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Small Molecule Therapeutics
UPARANT: A Urokinase Receptor–Derived Peptide Inhibitorof
VEGF-Driven Angiogenesis with Enhanced Stability andIn Vitro and In
Vivo Potency
Maria Vincenza Carriero1, Katia Bifulco1, Michele Minopoli1,
Liliana Lista2, Ornella Maglio2,3,Luigi Mele1, Gioconda Di
Carluccio1, Mario De Rosa4, and Vincenzo Pavone2
AbstractThis work is based on previous evidence showing that
chemotactic sequence of the urokinase receptor
(uPAR88-92) drives angiogenesis in vitro and in vivo in a
protease-independentmanner, and that the peptideAc-
Arg-Glu-Arg-Phe-NH2 (RERF) prevents both uPAR88–92- and
VEGF-induced angiogenesis. NewN-acetylated
and C-amidated peptide analogues containing a-methyl a-amino
acids were designed and synthesized tooptimize the biochemical
properties for therapeutic applications.Among
these,Ac-L-Arg-Aib-L-Arg-D-Ca(Me)Phe-NH2, namedUPARANT, adopts in
solution a turned conformation similar to that found for RERF, is
stable
to sterilization in 3 mg/mL sealed vials in autoclave for 20
minutes at 120�C, is stable in blood, and displays along-time
resistance to enzymatic proteolysis. UPARANT competes with
N-formyl-Met-Leu-Phe (fMLF) for
binding to the formyl-peptide receptor, inhibits VEGF-directed
endothelial cell migration, and prevents
cytoskeletal organization and avb3 activation in endothelial
cells exposed to VEGF. In vitro, UPARANTinhibits VEGF-dependent
tube formation of endothelial cells at a 100� lower concentration
than RERF. In vivo,UPARANT reduces to the basal level
VEGF-dependent capillary sprouts originating from the host vessels
that
invaded Matrigel sponges implanted in mice, and completely
prevents neovascularization induced by
subcorneal implantation of pellets containing VEGF in rabbits.
Both excellent stability and potency position
UPARANT as a promising new therapeutic agent for the control of
diseases fueled by excessive angiogenesis,
such as cancer and inflammation. Mol Cancer Ther; 13(5);
1092–104. �2014 AACR.
IntroductionAngiogenesis is a complex multistep process leading
to
the formation of new blood vessels from a preexistingvascular
network. During new vessel formation, endo-thelial cells degrade
their basement membrane, migrateinto the interstitial matrix, and
proliferate. An imbalancein this process contributes to numerous
malignant,inflammatory, ischemic, infectious, and immune disor-ders
(1).
The receptor for urokinase-type plasminogen activa-tor (uPAR)
plays an important role in controlling cellmigration (2, 3). uPAR
is a glycosylated glycosyl-phos-phatidyl-inositol–anchored protein
(4) formed by 3domains (DI, DII, and DIII) connected by short
linkerregions (5). Besides being responsible for
focalizinguPA-mediated plasminogen activation on cell surface(6),
uPAR also promotes intracellular signaling, thusregulating
physiologic processes such as wound heal-ing, immune responses, and
stem cell mobilization, aswell as pathologic conditions such as
inflammation andtumor progression (7–10). The role of uPAR in
angio-genesis is well documented. uPAR is able to focusurokinase
proteolytic activity on cell surface and tomodulate cell migration
(11, 12). We found that solubleforms of uPAR, containing the
88Ser-Arg-Ser-Arg-Tyr92
sequence (uPAR88-92), as well as the synthetic
peptideSer-Arg-Ser-Arg-Tyr (SRSRY), stimulates in vitro and invivo
angiogenesis in a protease-independent manner(13). The uPAR88-92
sequence interacts with the formylpeptide receptors (FPR) type 1
and 2, henceforth induc-ing cell migration in an integrin-dependent
manner(9, 14, 15). Upon binding to FPR, the synthetic peptideSRSRY
causes FPR internalization and triggers vitro-nectin receptor
activation with an inside–outside type ofmechanism (16).
Authors' Affiliations: 1Department of Experimental Oncology,
IstitutoNazionale Tumori "FondazioneG.Pascale"-IRCCS; 2Department
ofChem-ical Sciences, "Federico II" University of Naples;
3IBB-National ResearchCouncil Naples; and 4Department of
Experimental Medicine, SecondUniversity of Naples, Naples,
Italy
Note: Supplementary data for this article are available at
Molecular CancerTherapeutics Online
(http://mct.aacrjournals.org/).
Corresponding Authors: Vincenzo Pavone, Department of
ChemicalSciences, "Federico II" University of Naples, Naples,
Italy. Phone: 39-081674399; Fax: 39-081674090; E-mail:
[email protected]; andMaria Vincenza Carriero, Department of
Experimental Oncology, IstitutoNazionale Tumori "Fondazione G.
Pascale"-IRCCS, via M. Semmola,80131, Naples, Italy. Phone:
39-0815903569; Fax: 39-0815903814.
E-mail:[email protected]
doi: 10.1158/1535-7163.MCT-13-0949
�2014 American Association for Cancer Research.
MolecularCancer
Therapeutics
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Recently, we found that the residue Ser90 of uPAR iscritical for
uPAR signaling, and that S90P and S90Esingle amino acid
substitutions exert opposite effectson uPAR activities. Cells
expressing membrane-associ-ated uPAR carrying Ser90 substituted
with Glu exhibit areduced binding to and a decreased adhesion
ontovitronectin, an impaired agonist-induced FPR internal-ization,
and a dramatic reduction of in vitro and in vivocell migration and
invasion (17). To inhibit uPAR func-tions, we developed, a family
of penta-peptides carry-ing the S90E substitution in the uPAR88-92
sequence.These peptides block uPAR-dependent cell signaling
byinterfering with the complex cross-talk involving uPAR,FPR, and
integrins. The peptide containing the N-ter-minal pyro-glutamic
acid (pGlu)-Arg-Glu-Arg-Tyr-NH2 (pERERY-NH2) shares the same
binding site withSRSRY and competes with fMLF for binding to
FPR,thus preventing uPAR/FPR interaction. pERERY-NH2inhibits
migration of various tumor cell lines in culture(18). Subsequently,
new tetra-peptides having the gen-eral formula Ac-Arg-Glu-Arg-X-NH2
(X ¼ Phe, Tyr,Trp) were synthesized, and the peptide
Ac-Arg-Glu-Arg-Phe-NH2 (denoted RERF) was selected for its abil-ity
to potently prevent in vitro and in vivo cell migrationand invasion
(19). Recently, we found that RERF alsobehaves as an antiangiogenic
agent. It inhibits in vitroand in vivo responses promoted either by
uPAR88-92 orby VEGF. RERF also prevents cytoskeletal
organizationand the recruitment of avb3 integrin at the focal
adhe-sions in endothelial cells exposed to VEGF, by forcingavb3 in
an inactive state either directly or indirectly,through FPR
(20).Starting from these pieces of biologic information, we
intended to generate peptide analogues with optimizedproperties
for therapeutic applications. We synthesizednew peptides containing
a-methyl-a-amino acids, andthey were characterized from a biologic
point of view.All these new analogues are N-acetylated and
C-ami-dated, as being a common way of the N- and
C-terminalmodifications to enhance the stability to
exopeptidase-mediated proteolysis (21). Among the new series of
pep-tides, Ac-L-Arg-Aib-L-Arg-D-Ca(Me)Phe-NH2, showedthe best
desired activity in preliminary experiments ofVEGF-directed
endothelial cell migration.It adopts in solution a turned
conformation, it is quite
stable to trypsin and chymotrypsin digestion, inmice andrats
blood. It is also stable to sterilization in 3 mg/mLsealed vials in
autoclave for 20 minutes at 120�C, andremained stable at 25�C for 2
years.Ac-L-Arg-Aib-L-Arg-D-Ca(Me)Phe-NH2, named UPAR-
ANT, competeswith fMLF for binding to FPR, and inhibitsFPR
internalization in endothelial cells. It prevents
F-actinpolymerization and adhesion onto vitronectin in endothe-lial
cells exposed to VEGF. In vitro, UPARANT inhibitsVEGF-dependent
tube formation of endothelial cells at a�100 lower concentration
than RERF. In vivo, it preventsVEGF-dependent capillary sprouts
originating from thehost vessels that invaded Matrigel sponges
implanted in
mice and neovascularization induced by subcornealimplantation of
pellets containing VEGF in rabbits.
