Endothelial progenitor cell-dependent angiogenesis requires localization of the full-length form of uPAR in caveolae

VASCULAR BIOLOGY

Endothelial progenitor cell–dependent angiogenesis requires localization of thefull-length form of uPAR in caveolae*Francesca Margheri,1 *Anastasia Chilla,1 Anna Laurenzana,1 Simona Serratì,1 Benedetta Mazzanti,2 Riccardo Saccardi,2

Michela Santosuosso,2 Giovanna Danza,3 Niccolo Sturli,3 Fabiana Rosati,3 Lucia Magnelli,1 Laura Papucci,1 Lido Calorini,1

Francesca Bianchini,1 Mario Del Rosso,1,4 and Gabriella Fibbi1

1Department of Experimental Pathology and Oncology, University of Florence Istituto Toscano Tumori, Florence, Italy; 2Cord Blood Bank, Careggi UniversityHospital, Florence, Italy; 3Department of Clinical Physiopathology, University of Florence, Florence, Italy; and 4DENOTHE, Center for the study at molecular andclinical level of chronic, degenerative and neoplastic diseases to develop novel therapies, University of Florence, Florence, Italy

Endothelial urokinase-type plasminogenactivator receptor (uPAR) is thought toprovide a regulatory mechanism in angio-genesis. Here we studied the proangio-genic role of uPAR in endothelial colony-forming cells (ECFCs), a cell populationidentified in human umbilical blood thatembodies all of the properties of an endo-thelial progenitor cell matched with ahigh proliferative rate. By using caveolae-disrupting agents and by caveolin-1 si-lencing, we have shown that the angio-genic properties of ECFCs depend on

caveolae integrity and on the presence offull-length uPAR in such specialized mem-brane invaginations. Inhibition of uPARexpression by antisense oligonucleo-tides promoted caveolae disruption, sug-gesting that uPAR is an inducer of cave-olae organization. Vascular endothelialgrowth factor (VEGF) promoted accumu-lation of uPAR in ECFC caveolae in itsundegraded form. We also demonstratedthat VEGF-dependent ERK phosphoryla-tion required integrity of caveolae as wellas caveolar uPAR expression. VEGF activ-

ity depends on inhibition of ECFC MMP12production, which results in impairmentof MMP12-dependent uPAR truncation.Further, MMP12 overexpression in ECFCinhibited vascularization in vitro and invivo. Our data suggest that intratumorhoming of ECFCs suitably engineered tooverexpress MMP12 could have thechance to control uPAR-dependent ac-tivities required for tumor angiogenesisand malignant cells spreading. (Blood.2011;118(13):3743-3755)

Introduction

The term “angiogenesis ” connotes the process of new blood vesselformation from preexisting vessels.1,2 Identification of endothelialprogenitor cells (EPCs) in peripheral blood has highlighted analternative mechanism of vessel formation based on EPC recruit-ment from bone marrow.3 Similarly to endothelial cells (ECs), EPCs areable to migrate and differentiate to form primitive tubes in Matrigel.Moreover, EPCs can form endothelial-like colonies in vitro and show ahigh proliferative rate that is lacking in mature ECs.4

However, it is not yet clear how to define the EPCs that reallycontribute to vessel formation and their exact role in the process.5

In this study, we isolated cells from human umbilical cord blood(UCB), taking into consideration their capability to form a capillarynetwork in vitro coupled with a high proliferation potential. Such acell type was termed endothelial colony-forming cell (ECFC), aspreviously described.6-8

In the angiogenic process, ECs adhere to and degrade the extracellu-lar matrix (ECM), proliferate, and differentiate into tubular-like struc-tures.1,2,9 The membrane-associated plasminogen activation system(urokinase-type plasminogen activator [uPA]; uPA receptor [uPAR];uPA inhibitor type-1 [PAI-1]) is critical in angiogenesis, beinginvolved in uPA-mediated ECM degradation and uPAR-dependentcell adhesion.9 uPAR is a glycosylphosphatidylinositol-anchoredprotein that consists of 3 peptide domains (D1-D2-D3). It is presenton the cell membrane as a full-length (D1�D2�D3) and atruncated molecule (D2�D3), unable to bind uPA, but retaining the

property to interact with ECM molecules. We previously demon-strated that angiogenesis requires full-length uPAR. Indeed, insystemic sclerosis, a disease characterized by defective angiogen-esis, the impairment of vessel formation is related also to over-production of matrix metalloprotease-12 (MMP12), which trun-cates uPAR.10,11

The role of uPAR in ECFC-dependent vessel formation is lessknown. A single study indicated that EPCs display higher uPAactivity and uPAR levels than mature ECs.12

Like other glycosylphosphatidylinositol-anchored molecules,uPAR is present in “lipid rafts,” dynamic microdomains of the cellmembrane, rich in cholesterol, sphingolipids and glycolipids,trans-membrane protein receptors, integrins, and a large number ofsignaling molecules. However, uPAR can be found both in lipidrafts and nonraft fractions, and its distribution in different fractionsis associated with different signaling pathways and, consequently,biologic processes.13,14 Caveolae are functionally and morphologi-cally distinct forms of lipid rafts, characterized by the presence ofthe protein caveolin-1 and are particularly abundant in ECs,playing a fundamental role in their function.15 Vascular endothelialgrowth factor (VEGF) localizes in caveolae on interaction with itstype 2 receptor, forming a “functional platform” involved inangiogenesis,16,17 together with other signaling molecules.

It is known that caveolae disruption affects biologic functions ofthe ECs, including angiogenesis.18 The aim of our study was to

Submitted February 25, 2011; accepted July 7, 2011. Prepublished online asBlood First Edition paper, July 29, 2011; DOI 10.1182/blood-2011-02-338681.

*F.M. and A.C. contributed equally to this study.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2011 by The American Society of Hematology

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evaluate the relationship between caveolae and uPAR form/distribution on cell membrane in ECFC-dependent angiogenesis.

Here we show that ECFC angiogenesis depends on caveolaeintegrity and that uPAR induces caveolae formation. To assess therequirement of full-length uPAR in ECFC-dependent angiogenesis,we caused uPAR D1 truncation by transient transfection ofMMP12, which resulted in inhibition of angiogenesis in vitro andin vivo.

Methods

Isolation and characterization of EPCs

Human UCB samples (volume � 50 mL) were collected in citratephosphate dextrose solution from health newborns. We used cord bloodunits with a number of total nucleated cells � 1.3 � 109 (threshold ofsuitability for the banking established by the Umbilical Cord Bank ofCareggi, Florence, Italy) after maternal informed consent in accordancewith the Declaration of Helsinki and in compliance with Italian legislation.

