d-Peptide analogues of Boc-Phe-Leu-Phe-Leu-Phe-COOH induce neovascularization via endothelial N-formyl peptide receptor 3

Article abstract of DOI:10.1007/s10456-020-09714-0

N-formyl peptide receptors (FPRs) are G protein-coupled receptors involved in the recruitment and activation of immune cells in response to pathogen-associated molecular patterns. Three FPRs have been identified in humans (FPR1–FPR3), characterized by different ligand properties, biological function and cellular distribution. Recent findings from our laboratory have shown that the peptide BOC-FLFLF (l-BOC2), related to the FPR antagonist BOC2, acts as an angiogenesis inhibitor by binding to various angiogenic growth factors, including vascular endothelial growth factor-A₁₆₅ (VEGF). Here we show that the all-d-enantiomer of l-BOC2 (d-BOC2) is devoid of any VEGF antagonist activity. At variance, d-BOC2, as well as the d-FLFLF and succinimidyl (Succ)-d-FLFLF (d-Succ-F3) d-peptide variants, is endowed with a pro-angiogenic potential. In particular, the d-peptide d-Succ-F3 exerts a pro-angiogenic activity in a variety of in vitro assays on human umbilical vein endothelial cells (HUVECs) and in ex vivo and in vivo assays in chick and zebrafish embryos and adult mice. This activity is related to the capacity of d-Succ-F3 to bind FRP3 expressed by HUVECs. Indeed, the effects exerted by d-Succ-F3 on HUVECs are fully suppressed by the G protein-coupled receptor inhibitor pertussis toxin, the FPR2/FPR3 antagonist WRW4 and by an anti-FPR3 antibody. A similar inhibition was observed following WRW4-induced FPR3 desensitization in HUVECs. Finally, d-Succ-F3 prevented the binding of the anti-FPR3 antibody to the cell surface of HUVECs. In conclusion, our data demonstrate that the angiogenic activity of d-Succ-F3 is due to the engagement and activation of FPR3 expressed by endothelial cells, thus shedding a new light on the biological function of this chemoattractant receptor.

Full text of DOI:10.1007/s10456-020-09714-0

Angiogenesis
https://doi.org/10.1007/s10456-020-09714-0
ORIGINAL PAPER
d‑Peptide analogues of Boc‑Phe‑Leu‑Phe‑Leu‑Phe‑COOH induce
neovascularization via endothelial N‑formyl peptide receptor 3
Mohd I. Nawaz1,5 · Sara Rezzola1 · Chiara Tobia · Daniela Coltrini · Mirella Belleri · Stefania Mitola
1
1
1
1
·
Michela Corsini1 · Annamaria Sandomenico · Andrea Caporale
2
2,6
· Menotti Ruvo
2,3
· Marco Presta
1,4
Received: 4 November 2019 / Accepted: 17 February 2020
©
Springer Nature B.V. 2020
Abstract
N-formyl peptide receptors (FPRs) are G protein-coupled receptors involved in the recruitment and activation of immune
cells in response to pathogen-associated molecular patterns. Three FPRs have been identiꢀed in humans (FPR1–FPR3),
characterized by diꢁerent ligand properties, biological function and cellular distribution. Recent ꢀndings from our laboratory
have shown that the peptide BOC-FLFLF (l-BOC2), related to the FPR antagonist BOC2, acts as an angiogenesis inhibitor
by binding to various angiogenic growth factors, including vascular endothelial growth factor-A (VEGF). Here we show
1
65
that the all-d-enantiomer of l-BOC2 (d-BOC2) is devoid of any VEGF antagonist activity. At variance, d-BOC2, as well as
the d-FLFLF and succinimidyl (Succ)-d-FLFLF (d-Succ-F3) d-peptide variants, is endowed with a pro-angiogenic potential.
In particular, the d-peptide d-Succ-F3 exerts a pro-angiogenic activity in a variety of in vitro assays on human umbilical
vein endothelial cells (HUVECs) and in ex vivo and in vivo assays in chick and zebraꢀsh embryos and adult mice. This
activity is related to the capacity of d-Succ-F3 to bind FRP3 expressed by HUVECs. Indeed, the eꢁects exerted by d-Succ-
F3 on HUVECs are fully suppressed by the G protein-coupled receptor inhibitor pertussis toxin, the FPR2/FPR3 antagonist
WRW4 and by an anti-FPR3 antibody. A similar inhibition was observed following WRW4-induced FPR3 desensitization in
HUVECs. Finally, d-Succ-F3 prevented the binding of the anti-FPR3 antibody to the cell surface of HUVECs. In conclusion,
our data demonstrate that the angiogenic activity of d-Succ-F3 is due to the engagement and activation of FPR3 expressed
by endothelial cells, thus shedding a new light on the biological function of this chemoattractant receptor.
Keywords Angiogenesis · BOC2 · d-Peptide · Formyl peptide receptor · Huvecs
Introduction
N-formyl peptide receptors (FPRs) are implicated in the
Electronic supplementary material The online version of this
regulation of innate immune responses, inꢂammation and
article (https://doi.org/10.1007/s10456-020-09714-0) contains
tissue repair [1]. They recognize peptides containing N-for-
mylated methionine of bacterial and mitochondrial origin, as
well as a variety of microbial non-formyl peptides, danger-
associated molecular pattern host-derived peptides, small
molecules and eicosanoids [1, 2].
supplementary material, which is available to authorized users.
*
Marco Presta
marco.presta@unibs.it
1
Department of Molecular and Translational Medicine,
University of Brescia, Brescia, Italy
Three FPRs have been identified in humans
(FPR1–FPR3). They are characterized by diꢁerent ligand
properties, biological function and cellular distribution [3].
FPR1 and FPR2 are expressed by monocytes and neutro-
phils. In addition, FPR1 is expressed by microglial cells,
astrocytes, hepatocytes and dendritic cells, whereas FPR2
can be found in a larger variety of non-myeloid cells [2]. Dis-
tinct from the other members of the FPR family, FPR3 (for-
merly named FPRL2) remains relatively poorly investigated.