Materials and MethodsPeptide synthesis and purification
Peptides were synthesized by the solid-phaseapproach using
standard Fmoc methodology in a man-ual reaction vessel, and
purified by RP-HPLC-C18 col-umn to a 99% purity. Fluoresceinated
UPARANT (FITC-UPARANT) was synthesized by the on-resin
procedure,using e-aminocaproic acid as spacer (19). It retains
the93% of the inhibitory activity on cell migration (datanot
shown). Molecular weights were confirmed by massspectrometry.
Stability during sterilization in buffer and shelf lifeA total
of 90 sealed vials containing 0.5 mL of UPAR-
ANT in PBS at a concentration of 3 mg/mL were auto-claved for 20
minutes at 120�C. One sample was imme-diately analyzed by liquid
chromatography/mass spec-trometry (LC/MS) to ascertain the lack of
any modifiedcompound. The remaining samples were kept in a
ther-mostat at 25�C. Three vials were opened everymonth andanalyzed
by LC/MS.
Blood stabilityBlood stability studieswere performed at
IRBMScience
Park. Technical details are reported in the
SupplementaryMaterials and Methods.
Enzymatic digestion of peptidesThe peptides were subjected to
digestion by trypsin at a
peptide:trypsin ratio of 1,000:1 (w:w) for RERF and
Icm25,whereas a 10:1 (w:w) peptide:trypsin ratio was used forIcm25B
and UPARANT. The trypsin concentration is 0.6mg/L. The peptides
were also subjected to digestion bychymotrypsin at a
peptide:chymotrypsin ratio of 10:1(w:w). Icm25-4 was not studied
for enzyme digestion.Digestions were followed for 1 hour at 25�C in
0.1 M Tris-HCl buffer pH 8.5. The peptide consumption was fol-lowed
by RP-HPLCmeasuring the area peaks at differenttimes between 0 and
60 minutes. Quantification of theintact peptide peak over time
indicated that its disappear-ance seemed to follow first-order
kinetics (data notshown), and the t1/2 was estimated.
Nuclear magnetic resonance spectroscopy andstructure
calculations
Nuclear magnetic resonance (NMR) experiments wereperformed on
Bruker Avance 600 MHz spectrometer,equipped with triple resonance
cryo-probe. NMR char-acterization was performed in H2O/D2O 90:10
(v/v) andin H2O/CF3CD2OD 70:30 (v/v) at 298 K. Details arereported
in the Supplementary Materials and Methods.Restrained molecular
dynamic (RMD) simulations werecarried out using a hand-built
starting model with struc-tural data from NMR measurements [e.g.,
interprotondistances from Nuclear Overhauser Effect (NOE)
value].
Antiangiogenic Activity of UPARANT
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Details on structure calculations are reported in the
Sup-plementary Materials and Methods.
Cell culturesHuman umbilical vein endothelial cells (HUVEC)
were
purchased by Lonza (C2519A, Lot No. 0000115425),which provided a
certificate of analysis for each cell lot.This guarantees the
expression of CD31/105 and vonWillebrand Factor through 15
population doublings.HUVEC, used between the third to the seventh
passageaccording to Arnaoutova and colleagues, (22), weregrown in
Eagle basalmedium (EBM) supplementedwith4% FBS, 0.1% gentamicin, 1
mg/mL hydrocortisone, 10mg/mL EGF, and 12 mg/mL bovine brain
extract(Cambrex).
Migration assayCell migration of HUVEC was performed in
Boyden
chambers, using 8-mm-pore size
polyvinylpyrrolidone-free-polycarbonate filters at 37�C, 5% CO2 as
previouslydescribed (13). Briefly, 7 � 104 viable cells suspended
inserum-free EBM were allowed to migrate for 4 hourstoward EBM or
40 ng/mL VEGF165 (Pepro Tech), mixedwithdiluents or the
indicatedpeptides at 37�C ina5%CO2.At the end of assays, cells on
the lower filter surface werefixed, stained with hematoxylin, and
10 random fields/filter were counted at �200 magnification.
Binding assaysHUVECs (5 � 105 cells/sample) were
preincubated
with diluents or the indicated effectors for 60 minutes at4�C,
extensively rinsed with PBS and then exposed to 10nmol/L
N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein(FITC-fMLF) purchased
from Molecular Probes for 60minutes at 4�C. Quantification of
cell-associated fluores-cence was assessed by reading endothelial
cell lysateswith a fluorescence plate reader Victor 3 (Perkin
Elmer)using 485 nm excitation and 535 nm emission filters.
Analiquot of each sample was separated on 10% SDS-PAGEand subjected
to Western blot analysis for the a-tubulincontent.
Fluorescence microscopyHUVEC grown on glass slides to
semiconfluence were
incubated with the indicated effectors for 30 minutes at37�C and
then exposed to 10 nmol/L FITC-fMLF or 10nmol/L FITC-UPARANT
diluted in serum-free EBM for30 minutes at 37�C as described (19).
To analyze cytoskel-etal organization and avb3 localization, cells
grown onglass coverslips to semiconfluence, were starved for
60minutes in EBM. Then, cells were exposed to EBM alone,40 ng/mL
VEGF165, or 40 ng/mL VEGF165 mixed to 10nmol/L indicated peptides
for 30 minutes at 37�C inhumidified air with 5% CO2. Slides were
washed withPBS, fixed and permeabilized with 2.5% formaldehyde–0.1%
Triton X-100 in PBS for 10 minutes at 4�C. Afterseveral washes in
PBS, slides were incubated with2 mg/mL anti-vinculin mAb (clone
VIIF9 purchased from
Chemicon) and then with 1:800 goat Alexa Fluor 488antimouse
immunoglobulin G (IgG; Molecular Probes) at23�C for 60 and
45minutes, respectively. Thereafter, 2mg/mL LM609 anti-avb3
monoclonal antibodies (mAb; Che-micon) and then 1:800 diluted
rabbit Alexa Fluor 594antimouse IgG (Molecular Probes) were applied
to slidesat 23�C for 60 and 45 minutes, respectively. To
analyzecytoskeletal organization, coverslips were
incubatedwith0.1mg/mL rhodamine-conjugated phalloidin
(Invitrogen)at 23�C for 45 minutes. In all cases, slides were
mountedusing 20% (w/v) mowiol, cells visualized with a 510META-LSM
confocalmicroscopy (Carl Zeiss) and z-serieswith 0.38-mm intervals
were collected.