EPCs have been isolated from UCB as previously described.4,7 Bloodwas diluted 1:1 with Hanks balanced salt solution (EuroClone) and wasoverlaid on an appropriate volume of density gradient separation medium(Lympholyte; Cedarlane). Cells were centrifuged for 30 minutes at roomtemperature at 740g. Mononuclear cells were recovered, washed 3 timeswith Hanks balanced salt solution and resuspended in complete EGM-2medium (Lonza) supplemented with 10% FBS (Hyclone), 50 ng/mL VEGF(PeproTech) and 5 IU/mL heparin. Cells were seeded on gelatin-coated6-well tissue culture plates at a density of 5 � 105 cells/cm2 in a 5% CO2

humidified incubator at 37°C. On days 4 and 7, half of the medium wasexchanged with fresh medium. Then the medium was changed com-pletely with EGM2–10% FBS every 3 days. EPC colonies appeared incell cultures after 2-3 weeks and were identified as circumventedmonolayers of cobblestone-like cells. These cells will be referred to asECFCs, as previously described.7,19 The colonies were mechanicallypicked from the original plate and seeded on another gelatin-coated wellwith EGM2–10% FBS for expansion.

ECFCs were analyzed for the expression of surface antigens by flowcytometry. Washed cells were resuspended in flow cytometry buffer(CellWASH 0.1% sodium azide in PBS; BD Biosciences). Aliquots(0.1 � 106 cells/100 �L) were incubated with the following conjugatedmonoclonal antibodies: CD45-FITC, CD34-FITC, CD31-FITC (all fromBD Biosciences PharMingen); CD105/R-phycoerythrin (Ancell); ULEX-FITC (Vector Laboratories); phycoerythrin-labeled by Tetra-Tag phycoeryth-rin-labeling kit (StemCell Technologies) KDR (RELIATech), uPAR/R3(BioPorto Diagnostic A/S), and VWF (BD Biosciences PharMingen).

Nonspecific fluorescence and morphologic parameters of the cells weredetermined with isotype-matched mouse monoclonal antibodies (BDBiosciences PharMingen). All incubations were done for 20 minutes; and,after washing, cells were resuspended in 100 �L of CellWASH. 7-Amino-actinomycin (BD Biosciences PharMingen) was added to exclude deadcells from the analysis. Flow cytometric acquisition was performed bycollecting 104 events on a FACSCalibur (BD Biosciences) instrument, and

data were analyzed on DOT-PLOT biparametric diagrams using CellQuestpro OS X.1 software (BD Biosciences) on a Macintosh PC.

Caveolae isolation, characterization, modification, andsilencing with siRNA

Isolation of ECFC caveolar raft and nonraft fractions was performed using aCaveolae/Rafts isolation kit (Sigma-Aldrich), based on separation on anOptiPrep density gradient, according to the manufacturer’s instructions.After centrifugation (22 hours at 154 000g at 4°C), 9 fractions werecollected. Caveolar raft and nonraft fractions were determined by Westernblotting with anti–caveolin-1 antibodies (Sigma-Aldrich) for caveolarfractions. and anti-integrin�1 antibodies (Santa Cruz Biotechnology) fornonraft fractions.20 To disrupt caveolae,21 we used methyl-�-cyclodextrin(�-MCD) (Sigma-Aldrich). Cholesterol depletion was measured as de-scribed.22 Cells were pretreated with 5mM �-MCD for 20 minutes at 37°C,and the cell extract was prepared at 4°C in lysis buffer containing 1% TritonX-100. Caveolin-1 silencing was performed by transfection of ECFCs withsmall interfering RNA (siRNA) and negative control constructs (Dharma-con RNA Technologies/Euroclone), according to the manufacturer’sinstructions.

Western blot and RT-PCR analysis

Cell lysates were obtained, processed, and blotted as described.10 Theprimary antibodies were: anti–caveolin-1 (1:1000; rabbit polyclonal anti-body, Sigma-Aldrich); anti-uPAR R3 (1:500; mouse monoclonal antibody,BioPorto Diagnostics), which recognizes the full-length uPAR; anti-uPARM2 (1 �g/mL, rabbit polyclonal antibody, provided by Dr D’Alessio,Istituto Humanitas), which reveals both the full-length and truncated formof uPAR; anti-MMP12 (1:500; rabbit polyclonal antibody; ChemiconInternational); phospho-ERK (p42/p44; 200 �g/mL, 1:500) and ERK-2(200 �g/mL, 1:500; Santa Cruz Biotechnology). Aliquots of caveolar raftand nonraft fractions20 were subjected to Western blotting. After incubationwith HRP-conjugated anti–rabbit IgG (1:5000) for 1 hour (GE Healthcare),immune complexes were detected with the enhanced chemiluminescencedetection system (GE Healthcare). Membranes were exposed to autoradio-graphic films (Hyperfilm MP; GE Healthcare) for 1-30 minutes.

mRNA levels of relevant molecules were determined by an internal-based semiquantitative RT-PCR, as previously described.10 Primers, cy-cling conditions, and products size are reported in Table 1.

Treatments inducing uPAR up-regulation/inhibition

VEGF (50 ng/mL; Calbiochem-VWR International) was used to up-regulate uPAR in ECFCs. After overnight VEGF stimulation in EGM2-2%FCS, cells were recovered, and the supernatant was dialyzed againstdistilled water and lyophilized. To inhibit uPAR expression, we treatedECFCs with anti-uPAR aODNs in a complex with DOTAP (BoehringerMannheim), as reported.23,24 As a negative control, we used a scrambledODN. ECFCs were treated daily for 4 days; then the cells and the mediumwere recovered and used in in vitro studies. The term “control cells” refersto ECFCs cultivated in the presence of DOTAP, which does not affect cellviability.23

Table 1. PCR primers and cycling conditions

Primers Sequence Size, bp Cycling profile

uPAR 5�-AAAATGCTGTGTGCTGCTGACC-3�(sense)

5�-CCCTGCCCTGAAGTCGTTAGTG-3� (antisense)

910 94°C, 1 minute 56°C, 1 minute

72°C, 1 minute 35 cycles total

Caveolin-1 5�-CAACATCTACAAGCCCA-3�(sense)

5�-AAA CTT CTA CAC TAA CG-3� (antisense)

146 94°C, 30 seconds 50°C, 30 seconds

70°C, 30 seconds 30 cycles total

MMP-12 5�-CCACTGCTTCTGAGCTCTT-3�(sense)

5�-GCGTAGTCAACATCCTCACG-3�(antisense)



GAPDH 5�-CCACCCATGGCAAATTCCATGGCA-3�(sense)

5�-TCTAGACGGCAGGTCAGGTCCACC-3�(antisense)