2
3
4
Istituto Di Biostrutture e Bioimmagini, CNR, Napoli, Italy
AnBition srl, Napoli, Italy
Italian Consortium for Biotechnology (CIB), Unit of Brescia,
Trieste, Italy
5
6
Department of Ophthalmology, King Saud University,
Riyadh, Kingdom of Saudi Arabia
Present Address: Istituto Di Cristallograꢀa, CNR, Trieste,
Italy
Vol.:(0
1
123456789)
3

Angiogenesis
FPR3 is found in eosinophils, monocytes, macrophages and
dendritic cells, but its functional role remains elusive [4].
A few FPR3 ligands have been identiꢀed, including F2L,
an acetylated N-terminal fragment of human heme-binding
protein [5], and the neuroprotective peptide humanin [6].
Endothelial cells have been shown to express FPRs [7–9]
and scattered experimental evidence implicates FPRs in
neovessel formation during inꢂammatory responses. For
instance, the FPR ligands serum amyloid A (SAA), LL-37
and Hp(2–20) may regulate the angiogenic process under
inꢂammatory conditions [1]. In addition, FPR activation
appears to be involved in neovessel formation triggered by
the vitreous ꢂuid harvested from the eyes of patients aꢁected
by proliferative diabetic retinopathy [10]. In keeping with
these observations, high levels of the FPR ligand SAA are
detectable in vitreous and plasma of these patients [11] and
in eyes with macular oedema [12, 13].
of chiral centres in polypeptides might generate inactive
variants or variants with chirally inverted speciꢀcities [20,
21]. Retro-inversion is more often applied to improve the
stability of short bioactive all-l peptides, since this modiꢀ-
cation, when accompanied by reconstruction of the N- and
C-terminal groups, generates new molecules with a formida-
ble topological correlation, resulting from side chain spatial
overlap with the parent one [22–24].
On this basis, we investigated the possibility that all-d
peptide variants of l-BOC2 could represent VEGF antago-
nists more therapeutically relevant than their l-precursor.
They included the d-enantiomer of l-BOC2 (Boc-d-FLFLF,
from here on named d-BOC2), its d peptide derivative freed
of the Boc group (d-FLFLF, from here on named d-F3) and
a d peptide variant in which the Boc group was replaced by a
succinimidyl moiety (succinyl-d-FLFL, from here on named
d-Succ-F3). The structure of these peptides and of their all-l
enantiomers are shown in Supplementary Fig. S1.
The peptide N-tert-butyloxycarbonyl (Boc)-Phe-d-Leu-
Phe-d-Leu-Phe (BOC2) has been used extensively as a
FPR1/FPR2 antagonist to assess the role of FPRs in physi-
ological and pathological conditions (see [14–16] and ref-
erences therein). Recent ꢀndings from our laboratory have
shown that the BOC2-related peptide Boc-Phe-Leu-Phe-
Leu-Phe (BOC-FLFLF in single letter code, l-BOC2) [17],
having all-l conꢀgurations on its stereocenters, inhibits the
angiogenic activity of various heparin-binding angiogenic
growth factors, including vascular endothelial growth fac-
tor-A (VEGF), by interacting with their heparin-binding
Surprisingly, we found that, at variance with its l-enan-
tiomer, d-BOC2 is devoid of any VEGF antagonist activity.
On the contrary, d-BOC2, as well as d-F3 and d-Succ-F3,
are endowed with a signiꢀcant pro-angiogenic capacity. In
addition, we demonstrate that the angiogenic activity of
d-Succ-F3 is due to the engagement and activation of FPR3
expressed by endothelial cells.
Materials and methods
1
65
domain [9]. Thus, l-BOC2 appears to be endowed with a
multi-heparin-binding growth factor antagonist activity,
setting the basis for the design of novel l-BOC2-derived,
multi-target angiogenesis modulators [9].
Reagents
Reagents were from the following companies: M199, and
RPMI-640 media, foetal calf serum (FCS) and SYBR Green
PCR master mix (Gibco Life Technologies, Grand Island,
NY, USA); porcine gelatin, endothelial cell growth factor,
porcine heparin, 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-tri-
azole (MSNT), 1-methyl imidazole (MeIm), sym-collidine,
succinic anhydride, N,N,diisopropylethylamine (DIPEA),
Peptides are gaining a renewed interest as potential drugs
because of their generally high aꢃnity and selectivity deriv-
ing from rapid adaptation on the surface of targets enabled
by their structural ꢂexibility. Thus far, more than 60 peptide
drugs have been approved in the USA, Europe and Japan
and even more peptides are under clinical development
or have been tested in human clinical trials (reviewed in
tert-butyloxycarbonyl anhydride (Boc O), tetrahydrofuran
2
[
18]). However, despite several advantages compared to low
(THF) and dimethylsulfoxide (DMSO) (Sigma-Aldrich,
St. Louis, MO, USA); polyvinylpyrrolidone (PVP)-free
polycarbonate filters (Costar, Cambridge, MA, USA);
Diꢁ-Quik (Dade-Behring, Milan, Italy); TRIzol Reagent,
Moloney murine leukemia virus reverse transcriptase and
anti-rabbit IgG Alexa Fluor 594 (Invitrogen, Carlsbad, CA,
USA); anti-GAPDH and anti-Focal Adhesion Kinase (FAK)
antibodies (Santa Cruz Biotechnologies, Santa Cruz, CA,
USA); Matrigel (Cultrex BME, Gaithersburg, MD, USA);
l-BOC2 (Phoenix Europe GmbH, Karlsruhe, Germany);
anti-pVEGFR2 antibody (Cell Signaling Technology,
Beverly, MA); anti-FPR1, anti-FPR2, anti-FPR3 and anti-
FPR3(N-term) antibodies, and Trp-Arg-Trp-Trp-Trp-Trp-
CONH(2) (WRW4) (Abcam, Cambridge, Great Britain);
molecular weight organic therapeutics, short plasma half-life
and negligible oral bioavailability may represent two major
drawbacks intrinsic to the peptidic structure. The process
of depeptidization, consisting in the systematic alteration
of the peptidic scaꢁold to improve drug metabolism and
pharmacokinetics features while retaining bioactivity is a
major challenge in peptide-based therapeutics development
because of the strong correlation between structure, confor-
mation and biological properties of this class of molecules.