Western blottingHUVEC grown to semiconfluence were starved for
18
hours in 0.2% FBS EBM. Then, cells were exposed toEBM alone, 40
ng/mL VEGF165 with/without 10 nmol/L UPARANT at 37�C in humidified
air with 5% CO2. Atthe indicated times, cells were lysed in
radioimmuno-precipitation assay buffer (10 mmol/L Tris pH 7.5,
140mmol/L NaCl, 0.1% SDS, 1% Triton X-100, 0.5% NP40)containing 5
mmol/L Na3VO4 and a protease inhibitormixture (Sigma-Aldrich). Cell
lysate protein contentwas measured by a colorimetric assay
(Bio-Rad). West-ern blot analysis was performed as previously
described(17, 20). Briefly, 50 mg proteins were separated on
10%SDS-PAGE and transferred onto a polyvinylidene fluo-ride
membrane. The membranes were blocked with 5%nonfat dry milk and
probed with anti-phospho-Akt(Ser473; p-AKT) Ab or anti-p-ERK1/2
(Thr202/204) Ab(Cell Signaling) and then with anti-AKT mAb
(R&DSystem) or anti-ERK1/2 mAb (Millipore). In all cases,washed
filters were incubated with horseradish perox-idase–conjugated
antimouse or anti-rabbit antibody anddetected by Enhanced
Chemiluminescence Kit (GE-Healthcare).
Cell adhesion assayCell adhesion assays were performed using
16-well
plates coated with 5 mg/mL vitronectin, diluted in PBSand the
xCELLigence technology (Roche Diagnostics) asdescribed (23). HUVEC
(5� 103 cells/well) were plated ineach coatedwell, in serum-free
EBM in the presence or theabsence of 40 ng/mL VEGF165 with/without
10 nmol/LC-terminal amidated Ac-Glu-Arg-Phe-Arg-NH2 controlpeptide
(ERFR), 10 nmol/L RERF or 10 nmol/L UPAR-ANT and allowed to adhere
for 4 hours at 37�C, 5% CO2.Cell adhesion was automatically
monitored by the RealTime Cell Analysis xCELLigence system (Acea
Biosci-ence), which measures the impedance value of each welland
expressed as a cell index value.
Cell viabilityFor the MTS assay, the CellTiter 96 AQueous Cell
Pro-
liferation Assay Kit (Promega) was used following
themanufacturer’s instruction. Briefly,HUVEC (2� 103/well)were
seeded in 96-well tissue culture plates and left to
Carriero et al.
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adhere in complete media for 3 hours, then rinsed twicewithPBS
followedby the incubationwithEBMmixed to 40ng/mL VEGF165 with or
without peptides at the indicatedconcentration. Medium was replaced
every 24 hours. Atthe indicated times, suspended cells were removed
and 10mL of the MTS reagent was added into each well and cellswere
incubated at 37�C for 3 hours. The absorbance wasdetected at 490 nm
with a Microplate Reader (Bio-Rad).With a similar experimental
design, endothelial cell pro-liferation was assessed using 16-well
plates and the RealTime Cell Analysis xCELLigence technology. The
imped-ance value of each well was automatically monitored
andexpressed as a cell index.
Endothelial cell tube formation assayThe formation of
vascular-like structures was assessed
on Matrigel as described (13). HUVEC were suspended in300 mL
prewarmed EBM. Diluents or effectors were addedto the cell
suspension before seeding cells (4 � 104 cells/well) on plates
coated with 300 mL/well growth factorreducedMatrigel
(BectonDickinson).When indicated, cellswere preincubated for 30
minutes at 37�C with 1 mg/mLanti-humanVEGFAb,
2mg/mLLM609anti-avb3mAbs,ornonimmune serum. Complete capillary tube
networkswere assessed within a low-magnification field
observedunder light microscopy after 6-hour incubation at 37�C
inhumidified air with 5% CO2. To quantify tube formation,5 random
areas/well at �100 magnification were imagedand the number of tubes
formed by cord-like structuresexceeding 100 mm in length (24) were
visualized usingAxiovision 4.8 software (Carl Zeiss) and
counted.
Matrigel sponge angiogenesis assayTwelve 6- to 8-week-old
C57BL/6J male mice
(Charles River Laboratories) of 23 to 25 g 6 to 8 weeksold, were
maintained in accordance with institutionalguidelines complying
with national and internationallaws and policies. Briefly, VEGF165
(100 ng/mL) andheparin (50 U/mL) diluted in PBS, with or without 75
or150 mg/kg UPARANT, were added to unpolymerizedliquid Matrigel at
4�C, to a final volume of 500 mL. TheMatrigel suspension was slowly
injected subcutaneous-ly into the flanks of mice, using a cold
syringe, where itquickly polymerizes to form a solid gel. Matrigel
withbuffer alone was used as negative control. Five daysafter
injection, the animals were killed and the gels wereremoved,
minced, and then diluted in water for hemo-globin content
measurement with a Drabkin ReagentKit (Sigma). The final hemoglobin
concentration wasnormalized to 100 mg of recovered gel and
calculatedfrom a standard calibration curve after
spectrophoto-metric analysis at 540 nm.
Corneal pocket assayCorneal pocket assays were performed at
PRIMM.
Twelve female New Zealand White rabbits (CharlesRiver) weighing
2.5 to 3.0 kg were anesthetized byintramuscular injection of
acepromazina (1 mg/kg),
ketamine hydrochloride (35 mg/kg), and xylazinehydrochloride (5
mg/kg) and 3 to 4 drops of 0,4%ossibuprocain chlorohydrate solution
were topicallyapplied to the eye before micropocket surgery. A
wirespeculum was positioned in the eye, and a sucralfate-hydron
pellets containing PBS, 5 mg UPARANT, or 180ng VEGFwith/without 5
mg UPARANTwere implantedinto the cornea aftermaking amicropocket in
the stroma,using standard surgical tools (25, 26). Tobradex
(0.3%tobramycin–0.1% dexamethasone) was applied to thesurface of
the cornea after pellet implantation to preventinfection.
Observation and quantification of the angio-genic responses were
performed by a slit-lamp stereo-microscope. The angiogenic activity
was evaluated onthe basis of the number and growth rate of newly
formedcapillaries. A density value of 1 corresponded to 0 to
25vessels per cornea, 2 from 25 to 50, 3 from 50 to 75, 4 from75 to
100, and 5 for >100 vessels.
Statistical analysisThe results are expressed as the mean � SD
of the
number of the indicated determinations. Data were ana-lyzed by
one-way analysis of variance and post hoc Bon-ferroni modified t
test for multiple comparisons. P < 0.001was accepted as
significant.
Ethics statementThe research work with mouse model has been
approved by Institutional Ethical Committee of IstitutoNazionale
Tumori "Fondazione G. Pascale"-IRCCS,Naples, Italy (protocol no.
09, December 20, 2010).
