECs. Forthis purpose, siRNA107 was labeled with Cy3, a fluorescentmarker of siRNA duplex, and HMECs were transfected for 72hours with 25 nM Cy3-siRNA107 or scrambled Cy3-siRNA107.

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Fig. 4. Elastin-derived peptide-mediated angiogenesis is triggered byoccupancy of S-Gal (EBP). (A) Representative photomicrographs ofcapillary-like structure from HUVECs cultured on matrigel for 14hours in the absence or presence of elastin-derived peptides (100ng/ml KE or 200 ng/ml VGVAPG peptide). Cells were alsoconcomitantly incubated in the absence or presence of lactose (10–4

M) or V14 peptide (10 µg/ml). (B) Semi-quantitative evaluation ofpseudotube formation was performed as in Fig. 1. Results, eachbased on ten randomly selected fields, are expressed as mean±s.d. offour experiments. Insert shows a western blot analysis of EBP fromcell extracts of fibroblasts (FC), HUVECs and HMECs usingpolyclonal antibodies directed against the binding sequence of elastin(V14 peptide) on EBP. **P<0.01; ***P<0.001.

Fig. 5. KE increased proMMP-2 and proMT1-MMP expression andactivation in HMECs. (A) HMECs were cultured as monolayers andincubated in the presence of various concentrations of KE for 24hours. ProMMP-2 and MMP-2 in conditioned media (a) and in cellextracts (b) were assessed by gelatin zymography analysis. Arrowsindicate the positions of proMMP-2 and MMP-2. (c) TIMPs inconditioned medium of HMECs were analyzed by reversezymography analysis. Positions of TIMP-1 and TIMP-2 are shown.(d) Representative western blot of MT1-MMP from cell extracts ofHMECs cultured in the absence or presence of KE. Rabbitpolyclonal antibodies were used to reveal MT1-MMP (hinge region).Molecular masses of the bands are indicated, the 66-kDa speciescorresponds to proMT1-MMP, the 60 and 55-kDa species to MT1-MMP and the band at 44 kDa corresponds to a species processedfurther (Ellerbroek et al., 1999). (B) Semi-quantitative RT-PCRanalysis of proMMP-2 and proMT1-MMP was performed in HMECsincubated for various periods in the absence (white column) orpresence of 10 µg/ml KE (black column) and with variousconcentrations of KE for 24 hours. The time- and concentration-dependent histograms, each based on four experiments, display themean±s.d. of proMMP-2 and proMT1-MMP values that have beennormalized to the levels of GAPDH mRNA and expressed relative tocontrol cell value (time 0 and not KE treated). NS, not significant;*P<0.05; **P<0.01; P<0.001.

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As shown in Fig. 6A, scrambled Cy3-siRNA107 localizedpreferentially within the cytosol of cells, whereas Cy3-siRNA107 appeared mostly perinuclear. This localization wasoften associated with morphological changes, with cellsexhibiting a more condensed shape and less pseudopodeformation compared to HMECs transfected with scrambledsiRNA107. The influence of siRNA107 on proMT1-MMPmRNA expression was time- (Fig. 6C) and concentration-dependent (Fig. 6D). A 75-80% decrease of proMT1-MMPRNA level was reached by transfecting 25 nM and 50 nMsiRNA107, respectively, for 72 hours. Such siRNA transfectionled to a complete inhibition of EDP-induced proMT1-MMPexpression and proMMP-2 activation as determined by theBiotrak ELISA assay (Fig. 7A) and gelatin zymographyanalysis (Fig. 7B). Moreover, it abolished the effect of EDPson pseudotube formation (Fig. 7C,D). SiRNA107 is specific toMT1-MMP as no effect was observed on MT2-MMP, MT3-MMP and MT5-MMP expression (data not shown).

Altogether these data demonstrate that upregulation ofproMT1-MMP expression and activationwas the main proteolytic determinantinvolved in the EDP-mediated acceleratedrate of angiogenic phenotype.