Among the others, the inclusion of d-amino acids is a way
to induce resistance to the activity of endogenous proteases,
making d-peptide-based drugs more attractive and eꢃcient
than their l-peptide counterparts [19]. However, inversion
1
3

Angiogenesis
anti-CD31 antibody and HRP-labelled anti-rabbit and anti-
mouse polymers (Dako, Santa Clara, CA, USA); VEGFR2
kinase inhibitor SU5408 (MedChemExpress LLC, USA);
VEGF-A (VEGF) was kindly provided by K. Ballmer-
2489 UV/visible Detector, using a XBRIDGE Prep BEH130
OBDTM C18 column (5 μm, 50×19 mm ID). A gradient
from 20 to 70% of solvent B in solvent A over 10 min at a
10 mL/min ꢂow rate was used to purify the peptides. Sol-
1
65
Hofer (PSI, Villigen, Switzerland); reagents for peptide
synthesis, including resins, Fmoc-protected amino acids,
couping agents 1-[Bis(dimethylamino)methylene]-1H-
vent A was 0.1% TFA in H O; solvent B was 0.1% TFA in
2
CH CN. Puriꢀed peptides were characterized for purity and
3
identity by an Agilent 1290 Inꢀnity LC System coupled to
an Agilent 6230 time-of-ꢂight (TOF) MS System (Agilent
Technologies, Cernusco Sul Naviglio, Italy), using a C18
Waters xBridge (3 μm, 50 × 4.6 mm) column, applying a
1
,2,3-triazolo[4,5-b]pyridinium 3-oxid hexaꢂuorophosphate
(
HATU), ethyl (hydroxyimino)cyanoacetate (Oxyma), N,N′-
diisopropylcarbodiimide (DIC), triꢂuoroacetic acid (TFA)
and the solvents dichloromethane (DCM) and dimethyl-
formamide (DMF) (GL-Biochem, Shanghai, PRC; IRIS
Biotech, Marktredwitz, Germany; Carlo Erba Reagents,
linear gradient from 30 to 95% of 0.05% TFA in CH CN
3
over 15 min at a ꢂow rate of 0.2 mL/min. Yields of puriꢀed
peptides ranged between 65 and 75%. Peptides were>99%
pure and their MWs were consistent with the calculated
masses within the limits of the experimental error (see Sup-
plementary Table S1).
Cornaredo, Italy); HPLC-grade CH CN (Romil, Dublin,
3
Ireland).
Peptide synthesis
Cell cultures
The peptides utilized in this study and their abbrevia-
tions are listed in Supplementary Table S1. The synthesis
of the peptides was performed on a Wang Resin (loading
Human umbilical vein endothelial cells (HUVECs) were iso-
lated from umbilical cords as described [27]. Routinely, cells
were used at early (I–IV) passages. Cells were grown on
culture plates coated with porcine gelatin in M199 medium
supplemented with 20% FCS, endothelial cell growth fac-
tor (10 μg/mL), and porcine heparin (100 μg/mL). Human
THP-1 monocytic cells (ATCC® TIB-202™) and human
Jurkat leukemia cells (ATCC) were provided by S. Sozzani
0
.79 mmol/g) using the Fmoc solid-phase strategy. Resins
(
150 mg, 118.5 μmol) were swollen in DCM for 1 h, then
the ꢀrst amino acid was introduced by treatment with Fmoc-
Phe-OH or Fmoc-d-Phe-OH (5 eq)/MSNT (5.0 eq)/MeIm
(
3.5 eq) in DCM for 3 h and repeated overnight. Resin load-
ing was assessed according to standard procedures [25] and
was estimated to be 0.65 mmol/g. Fmoc deprotection of the
(
University of Brescia, Italy) and grown in RPMI-1640
ꢀ
rst amino acid was performed with a piperidine solution at
medium plus 10% FCS. Cells were tested regularly for
4
0% in DMF for 5 min and at 20% for 10 min. Each amino
Mycoplasma negativity.
acid (4.0 eq) was double-coupled using 0.5 M Oxyma, 0.
5
M DIC in DMF, for 30 min in the ꢀrst coupling and 0.5 M
HATU and 2.0 M sym-collidine in DMF for 30 min in the
second coupling [26]. Coupling eꢃciency was assessed
by Kaiser test. Each step was followed by resin washing
Western blot analysis
Cells were collected and washed in cold PBS and homog-
enized in lysis buꢁer containing 1% Triton-X100, 0.1%
polyethylene glycol dodecyl ether (BRIJ), 1.0 mM sodium
orthovanadate, and protease inhibitors cocktail. The cell
lysate was centrifuged, and the supernatant was collected.
Aliquots of each sample containing equal amounts of pro-
tein (20–50 μg) were subjected to SDS-PAGE. Gels were
transblotted onto PVDF membrane and blots were blocked
with 3.0% BSA in PBS for 1 h at room temperature. The
blotting analysis was performed with anti-pVEGFR2, anti-
FPR1, anti-FPR2, or anti-FPR3 antibodies. After treating
the membranes with appropriate secondary horseradish
peroxidase-labelled secondary antibody, blots were devel-
oped with enhanced chemiluminescence reagent (Bio-rad
Laboratories, Inc. Hercules, CA). Images were acquired
(ChemiDoc Touch; Bio-Rad Laboratories, Inc.), and band
intensity was evaluated (Image Lab 3.0 software; Bio-Rad
Laboratories, Inc.). Data were normalized to the levels of
GAPDH or FAK expression.
(
(
3 × 5 min) with DMF. N-terminal succinimidyl peptides
l-Succ-F3 and d-Succ-F3) were generated on resin after
chain assembly by treatment for 30 min with a solution of
succinic anhydride (10 eq) in DMF, containing 5% (v/v)
DIPEA. Peptides were cleaved from the resins by treatment
for 4 h with a freshly prepared TFA/H O solution (95:5,
2
v/v). TFA was evaporated under a mild nitrogen stream;
peptides were dissolved in H O/CH CN (75:25, v/v) and
2
3
lyophilized. Boc-protected peptides (l-BOC2 and d-BOC2)
were obtained from the corresponding unprotected crude l-
F3 and d-F3 by treatment in solution with Boc O. Peptides
2
were dissolved in 1.0 M NaHCO (10 mL) and a solution
3
of Boc O (1.2 eq) in THF (10 mL) was added dropwise for
2
3
0 min. The reaction mixture was stirred at room tempera-
ture overnight. The solution was acidiꢀed at pH 3.0 with
1
.0 M HCl and dried under vacuum. Peptides were puriꢀed
on a preparative WATERS 2545 Quaternary Gradient Mod-
ule HPLC (Waters, Milan, Italy) supplied with a WATERS
1
3

Angiogenesis
Fluorescence‑activated cell sorting (FACS) analysis
of DMSO, VEGF or d-Succ-F3. After 6 h of incubation,
the formation of angiogenic meshes was evaluated under an
inverted microscope.