ResultsDevelopment and synthesis of RERF analogues
Previously, we reported the NMR structure of RERFin solution
(19). RERF, although displaying some con-formational flexibility,
preferentially adopts an a-turn(type I-aRS) conformation (27) in
water/trifluoroethanol(TFE) mixed solvent. Ca-methyl-a-amino acids
withwell-defined stereo chemical properties impose
localrestrictions on backbone conformation, thus
conferringstructural stability (28–29). Aib residue (a-amino
iso-butyric acid) is the prototype of Ca-methyl-a-aminoacids, and
is well known to be characterized by arestricted conformational
freedom (30) in the 310-a-helical region of the Ramachandran plot.
OtherCa-methyl-a-amino acids, including Ca-methyl-phe-nylalanine
(31) are b-turn and helix inducers, muchstronger than the
unmethylated, phenyl-containing,protein amino acid Phe.
Ca-methy-a-amino acid, whenincorporated into peptide sequences
containing codedresidues, strongly facilitates and stabilizes type
IIIb-turns or helicogenic a-turns as previously described(32, 33).
In addition, Ca-methyl-a-amino acid substitu-tions could provide
enhanced binding to a moleculartarget, resistance to proteolytic
degradation and longerpersistence in circulation. On the basis of
this informa-tion, we have synthesized 4 peptide analogues of
RERF
Antiangiogenic Activity of UPARANT
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containing commercially available Ca-methyl-a-aminoacids,
namely:
Icm25-4 and Icm25-4c are both tetrapeptides analoguesof RERF in
which the Glu residue has been replaced byAib. Icm25-4c also
contains a second substitution of theterminal Phe residue with
D-Ca(Me)Phe (S-configura-tion). Icm25 and Icm25B are both
pentapeptides corre-sponding to N-terminal elongation of Icm25-4
and Icm25-4c with a second Aib residue.
Enzymatic digestionThe peptides were subjected to digestion by
trypsin
and chymotrypsin at different peptide:enzyme ratios,depending on
the digestion rates. The enzyme concen-tration is 0.6 mg/L, and is
similar to trypsin concentra-tion determined in plasma 117 to 637
mg/L (34). Icm25-4was not studied for enzyme digestion. The
peptideconsumption was determined by measuring the HPLCarea peaks
during digestion. It seemed to follow first-order kinetics (data
not shown), and the t1/2 wereestimated. We found that RERF and
Icm25 at a pep-
tide:trypsin ratio of 1,000:1 are rapidly digested, the
t1/2being 3.05 and 6.19 minutes, respectively. No digestionof
Icm25B and Icm25-4c was detected at a peptide:trypsin ratio of
1,000:1, whereas at a peptide:trypsinratio of 10:1 the t1/2 were
37.5 and 11.4 minutes forIcm25B and Icm25-4c, respectively. Both
RERF andIcm25, containing the natural Phe residue at the
C-terminus, were digested by chymotrypsin with t1/2being 23.6 and
20.0 minutes, respectively. Conversely,no digestion of Icm25B and
Icm25-4c, both containingthe unnatural C-terminal Ca(Me)Phe, was
detectedwithin 60 minutes.
Screening of the peptides as inhibitors of VEGF-dependent
endothelial cell migration
RERF has been shown to inhibit endothelial cell migra-tion
driven by VEGF (20). We compared the effects ofRERF and Aib
containing peptides in inhibiting endothe-lial cell migration
directed toward 40 ng/mLVEGF165. Byconventional Boyden chamber
assays, we found that 40ng/mL VEGF165 elicited endothelial cell
migration up to235% of the basal cell migration. When used as
chemoat-tractant at 10 nmol/L concentration, Aib containing
pep-tides, as well as RERF, slightly reduced basal cell migra-tion,
whereas the control peptides ARARY or ERFR wereineffective (Table
1). As expected, a 57% and 54%
Table 1. Peptide inhibitory activity on VEGF-dependent
endothelial cell migration
Effector Chemoattractants Cell migration (%) Inhibition (%)
EBM CTL 10040 ng/mL VEGF 235 � 1410 nmol/L ERFR 101 � 2a10
nmol/L ARARY 110 � 4b10 nmol/L RERF 99 � 5b10 nmol/L Icm25 95 �
9b10 nmol/L Icm25B 94 � 9b10 nmol/L Icm25-4 105 � 12b10 nmol/L
ICM25-4c 89 � 6b
1 mg/mL VEGF Ab EBM 90 � 340 ng/mL VEGF 102 � 2b 57
10 nmol/L ERFR 40 ng/mL VEGF 231 � 9 210 nmol/L Icm25-4 40 ng/mL
VEGF 121 � 8a 4910 nmol/L RERF 40 ng/mL VEGF 110 � 5b 5410 nmol/L
Icm25 40 ng/mL VEGF 96 � 5b 5910 nmol/L Icm25B 40 ng/mL VEGF 90 �
8b 6210 nmol/L Icm25-4c 40 ng/mL VEGF 75 � 3b 68NOTE: HUVEC (4 �
104 cells/well) were allowed to migrate toward chemoattractants, in
the presence of the indicated effectors for 4hours at 37�C,
5%CO2.Quantitative analysis of cellmigrationwas calculatedas
apercentageof cellmigration assessed in the absenceof anyeffector
or chemoattractant (CTL). The inhibitory effect of eachpeptidewas
reported relative to the extent of cellmigration towardVEGF (taken
as 100%).Data are themean�SDof 2 independent experiments, performed
in duplicate. Theywere analyzed byone-wayANOVA and post
hocBonferonnimodified t test for multiple comparisons. Statistical
significancewithP valueswas calculated against40 ng/mL
VEGF.aStatistical significance with P < 0.001.bStatistical
significance with P < 0.0001.
Icm25-4 Ac-Arg-Aib-Arg-Phe-NH2Icm25
Ac-Aib-Arg-Aib-Arg-Phe-NH2Icm25B
Ac-Aib-Arg-Aib-Arg-D-Ca(Me)Phe-NH2Icm25-4c
Ac-Arg-Aib-Arg-D-Ca(Me)Phe-NH2
Carriero et al.
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inhibition of VEGF-directed endothelial cell migrationwas
elicited by anti-VEGF Ab and by RERF, respectively.The combination
of VEGF165 with 10 nmol/L concentra-tion of Icm25, Icm25B, Icm25-4,
or Icm25-4c reducedendothelial cell migration by 59%, 62%, 49%, and
68%,respectively (Table 1). Because the peptide Icm25-4cshowed a
long t1/2 in enzyme digestions, and exerted thestrongest inhibition
of endothelial cell migration, it wasnamed UPARANT, and further
characterized for its bio-chemical properties, conformational
preferences and bio-logic activity.
Stability of UPARANTWe examined the stability of UPARANT during
stan-
dard autoclave sterilization. Ninety sealed vials contain-ing
0.5 mL of UPARANT in PBS at a concentration of 3mg/mL were
autoclaved for 20 minutes at 120�C. Onesamplewas immediately
analyzed by LC/MS to ascertainthe lack of any modified compound.
The remaining sam-ples were kept in a thermostat at 25�C. Three
vials wereopened everymonth and analyzed byLC/MS.As a result,no
degradation after autoclaving and standing at 25�C insealed vials
for 2 years was observed (data not shown).The stability of UPARANT
in physiologic solution, in
mice and rats blood was also studied at 0.3 and 1
mg/mLconcentrations. Blood stability was followed for 2
hours.UPARANT resulted undegraded as shown in Supple-mentary Tables
S1 and S2.