3744 MARGHERI et al BLOOD, 29 SEPTEMBER 2011 � VOLUME 118, NUMBER 13




Immunofluorescence confocal microscopy

Control and treated ECFCs were grown on coverslips, fixed, and permeabil-ized according to routine methods.25 The anti–human primary antibodiesused were: anti-uPAR R3 (1:40), anti-caveolin-1 (1:400). The secondaryantibodies were: CY3-conjugated anti–mouse IgG (1:800; Sigma-Aldrich)and FITC-conjugated anti–rabbit IgG (1:800; Sigma-Aldrich). For nuclearstaining, samples were incubated with 4,6-diamidino-2-phenylindole(2 �g/mL), for 15 minutes. In negative control slides primary antibody wasomitted. Coverslips were mounted with antifade mounting medium(Biomeda) and imaged on a Bio-Rad MRC 1024 ES confocal laser scanningmicroscope (Bio-Rad) equipped for fluorescence measurements. Imageswere acquired by using Bio-Rad confocal EZ-C1 3.40 Gold Versionsoftware. Maximum intensity projections of the acquired image stacks wereconstructed, analyzed, and color-combined by using ImageJ 1.44 software(www.rsb.info.nih.gov/ij; National Institutes of Health), also used forstatistical evaluation of caveolar cluster areas, uPAR/Cav-1 colocaliza-tion,26 fluorescence intensities, and background signals. Colocalization wasdetermined through the “Just Another Colocalization Plugin”27 (JACoP) ofImageJ 1.44 software. Caveolae for each cell were obtained by isolatinghigh fluorescent spots, restricting the fluorescent spot by size, andprocessing data with the “Analyze Particle” command contained in ImageJ1.44 software.

In vitro parameters of angiogenesis

Invasion was studied in Boyden chambers using porous filters coated withMatrigel (50 �g/filter).10,24 ECFCs (20 � 103) were placed in the topcompartment of the chamber and migration, evaluated after 6 hours, wasexpressed as the absolute number of migrated cells � SD. Where detailed,ECFC invasion was stimulated by 50 ng/mL VEGF in the bottom chamber.In other experiments ECFCs were pretreated with 5mM �-MCD for 20minutes at 37°C or subjected to caveolin-1 silencing. Some experimentswere performed in the presence of anti-uPAR R3 antibody or irrelevant IgG(all used at 1.5 �g/ml) or after anti-uPAR aODN treatment. Capillarymorphogenesis was performed in 13-mm tissue culture wells coated withMatrigel, as described.10,24 Experimental conditions were the same used forinvasion assay. ECFCs were plated (60 � 103/well) in complete EBM-2medium 2% FCS and incubated at 37°C, 5% CO2. Plates were photo-graphed at 6 hours and at 24 hours. Results were quantified by imageanalysis, giving the percentage of photographic field occupied by ECFC.Six to 9 photographic fields from 3 plates were scanned for each point.Results were expressed as percentage field occupancy with respect tocontrol taken as 100% � SD.

MMP-12 transient transfection

ECFCs (1 � 107 cells) were incubated in 500 �L EBM-2 medium with theaddition of recombinant vectors (15 �g each of either pCDNA3.1 � MMP12or pCDNA3.1 alone; Invitrogen) and electroporated using a Gene Pulserapparatus (0.260 KV, 0.975 �F; Bio-Rad). Production of recombinantMMP12 vector and the expression of MMP12 gene and protein werepreviously described.11 Expression of MMP12 gene was measured byRT-PCR, and the level of released protein was analyzed by Western blottingof culture medium.

MMP-12–dependent uPAR cleavage

To verify MMP12-dependent uPAR cleavage, 25 �g of recombinant humanuPAR (R&D Systems) was incubated overnight at 37°C with 15 �L ofEBM2–2% FBS (control) or conditioned medium (CM) derived fromempty vector or MMP-12 transiently transfected ECFCs. These experi-ments were performed in the absence and in the presence of anti-MMP12antibody (10 �g/mL), which inhibits the MMP12 catalytic activity, asshown in Table 2. CMs were dialyzed against distilled water, lyophilized,and reconstituted (10�). After incubation, the samples were processed andsubjected to Western blotting with anti-uPAR M2 antibody.

Matrigel-sponge assay in mice

Aliquots of 50 �L reconstituted CM from empty-vector, and MMP-12–transfected ECFCs, containing 50 U/mL heparin, were added to unpolymer-ized Matrigel at 4°C at a final volume of 0.6 mL, in the absence or in thepresence of anti-MMP12 antibody (10 �g/mL). The Matrigel suspensionwas injected subcutaneously into the flanks of C57/BL6 male mice (CharlesRiver) using a cold syringe (4 injections/animal). At body temperature, theMatrigel polymerizes to a solid gel, which becomes vascularized within4 days in response to CM. Pellets were removed, photographed, minced,and diluted in water to measure the hemoglobin content with a Drabkinreagent kit (Sigma-Aldrich). To verify the enzymatic activity of MMP12within the recovered Matrigel sponges, we solubilized the plugs,28 incu-bated aliquots of the solubilized plugs with standard uPAR and thenevaluated uPAR truncation by Western blotting, using the M2 antibody. Allmouse experiments were approved by the Italian Ministry of Health AnimalCare and Use Committee.

Statistics

Results were expressed as mean � SD of the indicated number ofexperiments. Multiple comparisons were performed by the Student-Newman-Keuls test, after demonstration of significant differences among medians bynonparametric variance analysis according to Kruskal-Wallis. Statisticalsignificances were accepted at P � .05.

Results

Isolation and characterization of ECFCs

ECFCs have been isolated from the blood of 10 of 12 umbilicalcords. ECFCs were considered as EPC-derived cells,7,19 whichoriginated from adherent cell population after 14-21 days ofculture. After 21 days, the median frequency of ECFCs was0.12 colonies/106 mononuclear cells (range, 0.03-0.57). UCB-ECFCs used showed a high proliferative potential and could beexpanded to at least P15 without signs of senescence. The meanproliferation index was 10.7 (SD 6.1) at P2, 10.2 at P5(SD 6.8), and 10.1 at P10 (SD 6.7). Immunophenotyping byFACS analysis revealed that CB ECFCs expressed endothelial cellsurface antigens CD31, ULEX, and CD105. Low levels of KDRand VWF were detected. In addition, uPAR was revealed (Table 3),whereas CD45 and CD34 were almost undetectable. In vitromorphology (Figure 1A left panel) and capillary morphogenesis(Figure 1A central and right panels) of ECFCs resembled that ofmature ECs.

Caveolae and uPAR distribution on cell membrane

ECFC extracts were subjected to density-gradient separation.Fractions were assayed by Western blotting for the presence of

Table 2. Enzyme-blocking activity of anti-MMP12 antibody

Conditioned media Enzymatic activity, pg/mL

Control* 84.2 � 11.6 (100%)

Control � anti-MMP12† 51.2 � 10.5 (60.8% of control)

pMMP12‡ 131.4 � 12.1 (156.05% of control)

pMMP12 � anti-MMP12§ 91.3 � 10.8 (108.4% of control)

MMP-12 enzymatic activity was measured in ECFC conditioned medium using afluorimetric kit (EnzoLyte 520 MMP-12 assay kit with 5-FAM/QXL 520 fluorescenceresonance energy transfer; AnaSpec, DBA-Italia), as previously described.11

*ECFC conditioned medium under basal conditions.†ECFC conditioned medium under basal conditions in the presence of anti-

MMP12 antibody.‡Conditioned medium from MMP12 transiently transfected ECFCs.§Medium from MMP12 transiently transfected ECFCs in the presence of

anti-MMP12 antibody.