For the FACS analysis of FPR1-3 protein expression in
HUVECs, cells were resuspended in MACS buꢁer con-
taining 1.0% FCS and incubated for 30 min at 4 °C with
HUVEC chemotaxis assay
1
:100 dilution of anti-FPR1, anti-FPR2, or anti-FPR3 anti-
6
body. Then, cells were washed, incubated with Alexa Fluor
47 anti-rabbit secondary antibody for 20 min at 4 °C and
HUVECs (1.0×10 cells/mL) resuspended in M199 medium
6
plus 2.0% FCS were seeded in the upper compartment of a
Boyden chamber containing 0.1% gelatin-coated PVP-free
polycarbonate ꢀlters (8.0 μm pore size). d-Succ-F3 diluted
in M199 medium plus 2.0% FCS, or HUVEC complete
medium [31] were placed in the lower compartment. When
indicated, cells were co-incubated for 30 min with the anti-
FPR3 antibody and then seeded in the upper compartment.
After 4 h of incubation, cells migrated to the lower side of
the ꢀlter were ꢀxed with methanol and stained with Diꢁ-
Quik. The number of migrated cells was determined by
counting ꢀve microscopic ꢀelds per well for each sample
in triplicate.
analysed with a MACSQuant cytofluorimeter (Milteny
Biotec) using FlowJo software. For the competition bind-
ing assay, cells were incubated with anti-FPR3 antibody in
the absence or presence of 1.2 mM d-Succ-F3 peptide or
0
.5 mM WRW4. For FPR3 desensitization experiments,
HUVECs were preincubated with 30 μM WRW4 for 48 h
before FACS analysis.
Real‑time PCR
Steady-state transcription levels of FPR1, FPR2, FPR3, and
GAPDH genes were evaluated in THP1 cells and HUVECs
isolated from diꢁerent donors by semi-quantitative real-time
PCR (RT-PCR). The expression levels of murine endothe-
lial Cd31 and pan-leucocyte Cd45 markers were determined
in harvested Matrigel plugs by quantitative RT-PCR (RT-
qPCR) as described [28]. Brieꢂy, total RNA was extracted
using TRIzol reagent according to manufacturer’s instruc-
tions (Invitrogen, Carlsbad, CA). Contaminating DNA was
digested using DNAse (Promega) and 2.0 μg of total RNA
were retro-transcribed with MMLV reverse transcriptase
HUVEC proliferation assay
4
2
HUVECs were seeded at 1.7 × 10 cells/cm and serum
starved overnight. Then, cells were stimulated with 30 ng/
mL VEGF or increasing concentrations of d-Succ-F3. After
48 h, cells were counted with a MACSQuant cytoꢂuorimeter.
HUVEC survival assay
(
Invitrogen) using random hexaprimers in a ꢀnal volume of
0 μL. Then, 1/10th of the reaction product of semi-quanti-
HUVECs were maintained overnight in complete medium
or in M199 medium plus 2.5% FCS in the absence or in
the presence of increasing concentrations of d-Succ-F3.
Apoptotic cell death was assessed by Annexin-V/Iodide
Propidium double staining (Immunostep) according to the
manufacturer’s instructions.
2
tative RT-PCR was analysed on the agarose gel. RT-qPCR
was performed with a ViiA™ 7 Real-Time PCR Detection
System (Applied Biosystems) using iQ™ SYBR Green
Supermix (Biorad) according to the manufacturer’s instruc-
tions. Primer sequences for the genes of interest are listed in
Supplementary Table S2.
Ex vivo murine aorta ring assay
HUVEC sprouting assay
The assay was performed as described [32]. Brieꢂy, 1.0-
mm mice aorta rings embedded in ꢀbrin gel were incubated
with increasing concentrations of l-Succ-F3 or d-Succ-F3
in serum-free endothelial cell basal medium (Clonetics).
After 5 days, vessel sprouts, morphologically distinguish-
able from scattering ꢀbroblasts/smooth muscle cells, were
counted under a stereomicroscope.
HUVEC aggregates (800 cells/spheroid) embedded in ꢀbrin
gel [29] were treated with VEGF and increasing concentra-
tions of the peptide under test. DMSO was used as a control
unless speciꢀed otherwise. Sprouts were counted after 24 h
and data were expressed as a fold change vs control.
Matrigel morphogenesis assay
Chick embryo chorioallantoic membrane (CAM)
assay
The assay was performed as described [30]. Brieꢂy, 10 μL of
Matrigel were added to the wells of a 15-well plate (IBIDI)
Alginate beads (4.0 μL) containing DMSO or d-Succ-F3
(8.0–150 ng/pellet) were placed on the top of the CAM of
fertilized white Leghorn chicken eggs at day 11 of incuba-
tion (7 eggs per experimental point) [33]. Pellets loaded with
4
and allowed to solidify for 30 min at 37 °C. Then, 1.0×10
HUVECs resuspended in 50 μL of M199 medium plus 5%
FCS were seeded on the top of the Matrigel in the presence
1
3

Angiogenesis
VEGF (0.1 μg/pellet) were used as a positive control. After
Statistical analysis
3
days, newly formed microvessels converging toward the
implant were counted under a stereomicroscope (STEMI-
SR, Zeiss) at×5 magniꢀcation.
Statistical analysis was performed with GraphPad Prism 6
(San Diego, CA, USA) using Student’s t-test or one-way
analysis of variance followed by Bonferroni multiple com-
parison post test. All data are the mean ± SEM of three
experiments unless speciꢀed otherwise and p values<0.05
were considered statistically signiꢀcant.