NMR analysis of UPARANTWe investigated the conformational
preferences of
UPARANT inwater and in water/TFE byNMR spectros-copy
(Supplementary Tables S3 and S4 and Fig. S1).ROESY spectra in water
and in water/TFE essentiallydisplay the samepattern of sequential
andmedium-range
NOE effects (see SupplementaryMaterials andMethods),indicating
that the peptide has a quite similar conforma-tional behavior in
both solvent systems. However,detailed analysis was performed in
water/TFE solutiononly, because of more dispersed amide signals and
fewerresonance overlaps. The pattern of NOEs and their rela-tive
intensities are summarized in Fig. 1A. RMD calcula-tions, using the
NMR experimental data as conformation-al restraints, gave an
ensemble of conformers quite similarin terms of backbone
conformation, underlining the pres-ence of one predominant and
stable structure forUPARANT. Figure 1B reports the backbone atoms
super-position of the ensemble of conformers along the
MDtrajectory. Figure 1C reports the average backbone torsionangles
of UPARANT as obtained fromRMDsimulation invacuo at 300 K.
Thepeptide conformationcorresponds toan incipient 310-helix
characterized by 2 consecutive type III b-turn, fol-lowed by a type
I b-turn. This structure is stabilized by thetypical COi–HNiþ3
hydrogen bonds (see Fig. 1C): a weakinteraction Ac-CO���NH-Arg3, a
H-bond Arg1-CO���NH-Ca(Me)Phe4, and Aib2-CO���NH-amide. Along the
trajec-tory of the RMD simulation, the Arg1 and Arg3
guanidinegroups experience intraresidue H-bonds, and the Arg1
guanidine group is also H-bonded to Ca(Me)Phe4-COgroup.
UPARANT competes with fMLF for binding to
theformyl-peptide-receptor
We have previously documented that RERF competeswith fMLF for
binding to FPR, being FPR the mainbinding site of RERF (19). To
test whether similar toRERF (20), UPARANT competes with fMLF for
bindingto FPR, HUVEC were preincubated at 4�C (to avoidreceptor
internalization) with diluents, 100 nmol/L
Figure 1. NMR analysis ofUPARANT. A, summary ofsequential,
short- and medium-range connectivities. Thethickness of the bar
indicates therelative intensities of theNOEs, thatis, weak, medium,
and strong. TheX in position 2 and the Z in position4 correspond to
Aib and Ca(Me)Phe, respectively.bR andbS refer tothe protons on the
Cb atoms at theproR and proS positions,respectively. Dd/DT refers
to thetemperature coefficients of theamide protons. B, ensemble
ofconformers along the RMDtrajectory. C, average
molecularconformation of the backbonetorsion angles f, c. H-bonds
areshown as broken lines with thecorresponding distances.
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concentration of fMLF, RERF, or UPARANT for 60 min-utes at 4�C,
and then exposed to 10
nmol/LN-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for
addi-tional 60 minutes at 4�C. Quantification of
cell-associatedfluorescence was assessed by reading endothelial
celllysates with a fluorescence plate reader. An aliquot ofeach
sample was subjected to Western blot analysis withanti-a-tubulin to
control that equal amount of proteins/sample has been read (inset
to Fig. 2A). Measurement ofcell-associated fluorescence revealed
that the control pep-tide ERFRdid notmodify binding of
FITC-fMLF,whereasan excess of unlabeled fMLF,RERF (used for
comparison),or UPARANT dramatically reduced binding of fluores-cent
FPR-agonist to endothelial cells at a similar extent(Fig. 2A). To
evaluate the effect of UPARANT on FPR
internalization in response to agonist-stimulation, bind-ing
experiments were also performed at 37�C. Uponexposure to FITC-fMLF,
FPR seemed mainly internalizedwith the appearance of fluorescent
spots localized in thecytoplasmas shownbyz-stack analysis of
confocal images(Fig. 2B and C). Similar to fMLF or RERF, 100
nmol/LUPARANT strongly reduced internalization of fluores-cent
agonist in all cell population (Fig. 2B). To assesswhether UPARANT
itself causes FPR internalization,HUVECs were preincubated with
diluents, or an excessof fMLF, RERF, or UPARANT for 30 minutes at
37�C, andthen exposed to 100 nmol/L FITC-UPARANT for addi-tional 30
minutes at 37�C. As shown by z-stack analysis ofconfocal images,
endothelial cells exhibited only a slightlyinternalization of
FITC-UPARANT,whichwas prevented
Figure 2. UPARANTprevents agonist-dependent FPR internalization
in endothelial cells. A, HUVECwere preincubatedwith diluents
(None), 100 nmol/L ERFR,100 nmol/L fMLF, 100 nmol/L RERF, or 100
nmol/L UPARANT for 60 minutes at 4�C, and then exposed to 10 nmol/L
N-formyl-Nle-or Leu-Phe-Nle-Tyr-Lys-fluorescein (FITC-fMLF) for 60
minutes at 4�C. Fluorimetric measurement of cell-associated
fluorescence was assessed using 485 nm excitation and535 nmemission
filters. Data, expressed as a percentage of the fluorescence
associated to cells exposed to FITC-fMLF (None¼ 100%),
representmean�SDfrom an experiment performed in triplicate,
representative of 2 replicates. Inset, Western blot analysis for
the a-tubulin content on an aliquot of eachcell lysate. B–E,
representative imagesofHUVEC incubatedwith diluents (None), 100
nmol/L fMLF, 100 nmol/LRERF, or 100 nmol/LUPARANT for 30minutesat
37�C, exposed to 10 nmol/L FITC-fMLF (B and C) or 10 nmol/L
FITC-UPARANT (D and E) for 30 minutes at 37�C and then visualized
using a Zeiss510 META LSM microscope. Z-series images represent
focal planes corresponding to 0.38 mm vertical interval of HUVEC
incubated with FITC-fMLF (C) orFITC-UPARANT (E) alone. Scale bar,
10 mm. Original magnifications, �630.
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by unlabeled fMLF, RERF, as well as by UPARANT (Fig.2D and E).
All together, these findings suggest that uPAR-ANT competes with
fMLF for binding to FPR on cellsurface and inhibits FPR activation
by preventing itsinternalization.
UPARANT inhibits cytoskeletal organization, avb3recruitment at
focal adhesions, and adhesion ontovitronectin of endothelial cells
stimulatedwithVEGFVEGF-triggered endothelial cell motility is the
result of
marked cytoskeletal reorganization and accumulation ofstress
fibers associated with new actin polymerization,and rapid formation
of focal adhesions, which do notoccur in the presence of RERF (20).
To analyze the effectsof UPARANT on cytoskeletal organization
induced byVEGF, HUVEC were exposed to 40 ng/mL VEGF165, inthe
presence/absence of 10 nmol/L UPARANT, and thendouble stained for
vinculin and F-actin. As expected,VEGF inducedamarked
reorganization of actin into stressfibers spanning the length of
the cells, most of which
colocalized with vinculin-positive focal adhesions, thatwas
prevented by 10 nmol/L RERF (Fig. 3A). A total of 10nmol/L UPARANT
fully abrogated VEGF-inducedeffects on cytoskeleton. In the latter
case, endothelial cellshad a condensed, rounded morphology; similar
tountreated cells, the F-actin was condensed into fewerfibers and
was completely absent from the leading edgesof the cells (Fig. 3A).
It is known that avb3 integrin has aprominent role in the activity
of VEGF. In response toVEGF, b3 integrin regulates
integrin-dependent actinreorganization, thus leading avb3-VEGFR2
complexes tolocalize at new formed focal adhesions (35).