uPAR IN EPC ANGIOGENESIS 3745BLOOD, 29 SEPTEMBER 2011 � VOLUME 118, NUMBER 13




caveolin-1, as caveolar marker (2-6) and of integrin-�1 as nonraftmarker (7-9, Figure 1B top and middle panels). Many studiespropose an important role of intact caveolae in the regulation ofangiogenesis.29,30 Among the methods available to modify themembrane raft composition,21 here we used �-MCD, whichinduces cholesterol depletion (Figure 1C right panel). Because

�-MCD could be toxic for the cells, we first checked for theexperimental nontoxic conditions together with the caveolar raftdisruption, evaluating ECFC vitality by morphology and trypanblue exclusion. After treatment with 5mM �-MCD for 20 minutes,the cells were still viable (Figure 1C left panel) and showed anormal morphology (data not shown), and caveolin-1 wasdistributed in all the fractions, thus demonstrating the disruptionof normal lipid organization (Figure 1B bottom panel). Incontrol conditions, uPAR was mainly present in nonraft frac-tions, whereas it redistributed in caveolar and nonraft fractionson �-MCD (Figure 1D). VEGF treatment up-regulated bothcaveolin-1 and uPAR (Figure 1E). A large amount of uPARshifted from nonraft to the caveolar fraction after VEGFtreatment (Figure 1F), and mainly the full-length uPAR waspresent in caveolae, whereas the truncated form was restricted tononraft fractions, indicating that VEGF also affects the mem-brane distribution of full-length uPAR on ECFCs. These resultsare representative of independent experiments in ECFC clonesexpressing different uPAR levels.

Figure 1. Relationship between caveolae and uPAR distribution in ECFC cell membrane. (A) Morphologic profile of ECFC colonies appeared after 2–3 weeks of culture(left panel). Capillary-morphogenesis in vitro at 6 and 24 hours (middle and right panel); (B) Caveolin-1 distribution. Cell lysates were layered on Optiprep solution andcentrifuged as described in “Methods” to separate lipid rafts (fractions 2-6) from (fractions 7-9) in control conditions and after treatment with �-MCD (5mM for20 minutes). Fractions obtained were probed by Western blotting with specific anti–caveolin-1 and anti-integrin �1 antibodies, as caveolar raft and nonraft markerrespectively. Data are representative of 5 independent experiments. (C) �-MCD-treatment: right panel shows the �-MCD-induced cholesterol depletion. Evaluation ofcholesterol amount was performed as previously described22; left panel shows vitality of the �-MCD-treated cells determined by trypan blue dye exclusion test.*P � .05, significantly different from control; (D) uPAR distribution: control and �-MCD–treated cells were subjected to Optiprep gradient separation and caveolar raft(CR) and nonraft (NR) fractions were analyzed by Western blotting with anti-uPAR antibody M2, which reveals both the full length and truncated uPAR. Numbers on theright indicates molecular weights expressed in kilodaltons. Data are representative of 5 independent experiments performed with clones expressing different levels ofuPAR; (E) VEGF treatment: expression of uPAR and caveolin-1. Left panels: PCR of uPAR and caveolin-1 cDNA in untreated (control; lane 1) and VEGF-treated (lane 2)ECFCs. Numbers on the left indicate the size of PCR products in base pairs (bp). GAPDH: glyceraldehyde-3-phosphate dehydrogenase used as housekeeping gene.Right panels show Western blotting for uPAR and caveolin-1. Numbers on the right indicate molecular weights expressed in kilodaltons; (F) uPAR distribution in controland in VEGF-treated ECFCs. Control and VEGF-treated cells were subjected to Optiprep gradient separation: caveolar raft (CR) and nonraft (NR) fractions werecollected and analyzed by Western blotting with anti-uPAR antibody M2. Numbers on the right indicate molecular weights expressed in kilodaltons; (G) silencing ofuPAR gene using anti-uPAR aODNs: cell cultures were treated with aODN for 4 days in the presence of the cationic phospholipid DOTAP. A scrambled ODN (sODN) wasused as a negative control. Top panels: PCR of ODNs-treated cells (on the left); Western blotting of ODNs-treated cells (-tubulin was used as a loading control; on theright). Bottom panels: distribution of caveolin in Optiprep-prepared caveolar rafts after ODNs treatment. Data shown in panels E, F, and G are representative of5 independent experiments.

Table 3. ECFC immunophenotyping by FACS analysis

Antigen ECFC

KDR 4.9 � 5.9

CD31 95.6 � 0.5

ULEX 98.2 � 0.5

vWF 2.3 � 0.5

CD105 99.9 � 0.0

uPAR-R3 10.3 � 6.1

CD45 0.4 � 0.1

CD34 0.2 � 0.2

Cell surface antigen expression. Results represent the mean percentage of cellsexpressing surface antigens � SD from 10 different clones.





The parallel increase of uPAR and caveolin-1 led us tohypothesize a potential role of uPAR in caveolae formation. Tocorroborate our hypothesis, we inhibited uPAR expression byanti-uPAR aODNs (Figure 1G), which resulted in a decrease ofuPAR mRNA and protein (top panels). This treatment also causedredistribution of caveolin-1 from caveolar rafts to all the fractions(bottom panels), similarly to �-MCD, thus demonstrating a loss ofnative lipid organization on cell surface in the absence of uPAR.

Immunofluorescence analysis: colocalization uPAR/caveolin-1

Because uPAR was found in caveolar rafts, we tested uPAR-caveolin colocalization by immunofluorescence in all our experi-mental conditions. Figure 2 shows that untreated ECFCs had ahomogeneous dotted distribution of uPAR and a partial colocaliza-tion of uPAR with caveolin-1, whereas VEGF treatment induced anincrease of fluorescence intensity of both caveolin-1 and uPARtogether with a more strong evidence of colocalization. Caveolarrafts disruption by �-MCD led to a loss of caveolin-1/uPARcolocalization, only partially recovered in VEGF-treated cells.Inhibition of uPAR expression by anti-uPAR aODN caused the lossof uPAR fluorescence signal but not of colocalization. ScrambledODN-treated cells maintained fluorescence levels and colocaliza-tion as in control cells.

To estimate the caveolar cluster number/size and colocalizationratio between caveolin-1 and uPAR, we performed the fluorescenceanalysis. As shown in Table 4, VEGF treatment increased caveolarcluster number and size, as well as uPAR/caveolin1 colocalizationcompared with control. The �-MCD treatment caused a decrease ofnumber and size of caveolar clusters and a reduction of uPAR/caveolin-1 colocalization, only partially recovered after VEGFtreatment. uPAR silencing by aODNs resulted in a drastic decreaseof number and size of caveolar clusters.