Zebraꢀsh yolk membrane (ZFYM) angiogenesis
assay
Zebraꢀsh (Danio rerio) adults (wild type AB strain) were
maintained at 28 °C on a 14 h light/10 h dark cycle under
standard laboratory conditions [34]. Embryos at 48 hpf
were anesthetized with 0.016% tricaine and injected into
the perivitelline space with 0.04% DMSO or increasing
concentrations of d-Succ-F3 in the proximity of developing
subintestinal vein vessels using an InjectMan IN2 microin-
jector (Eppendorf) equipped with FemtoJet. The angiogenic
Results
l‑BOC2‑derived d‑peptides induce HUVEC sprouting
Previous observations had shown the capacity of l-BOC2 to
inhibit the angiogenic activity of VEGF [9]. On this basis, its
d-enantiomer d-BOC2 (Supplementary Table S1) was syn-
thesized in the attempt to improve the stability and possibly
the activity of the precursor molecule. Then both molecules
were assessed for their capacity to aꢁect the pro-angiogenic
activity exerted by VEGF on HUVEC spheroids embedded
in a 3D ꢀbrin gel [29]. As anticipated, l-BOC2 inhibits the
sprouting of HUVEC spheroids mediated by VEGF in a
dose-dependent manner, with no eꢁect when administered
in the absence of VEGF. At variance, its all-d variant d-
BOC2 did not exert any inhibitory eꢁect on VEGF activity.
On the contrary, d-BOC2 was able to induce the sprouting of
HUVEC spheroids and to exert an additive pro-angiogenic
eꢁect when administered to endothelial cells in the presence
of a ꢀxed amount of VEGF (Fig. 1a). Unexpectedly, these
data suggest that the replacement of l-amino acids by their
d-enantiomers induced extensive structural modiꢀcations
that abolished the VEGF antagonist activity of the peptide
and conferred a pro-angiogenic potential to the d-enantiomer
d-BOC2.
response was evaluated at 72 hpf after alkaline phosphatase
+
(
AP) staining [35]. The total number of ectopic AP vessels
sprouting from the SIVs on both sides of the embryo body
was manually counted in each embryo (n=41 in two inde-
pendent experiments). Representative images were acquired
using a Leica MZ16F stereomicroscope equipped with a QI
ClicK Mono Uncooled Camera and Qcapture Pro software.
In vivo Matrigel plug assay
Matrigel was mixed at 4 °C with DMSO or with 5.0 μg
of d-Succ-F3 or l-Succ-F3 and injected subcutaneously
(
0.3 mL/mouse) into the ꢂank of 6–8 week-old C57BL6
female mice (Charles River, Calco, Italy). After 7 days,
plugs were harvested and processed for total RNA extrac-
tion after adding to each plug a ꢀxed number of human cells
as an internal standard [28]. Then, total RNA was extracted
using the TRIzol Reagent, samples were analysed for the
expression of murine Cd31 and Cd45 markers by RT-qPCR
and data were normalized for human GAPDH expression
levels [28]. In each experiment, an arbitrary value equal to
In order to conꢀrm these preliminary observations and to
elucidate the contribution of the N-terminal Boc group to the
observed pro-angiogenic activity of the all-d derivative, the
d-peptide d-F3 lacking the N-terminal Boc protecting group
was tested in the HUVEC spheroid assay. In parallel, we also
tested the d-Succ-F3 peptide in which the Boc group at the
N-terminus of the molecule was replaced by a succinic acid
moiety in order to achieve a pseudo-symmetrical compound
with the potential ability to interact with its target(s) in two
similar orientations, with a possible improved activity. The
corresponding l-enantiomers l-F3 and l-Succ-F3 (Supple-
mentary Table S1) were used as controls. In keeping with
the results obtained with d-BOC2, both d-F3 and d-Succ-
F3 induced the sprouting of HUVEC spheroids, whereas
their all-l variants were inactive (Fig. 1b). Thus, the pro-
angiogenic activity of these d-FLFLF peptides appears
to be independent of the presence or chemical nature of
their N-terminal moiety, but is strictly dependent on the
1
.0 was assigned to the levels of expression of the gene(s)
measured in one DMSO Matrigel sample that was used as
a reference.
Immunohistochemistry
Human umbilical cord fragments were formalin ꢀxed and
paraꢃn embedded. Serial sections (7 μm) were dewaxed,
hydrated, and processed for immunohistochemistry with
rabbit anti-human FPR3 (N-terminal) and mouse anti-
human CD31 antibodies. After incubation with Envision
HRP-labelled anti-rabbit or anti-mouse polymer, positive
signal was revealed by 3,3′-diaminibenzidine staining. Sec-
tions were counterstained with Mayer’s hematoxylin and
images were acquired with a Zeiss Axioplan 2 microscope
at 20×magniꢀcation.
1
3

Angiogenesis
Fig. 1 l-BOC2 and d-BOC2 derivatives exert a diꢁerent biological
activity in HUVECs. a HUVEC spheroids embedded in 3D ꢀbrin
gel were treated with vehicle or 30 ng/mL VEGF in the absence or in
the presence of the indicated concentrations of l-BOC2 (left panel)
vs vehicle. c HUVEC spheroids were treated with 30 ng/mL VEGF
or the indicated concentrations of d-BOC2, d-F3, or d-Succ-F3.
**p<0.01 vs vehicle. Representative images of HUVEC spheroids
treated with DMSO, 60 μM d-Succ-F3 or 30 ng/mL VEGF are
shown in the right panels. For all the experiments, radially growing
endothelial sprouts were counted after 24 h of incubation and data
(mean± SEM of three independent experiments) were expressed as
fold change vs vehicle
#
or D-BOC2 (right panel). **p<0.01 vs VEGF alone; p<0.01 vs
vehicle. b HUVEC spheroids were treated with vehicle (dashed
line), 30 ng/mL VEGF or the L- or D-enantiomers of BOC2 and
F3 or Succ-F3 peptides, all at 60 μM ꢀnal concentration. **p<0.01
conformation induced by the presence of the d-residues. In
particular, dose–response experiments indicated that d-F3
and d-Succ-F3 were able to induce a response in HUVECs
similar to that exerted by an optimal concentration of VEGF,
whereas the l-variants were ineꢁective (Fig. 1c). On this
basis, d-Succ-F3 was used in all the following experiments
due to its higher biological activity compared to d-BOC2
and its better solubility proꢀle compared to d-F3.