DiscussionInitial in vivo experiments, revealing anenhancement of angiogenesis by EDPs

prompted us to investigate, in vitro, the EDP-mediatedmechanism involved in such a process. Data obtained indicatedthat elastin fragments and peptides containing GXXPGsequence (VGVAPG) found as repeats in tropoelastin couldaccelerate the angiogenic phenotype of endothelial cells frommicro and macrovasculature in the matrigel assay. VGVAPG-containing elastin peptides were shown to be generated invivo from lung elastin by human leukocyte elastase inbronchoalveolar lavages of patients with emphysema(Maccioni and Moon, 1993); in addition, such a hexapeptidemotif was shown not to be cryptic in human skin elastin andliberated in the vicinity of vertical melanoma by elastin-degrading enzymes (Ntayi et al., 2004). Moreover, SGVAPG,AGGLPG and MGGIPG sequences as found in α1 chain oftype XV collagen, α2 chain of type V collagen and fibrillincovalent structures, respectively, mimicked the effects of EDPsin in vitro angiogenesis (data not shown). However, their actionas cryptic sites or liberated matrikines is currently purelyspeculative. We show that EDPs induced cytoskeleton

Fig. 6. Subcellular distribution of siRNA107 andtime-and concentration-dependent inhibition ofMT1-MMP expression in HMECs transfectedwith siRNA107. (A) HMECs were cultured to60% subconfluency in ECGM MV and thentransfected with Cy3-labeled siRNA107 for 72hours. (a,d) Confocal microscopy analysis ofscrambled siRNA107 (ssiRNA107) and siRNA107,respectively. (b,e) Phase-contrast microscopyanalysis of scrambled siRNA107 and siRNA107,respectively. (c,f) Overlays of fluorescence andphase-contrast photomicrographs. Perinucleardistribution of siRNA107 and nucleus membraneis indicated by the arrows. (B) HMECs werecultured in ECGM MV to 60% subconfluencyand then transfected with various concentrationsof siRNA107 or scrambled siRNA107 (ssiRNA107)for several periods in the presence ofoligofectamine as a transfecting reagent. MT1-MMP expression was determined by semi-quantitative RT-PCR and expressed relative toGAPDH expression. PCR products wereresolved by 1% (w/v) agarose gelelectrophoresis. Control cells (C) were incubatedwith oligofectamine alone. The φX174/HaeIIImarkers (M) were used to evaluate the DNAfragment length. (C,D) Time- and concentration-dependent inhibition of MT1-MMP expressionby siRNA. Semi-quantitative evaluation of MT1-MMP expression was performed by fluorometricscanning of the gel and computerized using Bio-Profile software (Vilbert-Lourmat, Marne laVallée, France). Data represent the mean±s.d. offour experiments. NS, not significant; *P<0.05;**P<0.01; ***P<0.001.

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reorganization in endothelial cells when cultured on matrigel,an effect that had already been reported for smooth musclecells (Mochizuki et al., 2002). This result is also in keepingwith the known effect of EDPs on calcium flux andmicrotubular cytoskeleton refolding, properties that areassociated with marked activation of the MT1-MMP/MMP-2system in several cell models (Sakim and Yana, 2003).Particularly, MT1-MMP activity was reported to control cellgeometry of tumor cells within the confines of the 3D ECM(Hotary et al., 2003). We have demonstrated that, under ourexperimental conditions, the EDP-mediated angiogenicphenotype was not associated with plasminogen activators orMMP-1 and MMP-9 upregulation (data not shown). However,at concentrations found in the circulation and lower (10–6 to10–2 mg/ml) (Kucich et al., 1983; Fülöp et al., 1990), thesepeptides could significantly enhance proMT1-MMP andproMMP-2 expression and activation in HUVECs andHMECs.

The EDP-mediated angiogenesis phenotype was triggeredby EBP as lactose and V14 completely abolished the effect ofEDPs on endothelial cell migration, capillary tube formationand proMT1-MMP and proMMP-2 expression and activation.Such results are in accordance with a previous report indicating

that the binding of galactosidase-bearing moietieslike lactose to EBP causes the loss of its ability tobind elastin and induces its dissociation from thecomplex (Hinek et al., 1998). In addition, theinhibition of EDP-mediated tubulogenesis bybatimastat, TIMP-2 but not TIMP-1, or by aninhibitor of furin convertase, is consistent with theputative contribution of MT1-MMP in the effectsobserved. To gain more convincing evidence of itsdirect involvement, we developed a siRNA approachfor selectively silencing MT1-MMP in HMECs.SiRNAs were synthesized by in vitro transcription