Role of caveolae and uPAR in in vitro ECFC angiogenesis andVEGF signaling

To evaluate the role of caveolae in ECFC-dependent angiogen-esis in vitro, we performed Matrigel invasion and capillarymorphogenesis after �-MCD treatment and caveolin-1 silenc-ing. �-MCD treatment impaired basal and VEGF-stimulatedECFC invasion (Figure 3A top panels) and capillary morphogen-esis (Figure 3A bottom panels). In some experiments, we added�-MCD for 20 minutes to preorganized capillary-like networksand observed the loss of preexistent capillary tubular structure(not shown).

On the basis of uPAR involvement in angiogenesis31,32 and ofour data showing its role in caveolae formation (Figures 1G and 2),we evaluated the function of caveolar uPAR in ECFC-dependentangiogenesis by uPAR up-regulation or uPAR inhibition. Toincrease uPAR, we stimulated ECFCs with VEGF (Figure 1E) inthe absence or in the presence of monoclonal anti-uPAR-R3. Asshown in Figure 3B, R3 reduced ECFC Matrigel invasion andcapillary morphogenesis in basal conditions, as well as theirVEGF-dependent increase, thus demonstrating that the proangio-genic role of VEGF involves uPAR up-regulation.

We silenced uPAR gene using anti-uPAR aODNs, shown here toabolish uPAR expression and caveolae formation (Figure 1G).Figure 3C shows that aODN treatment specifically impaired ECFCinvasion and capillary morphogenesis.

On caveolin-1 silencing (Figure 4A left), full-length andtruncated uPAR redistribution was similar to that obtained after�-MCD treatment (Figure 4A right). In addition, ECFC invasion

and capillary morphogenesis in basal conditions and after VEGFstimulation were impaired (Figure 4B).

We next investigated the effect of caveolae disruption onVEGF-induced signaling. We observed that VEGF stimulatedERK1/2 phosphorylation in ECFC, whereas �-MCD treatment andcaveolin-1 silencing markedly reduced VEGF-induced ERK1/2activation (Figure 4C). These results demonstrated the requirementof caveolar rafts integrity on VEGF signaling in ECFCs.

A correlation between uPAR expression/distribution and angio-genesis in control and VEGF-challenged ECFC was investigated.Clones expressing different uPAR levels were subjected to Western

Figure 2. Colocalization uPAR/caveolin-1 and caveolar rafts analysis by immu-nofluorescence. Immunostaining of uPAR and caveolin-1 in ECFCs in differentexperimental conditions. Panels on the left represent uPAR staining (red representsCy3) using anti-uPAR R3 antibody, which identifies full-length uPAR; central panelscorrespond to caveolin-1 staining (green represents FITC), using anti-caveolin-1antibody and right panels show the double staining of ECFCs with anti-uPAR (red)and anti–caveolin-1 (green). Nuclear staining is obtained by the use of 4,6-diamidino-2-phenylindole (blue). Cells were observed with an inverted confocalNikon Eclipse TE2000 microscope equipped with a Nikon S Fluor 60� oilimmersion lens (Nikon). Images were acquired at room temperature, using astandard 1024- by 1024-pixel image format and adjusting the zoom level to matchthe voxel size to the Nyquist criterion. The pinhole size was always set at 1 airyunit (airy disk), and each plane was Kalman averaged to reduce noise. In eachexperiment, the same instrumental settings were used for all image acquisitions.All images were Gaussian filtered to eliminate single-pixel noise before analysis(original magnification � 600).





blotting to assess uPAR distribution with M2 antibody in caveolarraft and nonraft fractions. On VEGF, full-length uPAR was mainlypresent in the caveolar fraction (Figure 5A), whereas the truncatedform was selectively present in nonraft fraction. ECFC clonesexpressing higher levels of uPAR were more prone to invadeMatrigel as well as to form capillary-like tubes in vitro (Figure 5Band C, respectively). In addition, the response to the VEGF wasdependent on uPAR levels. In addition, we determined caveolin-1expression in the 3 different clones of Figure 4A, finding a positivecorrelation between uPAR and caveolin-1 expression, providingfurther support to a functional link between uPAR expression andcaveolae organization.

Full-length uPAR is required to perform angiogenesis in vitroand in vivo

The data shown in Figure 1 demonstrate that VEGF up-regulatesuPAR and recruits its full-length form into caveolae, thus increas-ing angiogenesis in vitro (Figure 3). We have previously shown thatMMP12 uPAR D1 cleavage results in angiogenesis impairment.10

Thus, we investigated whether VEGF treatment of ECFCs alsodecreased MMP12 synthesis and consequently uPAR truncation,which could be in agreement with the increased angiogenicproperties of VEGF-treated ECFCs. Figure 6A shows PCR andWestern blotting analysis, indicating that VEGF-treated ECFCsexpressed lower amounts of MMP12 than untreated cells.

Then, we demonstrated that MMP12 is involved in in vitroangiogenesis of ECFCs, both in basal conditions and after VEGFtreatment (Figure 6B-C). Indeed, in the presence of MMP12antibody in the culture medium (10 �g/mL), both migrationand capillary morphogenesis were increased in control cells,whereas no differences were observed when MMP12 antibodywas added to VEGF-treated cells, yet producing very lowamounts of MMP12.

To demonstrate the consequence of uPAR cleavage on ECFCsangiogenesis, we transiently transfected ECFC with MMP12 and

evaluated enzyme release in the medium and the presence offull-length and truncated uPAR in cell lysates. Figure 7A (leftpanels) shows that MMP12 overexpressing ECFCs efficientlytruncated their own uPAR. MMP12 overexpression resulted ininhibition of ECFC Matrigel invasion and capillary morphogenesis(Figure 7A middle and right panels).

Because EPCs are recruited to sites requiring vascularization,such as ischemic tissues33 or tumors, where they contribute to the“angiogenic switch,”34 we evaluated whether ECFC MMP12overproduction also influenced in vivo angiogenesis.

First, we evaluated uPAR cleavage by MMP12 released into themedium from transfected cells. We incubated standard uPAR withdifferent CMs in the presence or absence of anti-MMP12 antibodyand evaluated full-length and truncated uPAR by Western blottingwith M2 antibody (Figure 7B). The control medium and themedium of empty vector-transfected ECFCs did not cleave uPAR,wheres CM of MMP12-transfected ECFCs produced truncateduPAR. Such truncation was inhibited by anti-MMP12 antibody, butnot by irrelevant IgG.

We performed the Matrigel sponge assay in mice, usingreconstituted ECFC CM from empty vector control (pCDNA3�

alone) or MMP12-transfected ECFCs, in the absence or presence ofanti-MMP12 antibody. Vascularization was evaluated in recoveredMatrigel sponges. We used the same medium that we experimen-tally demonstrated to contain high levels of active MMP12 and tobe able to truncate uPAR (Figure 7B). Groups of 4 pellets wereinjected for each treatment. Individual Matrigel sponges wererecovered at autopsy 5 days after implants. As shown in Figure 7C,whereas angiogenesis was stimulated by medium from emptyvector control, it was inhibited by MMP12-expressing ECFCs, asshown also by decrease in sponge hemoglobin content. Thisinhibitory effect was the result of the enzymatic activity of releasedMMP12 as it was reverted by anti-MMP12 antibody but not by anirrelevant IgG.