UPARANTcaused disappearance of avb3 at focal adhesions and
theappearance of thin, avb3-positive linings at the cell
edge,similar to untreated cells, whereas the addition of 10nmol/L
ERFR did not modify VEGF-dependent integrinredistribution. (Fig.
3B). RERF has been proven to inhibitVEGF-dependent signaling by
affecting avb3 integrin-dependent cell adhesion onto vitronectin
(20). We inves-tigated whether avb3 integrin is indeed involved in
the
Figure 3. UPARANT prevents cytoskeletal organization,
disappearance of avb3 from focal adhesions and cell adhesion onto
vitronectin in endothelial cellsstimulated with VEGF. A,
representative confocal images of HUVEC grown on glass slides to
semiconfluence exposed to EBM (None), or 40 ng/mL
VEGF165with/without 10 nmol/L RERF or 10 nmol/L UPARANT for 30
minutes at 37�C and double stained for vinculin and F-actin. Scale
bar, 20 mm. Originalmagnification, �630. B, representative confocal
images of HUVEC grown on glass slides to semiconfluence, exposed to
EBM (None), 40 ng/mL VEGF165mixed to 10 nmol/L ERFR or 10 nmol/L
UPARANT for 30 minutes at 37�C and double stained for vinculin and
avb3. Scale bar, 20 mm. Original magnification,�630. C, HUVEC (5 �
103/well) suspended in EBM mixed to diluents or 40 ng/mL VEGF165
with/without the indicated peptides were allowed toadhere for 4
hours at 37�Con 16-well plates coatedwith vitronectin. Cell
adhesionwas automatically monitored by the xCELLigence
system,whichmeasuresthe impedance value of each well, expressed as
cell index. In the graph, the black line shows the time of peptide
addition at which cell index valueswere normalized. Data, mean � SD
from a quadruplicate experiment representative of 2 replicates.
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inhibitory activity of UPARANT. HUVEC suspended inEBM mixed to
diluents or 40 ng/mL VEGF165 with/without 10 nmol/L ERFR, 10 nmol/L
RERF, or 10nmol/L UPARANT, were allowed to adhere for 4 hoursat
37�C on 16-well plates coated with vitronectin. Wefound that RERF
andUPARANT reduced endothelial celladhesion onto vitronectin at a
similar extent, either in thepresence or in the absence of VEGF,
whereas the controlpeptide ERFRwas almost ineffective (Fig. 3C).
All togeth-er, these findings show that UPARANT prevents
VEGF-driven avb3 relocalization and ligand-dependent
avb3activation.
UPARANT inhibits VEGF-dependent migration andintracellular
protein phosphorylation of endothelialcells without affecting cell
proliferation
To better characterize the inhibitory effect of UPAR-ANT on
VEGF-dependent cell migration, HUVEC wereallowed tomigrate in
Boyden chambers toward 40ng/mLVEGF165with/without increasing
concentrations of RERF
used as positive control, ERFRused as negative control,
orUPARANT.We found that unlike ERFR, bothUPARANTand RERF inhibit
VEGF-directedmigration of endothelialcells in a dose-dependent
manner. In both cases, theinhibition starts in the low fmol/L
concentration rangeand plateaus in the nmol/L range (Fig. 4A). RERF
hasbeen proven to prevent VEGF-induced phosphorylationof many
proteins, such as AKT and ERK1/2 (20). Toinvestigate the effects of
UPARANT on the phosphory-lation of these VEGF intracellular
targets, HUVEC wereexposed to EBM or 40 ng/mLVEGF165 with or
without 10nmol/L UPARANT from 0 to 60 minutes, and phosphor-ylated
AKT and ERK1/2 were detected by Western blot.As shown in Fig. 4B,
UPARANT consistently decreasedthe VEGF165-induced and
time-dependent increase of Aktand ERK1/2 phosphorylation. Moreover,
ANOVA statis-tical analysis revealed that growth of endothelial
cellsexposed to VEGF was not modified by 10 nmol/L or 10mmol/L
UPARANT up to 72 hours as shown by an MTSassay (Fig. 4C) as well as
by proliferation curves
Figure4. UPARANT inhibitsmigration andprotein phosphorylation of
endothelial cells stimulatedwith VEGFwithout affecting cell
proliferation. A,HUVECwereallowed to migrate in Boyden chambers for
4 hours at 37�C in 5% CO2 toward 40 ng/mL VEGF165 plus diluents
(None) or increasing concentrations of theindicated peptides. The
extent of cell migration was expressed as a percentage of the net
VEGF165-dependent cell migration, considered as 100%.Data, mean� SD
of 3 independent experiments, performed in duplicate. Statistical
significance with P values was calculated against 40 ng/mL VEGF
(None).�, statistical significance with P < 0.0001; ��,
statistical significance with P < 0.001. B, HUVEC grown on glass
slides to semiconfluence were exposedto 40 ng/mL VEGF165
with/without 10 nmol/L UPARANT at 37�C in humidified air with 5%
CO2 for the indicated times. Whole cell lysates (50
mg/sample)immunoblotted with anti-phospho-Akt (pAKT) or
anti-phospho-ERK1/2 (pERK1/2) Abs and then with total anti-Akt
(AKT) Ab or total anti-ERK1/2 (ERK) mAb.An experiment
representative of 2 replicates is shown. C and D, HUVEC (2 �
103/well) were seeded in 96-well tissue culture (C) or 16-well
plates (D),left to adhere in complete media for 3 hours at 37�C 5%
CO2, rinsed twice with PBS, and then allowed to grow in EBM mixed
to 40 ng/mL VEGF165 with orwithout the indicated peptides for 72
hours at 37�C 5% CO2. Medium was replaced every 24 hours. C,
absorbance of adherent cells was assessedby MTT assay. Plot
represents the mean � SD of 2 independent experiments, each
performed in quadruplicate. D, the impedance value of each well
wasautomatically monitored by the xCELLigence system and expressed
as cell index. Data, mean � SD from a quadruplicate experiment
representativeof 2 replicates.
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automatically monitored with the xCELLigence system(Fig. 4D).
This is in agreement with our previous datashowing that 10 mmol/L
RERF does not modify thegrowth of endothelial cells. Although we
have not iden-tified yet the exact molecular mechanism by which
RERFand UPARANT inhibit VEGF-triggered signaling, ourdata suggest
that RERF and UPARANT impinge on com-mon downstream signaling
mediators.
UPARANT inhibits tube formation of endothelialcells exposed to
VEGF in vitroTo investigate the ability ofUPARANT to inhibit
VEGF-
triggered angiogenesis in vitro, endothelial cells wereplated on
Matrigel in the presence of 40 ng/mLVEGF165 mixed to diluents or
increasing concentrationsof RERF, ERFR, or UPARANT. As expected,
endothelialcells exposed to VEGF, formed a 3-dimensional networkof
tubes resembling capillary-like structures, whichreached at 6 hours
the 257 � 10% of braches counted in
the absence of any angionenic stimulus (Fig. 5A and B).Unlike
ERFR control peptide or nonimmune serum, endo-thelial cell
incubation with anti-VEGF Ab or anti-avb3mAb reduced VEGF-dependent
tube formation to thebasal level (Fig. 5B). UPARANT reduced the
extent ofVEGF-dependent tube formation in a dose-dependentmanner.