as, according to a previous report (Brown et al., 2002), siRNAproduced by in vitro transcription is as much as 20-fold morepotent than chemically synthesized siRNA with a similarsequence. The perinuclear localization of siRNA107 wasconsistent with previous reports showing similar subcellulardistribution of siRNAs used to silence other genes (Byron etal., 2002; Nykanen et al., 2001). This localization couldrepresent sites of siRNA processing or sites where the RNA-induced silencing complex (RISC) resides (Montgomery et al.,1998). By contrast, scrambled siRNA107 localized essentiallyin the cytoplasm. HMECs transfected with siRNA107 adopteda condensed morphology that is consistent with the role ofMT1-MMP in the control of cell shape. Consequently EDP-mediated pseudotube formation on matrigel was drasticallyinhibited. These data further emphasized that MT1-MMP playsa pivotal function in EDP-mediated angiogenic phenotype.During the course of the present investigation, Ueda andcolleagues, using a similar approach to silence MT1-MMPexpression, demonstrated that downregulation of thisendopeptidase reduced the invasive capacity of HT-1080fibrosarcoma and gastric carcinoma cell lines in matrigel andalso decreased their motility on hyaluronan (Ueda et al., 2003).Given the importance of MT1-MMP in promoting EDP-

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Fig. 7. SiRNA107 decreases the amount of MT1-MMP,suppresses proMMP-2 activation and abolishespseudotube formation by HMECs cultured on matrigel.HMECs were cultured in six-well culture plates andtransfected at 60% confluency with 25 nM siRNA107 orscrambled siRNA107 (ssiRNA107) or oligofectamine alone(OL) for 72 hours. Then, the medium was replaced withfresh medium containing 2% (v/v) FCS and cells wereincubated in the absence (–KE) or presence (+KE) of 10µg/ml KE or 10–7 M phorbol myristate acetate (PMA) for24 hours. Total MT1-MMP (A) and proMMP-2activation (B) was analyzed by ELISA and zymographyanalysis, respectively. (C,D) HMECs were transfectedwith 25 nM siRNA107 or scrambled siRNA107(ssiRNA107) for 72 hours and then cultured on matrigelfor 24 hours in ECGM MV in the presence of 1 µg/mlKE. OF, HMECs incubated with oligofectamine alone.Non-transfected HMECs were cultured on matrigel in theabsence (control) or presence of 1 µg/ml KE (+KE).(D) Tube formation was observed with a phase-contrastmicroscope equipped with a digital camera. Images weretaken using a phase-contrast microscope with a ×10 lens.(C) Semi-quantitative evaluation of pseudotubes wasperformed after white and black pixellization of imagesand was expressed as black pixels relative to total pixelsfrom 20 selected fields. Data represent the mean±s.d. offour experiments. ***P<0.001.

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mediated angiogenic phenotype and also tumor progression(Brassart et al., 1998; Huet et al., 2002; Ntayi et al., 2004),such a siRNA-MT1-MMP silencing strategy might represent amore suitable alternative to the use of MMP inhibitors intreating tumors in clinical settings.

Angiogenesis estimated by microvessel density or otherendothelial cell markers is an independent prognostic variablein vertical growth phase melanomas and is associated withintense dermal elastolysis (Labrousse et al., 2004). Theimportance of MT1-MMP and MMP-2 in angiogenesis andtumor invasion has been firmly established through severalinvestigations. Angiogenesis impairment has been consistentlyobserved in MMP-2-deficient mice (Itoh et al., 1998) andMT1-MMP-deficient mice display reduced activation of latentMMP-2 and vascular inversion of calcified cartilage, and failto respond to bFGF in the corneal angiogenesis assay (Zhou etal., 2000). Moreover, MT1-MMP has been shown to be mainlyinvolved in endothelial cell migration, invasion and capillarytube formation in various angiogenesis models includingtubulogenesis in 3D collagen and fibrin gel invasion assays(Lafleur et al., 2002; Galvez et al., 2001). According toprevious reports, MT1-MMP may promote cell migration notonly through degradation and remodeling of cell-associatedECM, but also through its ability to process and activate severalcell surface molecules, such as CD44, integrin αVβ3 and tissuetransglutaminase (tTG), that are known to regulate cellmigration (Seiki, 2003). Indeed, CD44H is shed by MT1-MMPfurther promoting cell migration (Kajita et al., 2001). However,the precise mechanism involved in the stimulation of cellmotility is still unknown but it was speculated that theprocessing of CD44 by MT1-MMP could promote celldetachment from the ECM or generate cell signalling byactivating the ERK pathway, further inducing cell migration.Alternatively, it could trigger transcriptional activation of targetgenes through the cleaved cytoplasmic portion of the processedCD44 (Kajita et al., 2001; Gingras et al., 2001; Okamoto et al.,2001). Moreover, the alpha V chain of αVβ3 integrin isalternatively processed by MT1-MMP into a functional formthat stimulates migration through phosphorylation of focaladhesion kinase (FAK) (Deryugina et al., 2002; Ratnikov et al.,2002; Baciu et al., 2003), and cleavage of tTG by this enzymealso promoted cell migration on type I collagen (Belkin et al.,2001). On the other hand, the matrix-degrading activity ofMT1-MMP is required for the formation of an endothelial celltubular network in fibrin and collagen gels (Hiraoka et al.,1998; Lafleur et al., 2002; Haas et al., 1998; Galvez et al.,2001).