Table 4. Confocal microscopy analysis

Maximum caveolar raft,cluster area, �m2

(based on Cav-1 signal)

Mean of Cav-1,relative intensity

unit

Maximum Cav-1,relative intensity

unit

Caveolar raft,cluster number

(based on Cav-1 signal)

uPAR relativeintensity, units

(mean)

Colocalization(uPAR fraction

overlapping Cav-1)

CTRL 75 817 � 3271 14 016 � 2114 14 397 � 1733 91 � 4.015 36 129 � 3861 0.691

VEGF, % 100.5 205.2 505.1 127.5 16.8 0.976

�-MCD, % �65.9 �44.6 �39.9 �74.7 �6.9 0.305

VEGF � �-MCD, % 3.5 8.5 �7.3 12.1 �11.5 0.719

aODN, % �38.4 �48.0 �59.4 �80.0 �93.6 0.953*

sODN, % �15.2 �40.0 �7.4 �43.0 �39.9 0.708

Data are based on the analysis of rearrangement of membrane caveolar rafts and were obtained by pooling data from each single cell. Treatments operated onECFCs are shown in the first column. The targets of the statistical analysis are indicated in the top row. Control data (CTRL) are presented as raw data obtained byImageJ analysis of a wide pool of control cells. Other data are presented as percentage changes compared with control. The second column reports the percentagechange in the area of labeled contiguous structures that is taken as a measure of raft clusterization. These data are in agreement with results obtained by thebiochemical analysis performed with membrane fractionation. Quantitative analysis and colocalization of antigens were performed on acquired images analyzed asindividual channels. The Manders overlap coefficients (M1 and M2) were determined using JACoP plugin and also calculated using the intensity correlation analysisplugin of the open-source software WCIF-ImageJ as previously described.26 We used the same operating mode of image analysis: Manders overlap coefficientsindicate an overlap of the signals and thus represent the degree of colocalization between the red and green pixels: their values range from 0 (no overlap) to 1 (completeoverlap). Briefly, the background signal on each image was initially corrected using the ImageJ background subtraction function; and whenever possible, single cells onthe images were selected using the lasso tool (which defines a so-called region of interest). Colocalization was then calculated, after choosing the threshold values forthe green and red channels, with the aforementioned plugin on the regions of interest previously defined. Zero/zero pixel was excluded. The Manders coefficient relativeto the uPAR fraction that overlap Cav1 was reported. Quantization of caveolin-1 fluorescence and uPAR fluorescence was conducted on z stacks of 70 sectionscorresponding to a thickness of approximately 0.8 �m passing through the middle of the cells. Regions of interest were manually drawn around each cell, and theintegrated intensity after background correction was measured in caveolin-1 and uPAR channels. To account for differences in cell size, the integrated intensity in eachregion of interest was divided by its measured perimeter. ImageJ software was used for fluorescence quantization, and Origin 6.1, Version 6.1052 (B232) was used forstatistical analysis. Quantitative colocalization analysis of antigens was performed on acquired images analyzed as individual channels. Results are expressed asmean � SD or SE. Multiple comparisons were performed by 1-way ANOVA with Bonferroni correction. A P value � .05 was considered statistically significant.

*This data indicates that almost all uPAR molecules remaining after aODN treatment, although reduced, colocalized with caveolin-1.





To still further confirm these results, we tested the MMP12activity within the recovered Matrigel sponges. We demonstratedthat the standard uPAR cleaving activity persisted in the sponges

containing CM of MMP12-transfected ECFCs (Figure 7C inset).Such activity was inhibited by anti-MMP12 antibody but not byirrelevant IgG, thus demonstrating the specificity of this effect.

Figure 3. Role of caveolae and uPAR in in vitro ECFC-dependent angiogenesis. In vitro angiogenesis was measured by Matrigel invasion and by capillary morphogenesisof ECFCs after modification of caveolar rafts or of uPAR expression. In all the experiments, results are the mean of 3 different experiments performed in triplicate in 3 differentECFC preparations. The histograms on the right of each panel refer to the relative quantification and are expressed as mean value � SD. *P � .05, significantly different fromcontrol. In all the histograms, Matrigel invasion is expressed as the absolute number of migrated cells, by directly counting the migrated cells on the filters, whereas capillarymorphogenesis is quantified by measuring the percentage field occupancy of capillary projections, assuming the control as 100%. Six to 9 photographic fields from 3 plateswere scanned for each point. (A) Effects of caveolar raft modification. Top part: Matrigel invasion of ECFCs before and after treatment with 5mM �-MCD, in control and afterVEGF stimulation (50 ng/mL). Bottom part: Capillary morphogenesis at 6 hours in the same conditions (symbols as in Figure 1). (B) The experimental conditions were the sameas in panel A, but cells were treated with anti-uPAR antibodies (antibody R3) instead of �-MCD. Top panels: Matrigel invasion. Bottom panels: Capillary morphogenesis underthe same experimental conditions. (C) Silencing of uPAR gene using the anti-uPAR aODN. Top panels: Matrigel invasion of control (DOTAP) untreated and 4 days ODN-treatedECFCs in the presence of DOTAP. The same experimental conditions were used for capillary morphogenesis (bottom panels).





Discussion

In this study, we demonstrated that the angiogenic properties ofECFCs depend on the integrity of caveolae and on the presence offull-length uPAR in such specialized membrane structures. We alsoshowed that uPAR is an inducer of caveolar raft organization and

that uPAR integrity in caveolae is promoted by VEGF by adecrease of MMP12-dependent uPAR truncation. Further, MMP12overexpression inhibited vascularization in vitro and in vivo.

uPAR is anchored to the cell membrane through a glycosylphos-phatidylinositol tail, which accounts for its presence in “lipidrafts.” Caveolae are specialized lipid rafts, particularly abundant inECs. Our aim to investigate a possible correlation between caveolar

Figure 4. Caveolin-1 silencing. Effects on invasion, capillary morphogenesis and VEGF signaling. (A) Left panels: PCR and Western blotting of caveolin-1 silencing inECFCs. Numbers on the left: size of PCR products (bp); numbers on the right: molecular weights expressed in kilodaltons. Right panel: Western blotting of uPAR distributionbetween caveolar raft (CR) and nonraft (NR) fractions under control conditions (scrambled-sequence siRNA) and after caveolin-1 silencing (Cav-1 siRNA). uPAR was revealedwith M2 antibody. Numbers on the right: molecular weights expressed in kilodaltons. Results were similar to those Figure 1D, obtained with �-MCD. (B) Effects of caveolin-1silencing on basal and VEGF-stimulated ECFC Matrigel invasion (top panels) and ECFC capillary morphogenesis (bottom panels). For image acquisition and quantification,refer to the legends of Figures 1 and 3. Quantification is shown by histograms on the right: values refer to 3 experiments performed in triplicate in 3 different ECFC cell lines.Data are expressed as mean value � SD. *P � .05, significantly different from control. (C) Western blotting with anti-pERK1/2 antibodies in the experimental conditionsreported in the bottom captions (control conditions, �-MCD treatment, caveolin-1 silencing, and VEGF stimulation), showing that both �-MCD treatment and caveolin-1silencing inhibited VEGF-dependent ERK1/2 phosphorylation. ERK1/2 indicates loading control obtained with anti-total ERK1/2 antibodies. Numbers on the right: molecularweight expressed in kilodaltons. Histograms on the bottom show quantification of Western blotting experiments (as densitometric units) and refer to 3 experiments performed intriplicate in 3 different ECFC lines. Data are expressed as mean value � SD. *P � .05, significantly different from control.