respectively. As shown in Fig. 2a, b, d-Succ-F3 induces cap-
illary-like morphogenesis in HUVECs seeded on Matrigel
and their migration through a gelatin-coated filter in a
Boyden chamber assay. The response was similar to that
exerted by an optimal dose of VEGF or by HUVEC com-
plete medium that were used as positive controls in the two
assays, respectively. Accordingly, d-Succ-F3 induced a sig-
niꢀcant increase in the formation of endothelial cell sprouts
in an ex vivo murine aorta ring assay. When tested under the
same experimental conditions, l-Succ-F3 was ineꢁective,
thus conꢀrming the speciꢀcity of the eꢁect deriving from
the peculiar all-d peptide structure (Fig. 2c). Notably, the
d-peptide was unable to trigger a signiꢀcant proliferative
response or to support cell survival in HUVECs (Supple-
In keeping with the lack of anti-VEGF activity shown
by d-BOC2, d-Succ-F3 did not aꢁect VEGFR2 phospho-
rylation triggered by VEGF in HUVECs and exerted an
additive eꢁect on the sprouting of HUVEC spheroids when
co-administered with the growth factor (Supplementary
Fig. S2A, B). Notably, the capacity of d-Succ-F3 to induce
HUVEC sprouting was not aꢁected by the tyrosine kinase
VEGFR inhibitor SU5408 [36] (Supplementary Fig. S2C).
Accordingly, when compared to VEGF, administration of
d-Succ-F3 alone was unable to trigger VEGFR2 phospho-
rylation in endothelial cells (Supplementary Fig. S2D).
Thus, the pro-angiogenic activity of d-Succ-F3 appears to
be independent of the VEGF/VEGFR2 axis.
mentary Fig. S3).
In keeping with its pro-angiogenic ability, alginate beads
containing 150 ng/pellet of d-Succ-F3 induced a potent
neovascular response when placed on the top of the chick
embryo CAM similar to that exerted by an optimal dose of
VEGF (100 ng/pellet, [37]) (Fig. 2d).
The d‑Succ‑F3 peptide triggers angiogenic
responses in vitro and in vivo
Next, the capacity of d-Succ-F3 to stimulate neovessel
formation was assessed in vivo in the ZFYM angiogene-
sis assay [35]. As shown in Fig. 2E, 12–50 pg/embryo of
d-Succ-F3 injected into the perivitelline space of zebraꢀsh
embryos caused the sprouting of subintestinal vein vessels
in a dose-dependent manner, with a maximal eꢁect at the
In order to conꢀrm the pro-angiogenic potential of d-Succ-
F3, we tested its capacity to exert morphogenic and chemo-
tactic responses in HUVECs seeded on Matrigel or gelatin,
1
3

Angiogenesis
Fig. 2 Pro-angiogenic activity of d-Succ-F3. a HUVEC morphogen-
esis assay. HUVECs were seeded on Matrigel in the presence of vehi-
cle, 60 μM d-Succ-F3 or 30 ng/mL VEGF. Angiogenic meshes was
counted after 6 h of incubation and data (mean± SEM of four inde-
pendent experiments) were expressed as the number of meshes per
VEGF were placed on the top of the CAM at day 11 of incubation.
Newly formed vessels converging toward the implant were counted
after 3 days (mean± SEM of three independent experiments). Rep-
resentative images of CAMs grafted with vehicle or d-Succ-F3 are
shown in the right panels. e ZFYM angiogenesis assay. Two nL of
0.04% DMSO or of the indicated concentrations of d-Succ-F3 were
injected into the perivitelline space of zebraꢀsh embryos at 48 hpf.
The angiogenic response was evaluated at 72 hpf following alkaline
phosphatase (AP) staining. Data are represented as a total number
ꢀ
eld. Representative images of HUVEC morphogenesis on Matrigel
following the diꢁerent treatments are shown in the right panels. b
HUVEC migration assay. Cells were seeded in the upper compart-
ment of a Boyden chamber, whereas vehicle, 60 μM d-Succ-F3, or
HUVEC complete medium were placed in the lower compartment.
Migrated cells were counted after 4 h of incubation and data were
expressed as the number of migrated cells per ꢀeld (mean± SEM of
three independent experiments). c Murine aorta ring assay. Murine
aorta rings were embedded in the 3D-ꢀbrin gel and treated with
l-Succ-F3 or d-Succ-F3. After 5 days, vessel sprouts were counted
under a stereomicroscope and data were expressed as the number of
sprouts per aorta ring (mean± SEM of two independent experiments).
Representative images of aorta rings treated with the two compounds
+
of ectopic AP SIV sprouts/embryo (mean± SEM of 41 embryos
in two independent experiments). Representative images of the for-
mation of ectopic SIVs (arrows) in embryos treated with vehicle or
50 pg d-Succ-F3. Scale bar: 200 µm. f Matrigel plug angiogenesis
assay. Matrigel plugs containing vehicle and 5.0 μg of l-Succ-F3 or
of d-Succ-F3 were injected in the ꢂank of female mice. After 7 days,
quantiꢀcation of the expression of endothelial cell Cd31 and leuko-
cyte Cd45 markers was performed by RT-qPCR in harvested plugs.
Data are mean± SEM of three experiments (n=5–10 mice for each
experiment) and are expressed as Cd31/GAPDH and Cd45/GAPDH
mRNA ratios. *p<0.05 or **p<0.01 vs vehicle
(
60 μM) are shown in the right panels. d Chick embryo CAM assay.
Alginate beads containing vehicle, 150 ng d-Succ-F3, or 100 ng
dose of 50 pg/embryo. Finally, to get further insights about
the d-Succ-F3-driven neovascular response, d-Succ-F3
was embedded in Matrigel and implanted subcutaneously
in mice. RT-qPCR analysis of the plugs harvested 7 days
after implantation demonstrated a signiꢀcant increase of the
expression of the endothelial marker Cd31 and of the pan-
leukocyte marker Cd45 in d-Succ-F3 plugs when compared
to vehicle- or l-Succ-F3-loaded plugs (Fig. 2f).
1
3

Angiogenesis
Together, these data demonstrate the capacity of d-Succ-
by HUVECs [9]. On this basis, RT-PCR analysis was per-
formed to assess the levels of FPR1, FPR2 and FPR3 tran-
scripts in 5 independent HUVEC preparations established
from healthy donors. When tested at the ꢀrst cell passage,
all HUVEC preparations express FPR3, but not FPR1 and
FPR2, whereas monocytic THP-1 cells express all three
FPR genes (Fig. 3a). Similar results were obtained when
HUVECs were analysed up to the fourth cell passage (data
not shown). FACS analysis conꢀrmed the expression of
FPR3 protein on the cell surface of our primary HUVEC cul-
tures, whereas FPR1 and FPR2 were undetectable (Fig. 3b,
c). When HUVEC lysates were analysed by Western blot-
ting, the same anti-FPR3 antibody used for FACS analy-
sis recognized an immunoreactive band that migrated with
the predicted apparent molecular weight, identical to that
F3 to act as a pro-angiogenic compound in vitro and in vivo.