Importantly, the link between elastin degradation, MT1-MMP upregulation and angiogenesis, may be related to severalcardiovascular diseases where enhanced angiogenesis(Einstein, 1991; Thompson et al., 1996), degradation of elasticfibers (Robert and Robert, 1980; Nakashima et al., 1990;Nakashima and Sueishi, 1992; Jacob, 2003) and/or MT1-MMPoverexpression (Rajavashist et al., 1999; Hong et al., 2000) areassociated. As a key example, atherosclerotic abdominal aorticaneurysm (AAAA) is associated with neovascularization in allthree layers (Thompson et al., 1996; Holmes et al., 1995;Ferrara et al., 1991), VEGF overexpression (Kobayashi et al.,2002) and tropoelastin accumulation (Krettek et al., 2003).Neovascularization in AAAA could also be related to enhancedelastin degradation, as EDP (VGVAPG) was shown to induce

several characteristic features of aneurysmal disease such as anincrease of vessel density in a rat aneurysmal model (Nackmanet al., 1997) and an increased concentration of soluble elastinfragments in human serum from patients with aneurysmal,ulcerative manifestations of atherosclerosis (Petersen et al.,2002) and acute aortic dissection (Shinoara et al., 2003). Inaddition, as MT1-MMP is overexpressed by proinflammatorymolecules, it might also contribute to the enhanced local matrixdegradation in human atherosclerotic plaques as previouslyreported (Rajavashisth et al., 1999; Hong et al., 2000). On theother hand, decreased arteriolar and capillary density in aspontaneous hypertensive rat (SHR) model correlated withdecreased MT1-MMP and VEGF receptor expression(VEGFR-2) (Wang et al., 2004). These data were in keepingwith previous reports showing an increase of aortic elastincontent in hypertensive animals (Keeley and Alatawi,1991); hypertension was also associated with elastinhaploinsufficiency in human and mice (Faury et al., 2003). Bycontrast, angiogenesis was restored by increasing the levels ofVEGFR-2 and MT1-MMP using a sponge implantation modelassociated to a VEGF gene transfer technology in SHR (Wanget al., 2004). However, the aortic elastin content was notappreciated in this study.

Recently, MT1-MMP overexpression has been associatedwith increased VEGF expression in breast carcinoma cell lines(Sounni et al., 2004). Under our experimental conditions EDPswere found not to influence VEGF or bFGF production byendothelial cells (data not shown). However, as EDP-mediatedMT1-MMP upregulation triggered the PI3-kinase/Akt pathwayin endothelial cells (data not shown), we cannot exclude thefact that those peptides might transactivate VEGFR-1 throughEBP as this receptor was reported to inhibit VEGFR-2-mediated proliferation but not migration via PI3-kinaseactivation (Zeng et al., 2001). Such a receptor transactivationmechanism was reported in aortic smooth muscle cells incultures where EBP occupancy by EDPs activates PDGFreceptors (Mochizuki et al., 2002). Experiments to test thishypothesis are currently underway.

Thus, EDP-mediated MT1-MMP expression on the cellsurface of endothelial cells, by triggering matrix pericellularproteolysis, MT1-MMP endocytosis, MMP proteolyticactivation cascades and/or cell surface receptor shedding, maybe of importance in processes where those mechanisms arerequired for angiogenesis. Overall, our results ascribed a newfunction to EBP as a mediator of angiogenesis.

We thank Hervé Kaplan for technical assistance with microscopy.This work is part of an Interregional Research Program Régulation dela Matrice Extracellulaire et Pathologie on angiogenesis and wassupported by a grant of the ‘Ministère de l’Education Nationale et dela Recherche’, and by CNRS (Centre National de la RechercheScientifique), France.

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