rafts, distribution of full-length and truncated forms of uPAR, andangiogenesis in ECFCs relies on its identification in caveolae13 andon the recognized role of full-length uPAR in angiogen-esis.10,11,31,32,35 Here, we used UCB ECFCs because of theirbiologic properties5: ECFCs display a higher proliferative ratecompared with mature ECs, form secondary and tertiary colonieson replating and de novo blood vessel formation in vivo,36 and canbe obtained by a noninvasive method and then expanded ex vivo toachieve a considerable number of cells.

We observed that in unstimulated ECFCs uPAR distributesmainly in nonraft fractions of the membrane. VEGF stimulationup-regulated both caveolin-1 and uPAR inducing, at the same time,a shift of full-length uPAR from the nonraft fraction to caveolae.Integrity of caveolae is essential in many cellular functions,29,30

including angiogenesis. We observed that ECFC caveolae disrup-tion by �-MCD or caveolin-1 silencing induced cell membraneuPAR redistribution outside of caveolae and that uPAR silencingimpaired a normal caveolae organization, thus indicating a role ofuPAR as a caveolar raft organizer.

Studies on caveolin-1 emphasize a paradoxical function ofcaveolin in angiogenesis promotion/inhibition18 in given cellularand animal models, which are restriction of EC hyperproliferation

in undifferentiated EC and promotion of migration/differentiationof EC in mature vessels. In line with a positive role of caveolin-1 inthe migration/differentiation steps of angiogenesis, our data showthat VEGF stimulation of ECFCs induces a caveolin-dependentERK1/2 phosphorylation, which is considered the terminal signal-ing event in EC migration/differentiation after VEGF challenge.Moreover, our data further support the principle that integrity ofcaveolae structure is critical for accurate VEGF-induced signaling.37

Further, immunofluorescence has indicated that the number andsize of caveolar clusters and uPAR/caveolin-1 colocalizationincreased under the effect of VEGF, whereas the same parametersdecreased on chemical disruption of caveolae.

We observed that the treatment of ECFCs with �-MCD, as wellas caveolin silencing, impaired Matrigel invasion and capillarymorphogenesis, thus demonstrating that integrity of caveolaeaffects the angiogenic properties of ECFCs in vitro. To evaluate therole of caveolar raft uPAR, we performed angiogenic assays byuPAR gain/loss of function. Stimulation of ECFCs with VEGF,which we demonstrated to up-regulate caveolin-1 and uPAR and topromote partition of full-length uPAR in caveolae, caused an increase ofMatrigel invasion and capillary morphogenesis, inhibited by anti-uPARantibodies. In addition, silencing of uPAR gene using anti-uPAR

Figure 5. Relationship between uPAR levels and in vitro angiogenesis. (A) Different clones, displaying different levels and distribution of uPAR, evaluated by Westernblotting analysis for uPAR and caveolin-1 expression in basal conditions (�VEGF) and after VEGF challenge (�VEGF). Numbers on the right indicate molecular weight. Thepictures show the results of a typical experiment of 3 that gave similar results. (B) Matrigel invasion of different ECFC clones in control conditions and in VEGF-stimulated cells.The histograms refer to quantification, expressed as number of migrated cells, of 3 different experiments performed in triplicate with each clone. Results are shown as meanvalue � SD. *P � .05, significantly different from control. (C) In vitro capillary morphogenesis of different ECFC clones displaying different uPAR levels. The numbers in theframe represent the percentage of uPAR-positive cells. Histograms on the right show quantification of capillary morphogenesis experiments (under control conditions an VEGFstimulation, respectively). Image acquisition and quantification were performed as reported in Figures 1 and 3. Results are representative of a typical experiment of3 experiments performed in triplicate with each clone and are quantified by measuring the absolute percentage field occupancy of capillary projections. *P � .05, significantlydifferent from control.





aODNs, which inhibit uPAR expression and caveolae formation,resulted in impairment of ECFC Matrigel invasion and capillarymorphogenesis. These results demonstrated that integrity of cave-olae influences the angiogenic properties of ECFCs and thatcaveolar uPAR is critical in ECFC-dependent angiogenesis.

Overall, these results demonstrate that uPAR-mediated cave-olae formation is required for ECFC angiogenesis, which is

inhibited by uPAR silencing, by �-MCD treatment which disruptcaveolae and induces uPAR redistribution outside of caveolae, andby caveolin-1 silencing.

uPAR has been shown to play a major role in VEGF-inducedEC migration/invasion, as VEGF leads to uPA–PAI-1–uPARcomplex formation, which is internalized via low-density lipopro-tein receptor-like proteins and is thereafter redistributed to the

Figure 6. In vitro MMP12-dependent angiogenesis. (A) Left panel: RT-PCR of MMP12 in control and VEGF-treated ECFCs. Right panel: Western blot analysis of MMP12revealed in the culture medium of control and VEGF-treated ECFCs. (B) Matrigel invasion of control and VEGF-treated ECFCs, in the absence and in the presence ofanti-MMP12 antibody (10 �g/mL), under basal conditions (control), and after VEGF stimulation. Histogram refers to quantification of Matrigel invasion assay obtained bycounting the total number of migrated cells/filter. Results are the mean of 3 different experiments performed in triplicate in 3 ECFC preparations and are shown as meanvalue � SD. *P � .05, significantly different from control. (C) Capillary morphogenesis of ECFCs using the same experimental conditions described for panel B. The figures arerepresentative of a typical experiment of 3 experiments performed in triplicate in 3 ECFC preparations. Quantification was performed by measuring the percentage fieldoccupancy of capillary projections, taking the control as 100%. *P � .05, significantly different from control. Image acquisition and quantification were performed as describedin Figures 1 and 3.





leading edge of migrating cells. Our results open the question ofwhether uPAR redistribution to caveolae is performed via lateralshift or via an low-density lipoprotein receptor-dependent internal-ization and recycling process. The latter one is suggested by theobservation that truncated uPAR is not found in lipid rafts and thatuPAR needs to be in its full-length to recycle through thispathway.38

On the basis of our previous studies10,11 showing that theMMP12-dependent uPAR cleavage severely impaired angiogen-esis, we evaluated a possible role of MMP12 in ECFC angiogen-esis. We demonstrated that the increased angiogenic ability ofVEGF-treated ECFCs is related both to uPAR up-regulation and toits lower cleavage because of the decreased levels of MMP12 andthat MMP12 overproduced by transfected ECFCs cleaves uPARand reduces ECFC angiogenic performance in vitro.