FPR3 mediates the biological activity of d‑Succ‑F3
in HUVECs
The strong structural correlation between l-BOC2 and its
all-d variants led to the hypothesis that the angiogenic activ-
ity shown by d-Succ-F3 might be related to its capacity to
interact with FPRs expressed by HUVECs.
Previous observations had described the expression of
FPR2 in HUVECs, with no information about the expres-
sion of the other members of the FPR family in these cells
[
7, 8]. On the other hand, preliminary data from our labo-
ratory had identiꢀed FPR3 as the only receptor expressed
Fig. 3 HUVECs express N-Formyl Peptide Receptor 3. a HUVECs
were isolated from ꢀve donors and the expression levels of FPR1,
FPR2, and FPR3 transcripts were evaluated by semi-quantitative RT-
PCR in parallel with a human monocytic THP-1 cell extract here used
as positive control. Similar results were obtained when HUVEC cul-
tures were tested at I–IV cell passage (not shown). b HUVEC prepa-
ration from Donor 5 was assessed for the expression of cell surface
FPR1, FPR2, and FPR3 proteins by FACS analysis. c HUVEC prepa-
rations from Donors 1–3 were assessed for the expression of cell sur-
face FPR3 protein by FACS analysis. d Total FPR3 protein levels in
the extracts of HUVEC preparations were evaluated by Western blot
analysis. THP-1 and Jurkat cell extracts were used as positive con-
trols. e Immunohistochemical analysis of human umbilical vein.
Paraꢃn-embedded serial sections of an isolated human umbilical
vein were immunostained with anti-FPR3 or anti-CD31 antibodies.
Endothelial cells express both antigens. No immunoreactivity was
observed in the absence of the primary antibody (not shown). Scale
bar=50 μm
1
3

Angiogenesis
Fig. 4 FPR3 mediates the biological activity of d-Succ-F3 in
HUVECs. a HUVEC sprouting assay. HUVEC spheroids were
treated with vehicle or with 100 ng/mL pertussis toxin (PTX) or
experiments). c HUVEC spheroids were treated with vehicle, 30 μM
d-Succ-F3 or 30 ng/mL VEGF in the absence or in the presence of
an anti-FPR3 antibody (1:100 dilution, vol:vol). Radially growing
endothelial sprouts were counted after 24 h of incubation and data
(mean± SEM of three independent experiments) were expressed as
5
0 μM WRW4 in the absence or in the presence of 30 μM d-Succ-
F3 or 30 ng/mL VEGF. Radially growing endothelial sprouts were
counted after 24 h of incubation and data (mean± SEM of three
independent experiments) were expressed as fold change vs vehi-
cle. b HUVEC migration assay. Untreated and FPR3-desensitized
HUVECs were seeded in the upper compartment of a Boyden
chamber, whereas vehicle, 60 μM d-Succ-F3 or HUVEC complete
medium were placed in the lower compartment. Migrated cells were
counted after 4 h of incubation and data were expressed as the num-
ber of migrated cells per ꢀeld (mean± SEM of three independent
4
fold change vs vehicle. d HUVECs (1.0×10 /100 μL) were incubated
for 30 min at 4 °C with vehicle (blue line), 1.0 mM WRW4 (red line)
or 0.5 mM d-Succ-F3 (green line) in MACS buꢁer. Then, cells were
evaluated for the capacity to bind the anti-FPR3 antibody by FACS
analysis. Omission of the primary antibody represented the negative
#
control (black line). *p<0.05 or **p<0.01 vs vehicle; p<0.01 vs
d-Succ-F3 alone
observed for THP-1 and lymphoblastic Jurkat cell extracts,
here used as positive controls (Fig. 3d). Accordingly, immu-
nohistochemical analysis performed with a distinct anti-
FPR3 antibody, demonstrated that FPR3 is expressed in vivo
eꢁect on VEGF activity, thus supporting the hypothesis that
the activity of d-Succ-F3 is mediated by G protein-coupled
FPRs. Accordingly, the FPR2/FPR3 antagonist WRW4 [38,
39] abrogated the activity of d-Succ-F3 (Fig. 4a). These
data, together with the observation that HUVECs express
FPR3, but not the other members of the FPR family, impli-
cate FPR3 in mediating the pro-angiogenic activity of
d-Succ-F3 in HUVECs by a mechanism that involves the
binding site of the FPR inhibitor WRW4.
+
by CD31 endothelial cells in human umbilical cord blood
specimens (Fig. 3e). Together, these observations unambigu-
ously identify FPR3 as the only detectable member of the
FPR family expressed by HUVECs under our experimental
conditions.
As shown in Fig. 4a, the G protein-coupled receptor
inhibitor pertussis toxin hampers the capacity of d-Succ-
F3 to induce the sprouting of HUVEC spheroids with no
To further substantiate this hypothesis, HUVECs were
pre-incubated for 48 h with WRW4, thus leading to desen-
sitization and downregulation of FPR3 receptors on the
1
3

Angiogenesis
cell surface [4] (Supplementary Fig. S4). As anticipated,
FPR3 desensitization caused a decrease in the capacity of
HUVECs to migrate in response to d-Succ-F3 in a Boyden
chamber assay, with no eꢁect on the migration induced
by the complete HUVEC medium here used as a positive
control (Fig. 4b). Accordingly, the anti-FPR3 antibody
hampered the capacity of d-Succ-F3 to induce the sprout-
ing of HUVEC spheroids with no eꢁect on VEGF activity
[9]. In the present work, we conꢀrmed and extended these
preliminary ꢀndings by showing that FPR3 is expressed in
HUVECs at both transcript and protein levels, as demon-
strated by RT-PCR, FACS and Western blot analyses. In
contrast, FPR1 and FPR2 expression were below the limits
of detection in our cells. These ꢀndings were conꢀrmed on
ꢀve independent HUVEC preparations from distinct donors,
with no signiꢀcant diꢁerences between primary cell isolates
and HUVECs maintained in culture up to the fourth cell
passage. Accordingly, immunohistochemical analysis dem-
onstrated that HUVECs express FPR3 in vivo. Together,
these observations unambiguously identify FPR3 as the only
member of the FPR family expressed by HUVECs under
our experimental conditions. Further experiments will be
required to investigate how cell origin and experimental
conditions may aꢁect the expression of diꢁerent FPRs in
endothelial cells.