Similar to RERF, the inhibitory effect of UPAR-ANT starts in the
femtomolar concentration range andlevels off in the nanomolar
range. Remarkably, UPAR-ANT caused an overall 75% inhibition of
VEGF-depen-dent tube formation above 10 nmol/L, when comparedwith
RERF, which caused a 54% inhibition. Also, the IC50of UPARANT seems
to be �100-fold lower as comparedwith RERF (Fig. 5C), indicating
again that UPARANTinhibits VEGF signaling more efficiently than
RERF.
UPARANT inhibits angiogenesis in vivoTo assess the effect of
UPARANT on angiogenesis in
vivo, first a quantitative Matrigel sponge assay was
Figure 5. UPARANT inhibits VEGF-induced angiogenesis in vitro
and in vivo. A–C, HUVEC were suspended in prewarmed EBM, with or
without 40 ng/mLVEGF165 in the presence or the absence of 5 mg/mL
nonimmune serum (NIS), 1 mg/mL anti-human VEGF Ab, 5 mg/ml
anti-avb3 mAb, clone LM609,or the indicated peptides at 10nmol/L
concentration and seededonMatrigel-coatedplates for 6 hours at
37�C, 5%CO2. A, representative pictureswere takenwith an inverted
microscope at �200 magnifications. B, quantitative analysis of tube
formation was indicated as a percentage of tubes formed bycord-like
structures exceeding 100 mm in length, counted in the absence of
any angiogenic stimulus considered as 100% (CTL). Data, mean � SD
of 3independent experiments performed in duplicate. �, P <
0.0001 in respect to VEGF (None). C, quantitative analysis of tube
formation was indicated as apercentage of tubular branches counted
in the presence of VEGF165 alone, considered as 100%. Data, mean �
SD of 2 independent experimentsperformed in duplicate. D, Matrigel
sponge assay in mice. Matrigel-containing sponges loaded with PBS
(CTL), 100 ng/mL VEGFmixed to PBS (None), 75 or150 mg UPARANT were
implanted subcutaneously into the dorsal flank of 12 C57BL/6J male
mice. Five days after injection, the animals were killedand the
gels were removed,minced, and diluted inwater. The hemoglobin
concentrationwas determinedwith a Drabkin Reagent Kit, normalized
to 100mg ofrecovered gel and calculated from a standard calibration
curve after spectrophotometric analysis at 540 nm. �, P < 0.0001
in respect to VEGF alone(None). E and F, corneal pocket assay in 12
female New Zealand White rabbits. Sucralfate-hydron pellets
impregnated with diluents (PBS), 5 mg UPARANT,180 ng VEGF, or a
mixture of 180 ng VEGF and 5 mg UPARANT were implanted in rabbit
corneas. E, representative stereomicroscopy
imagesrecorded15dayafter pellet implantation. F, quantification of
the angiogenic score (1 corresponds to0–25vessels per cornea, 2
from25 to50, 3 from50 to75, 4from 75 to 100, and 5 for >100
vessels). Plots represent data from a total of 6 eyes/sample. The
median values are indicated.
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performed. A cocktail of VEGF165 (100 ng/mL) and hep-arin (50
U/mL) promoted a hemorrhagic vascularizationof the Matrigel sponge,
which was clearly detectable at 5days postimplantation (Fig. 5D).
The presence in thesponge of 75 or 150 mg UPARANT produced a
markedreduction of capillary sprouts originating from the
hostvessels that invaded Matrigel sponges as detected byvisual
inspection of the gels and quantification of hemo-globin recovered
from sponges (Fig. 5D). A 64% and 72%reduction of hemoglobin was
found in sponges loadedwith 75 and 150 mg, respectively.
Furthermore, antiangiogenic activity of UPARANTwas investigated
by a corneal pocket assay in rabbits.Neovascular growth was
evaluated in 12 rabbits uponsubcorneal implantation of slow release
pellets containingPBS, 5 mg UPARANT, or 180 ng VEGF with/without 5
mgUPARANT. Implantation of pellets containing 180 ngVEGF stimulated
ingrowth of blood vessels in the rabbitcorneas starting from day 2,
and continuing to growprogressively up to 15 days before
regressing. At day15, rabbit corneas probed with pellets
impregnated withVEGF exhibited higher angiogenic score then the
averagescore of those impregnated with vehicle only (PBS; 3.3 �0.6
vs. 0.1 � 0.1 with P < 0.0001; Fig. 5E and F). WhenUPARANTwas
tested at 5 mg/pellet alone, it did not elicitinflammatory
response. Conversely,UPARANTelicited a75% inhibition of
VEGF-induced vascularization (0.84 �0.4 vs. 3.3 � 0.6 with P <
0.0001; Fig. 5E and F). Takentogether, our findings indicate
thatUPARANTbehaves asan antiangiogenic factor, which prevents
VEGF-inducedangiogenesis in vivo more effectively than RERF.
DiscussionThis work is based on previous evidence showing
that
the chemotactic sequence of the human urokinase recep-tor
(uPAR88-92) drives in vitro and in vivo angiogenesis in
aprotease-independent manner (13). A few years ago wedeveloped the
peptide Ac-Arg-Glu-Arg-Phe-NH2, name-ly RERF, that prevents, in
vitro and in vivo, migration andinvasion of tumor cells by
inhibiting the uPAR88-92-depen-dent signals (19).More
recently,wehavedocumented thatRERF prevents both uPAR88–92- and
VEGF-inducedangiogenesis in vitro and in vivo (13). However, RERF
isexpected to have some pharmacokinetic drawbacks as
drug candidate, which are common to peptides: they areusually
very susceptible to proteolytic degradation in vivoand are rapidly
cleared from the circulation in minutes(36). To overcome these
drawbacks, we have generated aseries of peptide analogues of RERF
containing Ca-meth-yl-a-amino acids, a structural modification used
in thepast to overcome these limitations (37). Among the enor-mous
scenario of possible peptide modifications devel-oped in the last
50 years, we have chosen Ca-methyl-a-amino acid substitutions on
the basis of the previouslyreportedNMR solution structure of RERF.
In fact, when itis necessary to modify the peptide composition
toimprove the pharmacologic profile, it is also mandatoryto keep,
and possibly reinforce, the bioactive conforma-tion, and henceforth
the pharmacophore orientation as inthe original bioactive peptide.
The NMR solution struc-ture of RERF revealed that this peptide,
although display-ing some conformational flexibility,
preferentially adoptsa turned structure, therefore Ca-methyl
a-amino acidsubstitutions were selected for their well-known
propen-sity to strongly induce turned structures. Among theseries
of newly synthesized analogues (Icm25, Icm25B,Icm25-4, and
UPARANT), UPARANT displayed in apreliminary evaluation of the
biologic activity the stron-gest inhibition of endothelial cell
migration and showed along t1/2 in enzyme digestions. UPARANT was
firstcharacterized from a structural point of view by
NMRspectroscopy.
The solution structures of UPARANT and RERFreveals common
features as can be seen by comparisonof Fig. 6A–C. The backbone
atoms superimposition for allresidues of RERF and UPARANT gave
root-mean-squaredeviation (RMSD) values of 0.76 Å, which
underlinestheir structural similarity. Both RERF and UPARANTadopt
in solution a helicogenic-turned structures; ana-turn (type I-aRS)
is found for RERF and an incipient310-helix for UPARANT (Fig. 1C).