In vivo experiments demonstrated that culture medium ofMMP12-transfected ECFCs inhibited vascularization of the Matri-gel sponge in mice, which was counteracted by anti-MMP12antibody, suggesting that ECFC-induced angiogenesis in vivodepends on the presence of the full-length uPAR.

Although many studies evaluating the therapeutic potential ofEPCs in neovascularization in cardiovascular disease39 and insystemic sclerosis40,41 have reported conflicting results, these cellsrepresent a hope for therapy. Loss of function of MMP12 ininjected EPCs, alone or together with induction of uPAR up-regulation by VEGF stimulation, could be evaluated as goodstrategy for vascular therapy.

The possibility to reduce angiogenesis through the “loss offunction” of uPAR could be important to control excessiveangiogenesis, such as in tumors. Indeed, many studies suggest animportant role of EPCs in tumor vascularization and metastasis byincorporation in vessels and/or by supporting angiogenesis viaparacrine secretion of proangiogenic cytokines.34,42 Limited workhas been performed on the ability of human ECFCs to regeneratethe vascular system in preclinical studies,43 and on their role intumor angiogenesis. However, our Matrigel sponge data indicatethat, if ECFCs are able to reach a site of vascularization, they cansignificantly influence EC angiogenesis. Therefore, ECFCs couldbe used as cellular vehicles to deliver anticancer agents.44 Apossibility for cancer therapy could be to manipulate ECFCs to

Figure 7. Effect of MMP12 transfection on in vitro and in vivo angiogenesis. ECFCs were transiently transfected with the recombinant vector pCDNA3.1 � MMP12. Ascontrol of transfection, empty vector pCDNA3.1 was used. (A) Left panel (top part): Western blotting analysis of MMP12 released in the medium from control and MMP12transiently transfected ECFCs. Left panel (bottom part): truncation of uPAR in MMP12-transfected ECFCs. Numbers on the right show molecular weight expressed inkilodaltons. This figure shows typical results of 3 different experiments that gave similar results. The middle part of the figure shows Matrigel invasion (top panels) and capillarymorphogenesis (bottom panels) of MMP12 transiently transfected ECFCs. Results are the mean of 3 different experiments performed in triplicate with 3 ECFC preparations.Histograms: Quantification of Matrigel invasion and capillary morphogenesis performed by counting migrated cells and percentage field occupancy of capillary projections,respectively. Image acquisition and quantification were performed as described in Figures 1 and 3. Results are shown as mean value � SD. *P � .05, significantly differentfrom control. (B) Western blotting showing MMP12-dependent uPAR cleavage. Standard uPAR (st) was incubated overnight with culture medium from control, empty vectorand MMP12 transiently transfected ECFCs, in the absence or presence of anti-MMP12 antibody. st indicates standard uPAR incubated in EBM-2 medium with 2% FBS.Full-length and truncated forms of uPAR were evaluated by Western blotting analysis with the specific monoclonal antibody anti-uPAR M2. The figure shows the results of atypical experiment of 5 different experiments. (C) In vivo Matrigel sponge assay. Flanks of mice were injected with Matrigel (4 injections/animal) containing aliquots of 50 �L ofreconstituted culture medium of ECFCs transfected with recombinant vector pCDNA3.1 � MMP12 or with pCDNA3.1 (empty vector), in the absence or in the presence ofanti-MMP12 antibody (10 �g/mL), or of an irrelevant Ig. Top panels: Quantification of the experiment by evaluating hemoglobin (Hb) content of the sponges under the variousexperimental conditions (4 injections/animal; 2 animals for each condition). Graphs are shown as mean � SD. *P � .05. Bottom panels: A representative photograph, takenwith the Zeiss SR Stemi stereomicroscope, of the vascularization of individual Matrigel sponges recovered at autopsy for the corresponding condition and representative of atypical experiment. Image acquisition and quantification was performed as described in Figures 1 and 3. Inset (C top part): Western blotting of standard uPAR (st) incubatedwith aliquots of the solubilized plugs and then evaluated by Western blotting, using the M2 antibody (which recognizes both native and truncated uPAR forms). Onlyplug-extracted CM of pMMP12-ECFC cleaved uPAR. Such inhibition was counteracted by anti-MMP12 antibody but not by an irrelevant Ig, thus showing the specificity ofanti-MMP12 antibodies. The figure shows a typical Western blotting pattern, of 5 experiments performed with different plugs extracts, that gave similar results.





deliver MMP12 into the tumor mass with the aim to truncate uPARboth in ECs and tumor cells, thus inhibiting tumor angiogenesis,growth, and metastasis, features that require an intact native uPAR.Our data showing that caveolae concentrate a functional form ofuPAR, that is critical for the invasion process, provide a possibleexplanation to new attempts to control cancer cell spreading basedon administration of statins that modify lipid rafts by reducingmembrane cholesterol.45 Because statins are safe and orally avail-able agents, they may enlarge the range of their indications alsothrough their angiogenic modulating effect.46,47

Our data suggest that a combined therapy based on cholesterol-depleting agents and uPAR loss of function could be a promisingapproach in the control of tumor spreading and growth.

Acknowledgments

This work was supported by Ministero Italiano dell’Universita edella Ricerca Scientifica, Ente Cassa di Risparmio di Firenze,Toscana Life Sciences, and Istituto Toscano Tumori.

This work is in memory of Professor Mario Serio.

Authorship

Contribution: F.M., A.C., and S.S. performed in vitro experiments;A.L., L.C., and F.B. performed in vivo experiments; B.M., R.S.,and M.S. prepared ECFCs; L.M. and L.P. performed immunofluo-rescence experiments; G.D., N.S., and F.R. performed immunoflu-orescence analysis; and M.D.R. and G.F. designed the research andwrote the paper.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Gabriella Fibbi, Department of Experimental,Pathology and Oncology, Viale G.B. Morgagni 50, 50134 Florence,Italy; e-mail: [email protected]; and Mario Del Rosso, Department ofExperimental, Pathology and Oncology, Viale G.B. Morgagni 50,50134 Florence, Italy; e-mail: [email protected].

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online July 29, 2011 originally publisheddoi:10.1182/blood-2011-02-338681

2011 118: 3743-3755

Magnelli, Laura Papucci, Lido Calorini, Francesca Bianchini, Mario Del Rosso and Gabriella FibbiRiccardo Saccardi, Michela Santosuosso, Giovanna Danza, Niccolò Sturli, Fabiana Rosati, Lucia Francesca Margheri, Anastasia Chillà, Anna Laurenzana, Simona Serratì, Benedetta Mazzanti, localization of the full-length form of uPAR in caveolae

dependent angiogenesis requires−Endothelial progenitor cell

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Endothelial progenitor cell-dependent angiogenesis requires localization of the full-length form of uPAR in caveolae