However, UPARANTshows a more stable and compact turned structure
withrespect to RERF. As anticipated from the design, thepresence,
in the tetrapeptide UPARANT, of Ca-meth-yl-a-amino acids, such as
Aib and Ca(Me)Phe, induces asignificant stabilization of the turned
structure. UPAR-ANT and RERF share a common structural motif:
thearomatic ring of the Phe residue is flanked by 2 positively
Figure 6. Comparison of thesolution structures of UPARANTand
RERF. A, simplifiedrepresentation with backboneatoms in ribbon
drawing ofUPARANT average structure. B,averagemolecular
conformation ofRERF as derived from NMRanalysis in water/TFE
solution,backbone atoms are in ribbondrawing. C, backbone
atomssuperposition of RERF (purple) andUPARANT (cyan).
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charged residues. A major difference regards the orien-tation of
Arg1 side chain. In the structure of RERF, theguanidinium group is
involved in ion pairing with Glu2,whereas in UPARANT the same group
is involved in H-bonding with the amide terminal CO group. In
addition,it is worth mentioning that RERF and UPARANT wereboth
studied by NMR spectroscopy in 2 solvent systems,water and
water/TFE, for the well-known secondarystructure inducing effect of
TFE. RERF is poorly struc-tured in water and its turned structure
clearly appears inthe presence of TFE only. Conversely, UPARANT is
wellstructured in both solvent systems. We have foreseenthat
stabilization of the turned conformation for UPAR-ANT, similar to
that found for RERF, would have ben-eficial effects on biologic
activity. We found that UPAR-ANT inhibits VEGF-driven angiogenesis
in vitro and invivo more efficiently as compared with RERF. In
vitro,UPARANT inhibits VEGF-dependent tube formation ofendothelial
cells at a 100� lower concentration thanRERF. Remarkably, a 75%
reduction of tubes formed byendothelial cells exposed to VEGF is
achieved by UPAR-ANT at 10 nmol/L concentration whereas only a
54%inhibition has been observed in the presence of 10 nmol/L RERF.
In vivo, UPARANT reduces to the basal levelVEGF-dependent capillary
sprouts originating from thehost vessels that invaded Matrigel
sponges implanted inmice and prevents neovascularization induced by
sub-corneal implantation of pellets containing VEGF inrabbits.Our
previous work has documented that RERF pre-
vents uPAR/FPR and fMLF/FPR interactions, beingFPR the main
binding site of RERF (19). Although wehave not investigated
UPARANT/FPR association anddisassociation process, we found that an
excess ofUPARANT prevents fMLF binding to FPR, suggestingthat fMLF
and UPARANT share the same binding site.The pivotal role of avb3 in
mediating VEGF-dependentproangiogenic stimulus is well established
(35). Theinvolvement of avb3 integrin in the UPARANT inhib-itory
effect is supported by the findings that cell adhe-sion to
vitronectin is greatly reduced in the presence ofUPARANT. Taken
together, our results confirm theinvolvement of FPR in binding and
mediating inhibitionby UPARANT and suggest a mechanism by which
avb3is forced into an inactive state by UPARANT and,consequently,
VEGF downstream signaling mediatorsare not activated.Our findings
unravel a complex picture of the mech-
anistic effects of UPARANT on endothelial cells and,more
generally, highlight the role of uPAR, integrins,and VEGF-induced
signaling events in the regulation ofendothelial cell functions. At
molecular level, inhibitoryeffect of UPARANT on VEGF signaling is
shown by thedecreased amount of phospho-AKT and phospho-ERK1/2 in
VEGF-treated endothelial cells.Pharmacologic control of
angiogenesis is considered
one of the most promising approaches for the treatmentof
diseases sustained by excessive angiogenesis, such as
cancer and inflammation. However, several side effectshave been
ascribed to antiangiogenic drugs such asbevacizumab that
significantly increases the risk ofcardiac ischemic events in
cancer patients (38). Withrespect to this, UPARANT may be
considered a gooddrug by virtue of its ability to selectively
inhibit FPRunlike bevacizumab, indicating that UPARANTbehaves
differently from other VEGF inhibitors. Mech-anistically, UPARANT
blocks VEGF-triggered signalingby preventing FPR and avb3 integrin
activities withoutaffecting cell survival. In this respect,
although we havenot ascertain whether a direct interaction
betweenUPARANT and VEGF occurs, it is possible to assumethat
UPARANT bocks integrin avb3 activity, thus inhi-biting only a part
of the VEGF/VEGFR signaling. Thissuggests a potentially wider, but
still target-specificactivity for UPARANT.
Furthermore, unlike RERF, UPARANT is stable to ster-ilization,
is stable in blood, and displays a long-timeresistance to enzymatic
digestion. Peptide-based thera-peutics is one of the fastest
growing class of new drugs(39). Peptides have unique advantages as
therapeuticagents. They have high activity per unit mass, and
lowmanufacturing costs. They also offer great potency,
selec-tivity, and specificity, have low off-target toxicity and
lowdrug–drug interactionpotential. In this frame,UPARANTcould be
considered a promising therapeutic agent for thecontrol of diseases
fueled by excessive angiogenesis, suchas cancer and
inflammation.
Disclosure of Potential Conflicts of InterestM.V. Carriero has
ownership interest (including patents) in Pharma-
phelix s.r.l. V. Pavone is the President of Pharmaphelix s.r.l.
and also hasownership interest (including patents) in the same. No
potential conflictsof interest were disclosed by the other
authors.
Authors' ContributionsConception and design: M.V. Carriero, V.
PavoneDevelopment of methodology: K. Bifulco, L. Lista, L. Mele,
G.Di CarluccioAcquisition of data (provided animals, acquired and
managed patients,provided facilities, etc.): O. MaglioAnalysis and
interpretation of data (e.g., statistical analysis, biostatis-tics,
computational analysis): M.V. Carriero, K. Bifulco, M. Minopoli,O.
Maglio, V. PavoneWriting, review, and/or revision of the
manuscript: M.V. Carriero,O. Maglio, V. PavoneAdministrative,
technical, or material support (i.e., reporting or orga-nizing
data, constructing databases): K. Bifulco, L. ListaStudy
supervision: M.V. Carriero, M. De Rosa, V. Pavone
AcknowledgmentsThe authors thank M. Paro at PRIMM for corneal
pocket assays in
rabbits and EdithMonteagudo, Head of Preclinical Research, and
the staffof IRBM Science Park for blood stability studies.
Grant SupportThis work was supported by Associazione Italiana
per la Ricerca sul
Cancro 2013, project 14225 (M.V. Carriero) and by Italian
Ministry ofHealth RF-2010-2316780 (M.V. Carriero).
The costs of publication of this article were defrayed in part
by the pay-ment of page charges. This article must therefore be
hereby marked adver-tisement in accordance with 18U.S.C. Section
1734 solely to indicate this fact.
Received November 5, 2013; revised February 13, 2014; accepted
March4, 2014; published OnlineFirst April 4, 2014.
Antiangiogenic Activity of UPARANT
www.aacrjournals.org Mol Cancer Ther; 13(5) May 2014 1103
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PotencyIn Vivoand In VitroVEGF-Driven Angiogenesis with Enhanced
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Derived Peptide Inhibitor of−UPARANT: A Urokinase Receptor
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