(
Fig. 4c). Conversely, similar to WRW4, d-Succ-3 prevented
the binding of the anti-FPR3 antibody to the surface of
HUVECs as assessed by FACS analysis (Fig. 4d).
Together, these observations provide experimental evi-
dence about the role of FPR3 in mediating the pro-angio-
genic activity of d-Succ-F3 in HUVECs.
Discussion
Several experimental evidences support the involvement
of FPR3 in endothelial stimulation by d-Succ-F3. Indeed,
the eꢁects exerted by d-Succ-F3 on HUVECs are fully sup-
pressed by the G protein-coupled receptor inhibitor pertussis
toxin, the FPR2/FPR3 antagonist WRW4 and by an anti-
FPR3 antibody. A similar inhibition was observed following
WRW4-induced FPR3 desensitization in HUVECs. Finally,
d-Succ-F3 prevented the binding of the anti-FPR3 antibody
to the cell surface of HUVECs. Together, these results show
that FPR3 expressed by HUVECs mediates the angiogenic
activity of d-Succ-F3. Clearly, we can not rule out the pos-
sibility that d-Succ-F3 may interact also with other mem-
bers of the FPR family to exert its pro-angiogenic action.
In particular, further studies will be required to identify the
FPR orthologues responsible for the neovascular responses
elicited by d-Succ-F3 in zebraꢀsh and chick embryos, as
well as in adult mice.
The present work stemmed from the observation that the
l-BOC2 peptide, structurally related to the FPR inhibitor
BOC2 [14], is endowed with an angiosuppressive poten-
tial due to its capacity to bind VEGF, as well as a variety
of other heparin-binding pro-angiogenic mediators, setting
the basis for the design of novel multi-target angiogenesis
inhibitors [9].
In this frame, given the higher stability of d-peptides
when compared to their l-enantiomers, we investigated the
possibility that d-BOC2, the d-mirror image of l-BOC2,
might represent an interesting anti-angiogenic compound.
Surprisingly, we found that d-BOC2 was devoid of any
VEGF antagonist activity. On the contrary, d-BOC2 showed
a significant pro-angiogenic potential when tested in a
HUVEC sprouting assay. Similar results were obtained with
the d-peptides d-F3 and d-Succ-F3 that share with d-BOC2
the same d-amino acid sequence. Accordingly, the d-peptide
d-Succ-F3 exerts a pro-angiogenic activity in a variety of
in vitro assays on HUVECs and in ex vivo and in vivo assays
in chick and zebraꢀsh embryos and adult mice.
A few natural FPR3 ligands have been identiꢀed, includ-
ing F2L [5] and humanin [6], that can bind also FPR2
although with diꢁerent potency [5, 6]. Notably, both F2L
and humanin modulate the angiogenic process even though
with opposite eꢁects. F2L, a proteolytic fragment of the
heme-binding protein originally identiꢀed as an endogenous
ligand for FPR3 [5], induces the chemotactic migration of
HUVECs, which is inhibited by the FPR2/FPR3 antagonist
WRW4 [40]. In apparent contradiction with these observa-
tions, F2L inhibits HUVEC proliferation and tube formation
triggered by the human cathelicidin antimicrobial peptide
LL-37/hCAP-18 [40], a pro-angiogenic FPR2 ligand [42]. At
variance with F2L, humanin inhibits angiogenesis by modu-
lating the expression of anti-angiogenic and pro-angiogenic
mediators and preventing pathological renal microvascular
remodelling [43]. However, the direct eꢁect of this FPR ago-
nist on endothelial cells remains unknown.
Given the above-mentioned role of FPRs in angiogenesis
and the capacity of BOC2 and related peptides to exert FPR
agonist/antagonist activities [14–16, 40, 41], we addressed
the possibility that Succ-d-F3 may exert its pro-angio-
genic activity by interacting with FPRs on the endothelial
cell surface. Three FPRs have been identiꢀed in humans
(
FPR1–FPR3), characterized by diꢁerent ligand properties,
biological function and cellular distribution [3]. Among
them, FPR3 has been poorly investigated and its functional
role is uncertain [4].
Even though previous observations had shown the expres-
sion of FPR2 in HUVECs, no data were available about
FPR3 expression in these cells [7, 8]. At variance, prelimi-
nary data from our laboratory had identiꢀed FPR3 as the
only member of the FPR family expressed by HUVECs
The contribution of FPRs to the angiogenic process is
substantiated by the capacity of the FPR2 agonist SAA to
1
3

Angiogenesis
induce pro-angiogenic responses in diꢁerent experimen-
tal models in vitro and in vivo [44–47]. In addition, the
helicobacter pylori-derived peptide Hp(2–20) upregulates
VEGF production, implicating this FPR1/FPR2 ligand in
the vascularization and healing processes of gastric mucosa
heparin-binding angiogenic factors [9], whereas the corre-
sponding d enantiomers induce angiogenesis via FPR3 with
no eꢁect on the VEGF/VEGFR2 axis. This observation sug-
gests that the chirality of these molecules has a tremendous
impact on the exposed chemical groups and surfaces used to
interact with the accepting targets. In addition, we found that
the N-terminally free and succinimidylated peptides have a
potency signiꢀcantly higher than that shown by d-BOC2.
This indicates that the N-terminal charge, opposed in the two
most active peptides, has a limited role in receptor recogni-
tion and that the Boc group might disturb the interaction
with FPR3 by sterically interfering with its recognition.
Thus far, most of the pro-angiogenic peptides are growth
factor mimetics that target their cognate receptors. For
instance, various pro-angiogenic peptides have been char-
acterized as ꢀbroblast growth factor, angiopoietin-1, platelet
growth factor or VEGF mimetics [55–57]. Here, we describe
d-Succ-F3 as a novel ꢀve-amino-acid-long, pro-angiogenic
d-peptide able to induce neovascular responses in various
experimental settings. Its pro-angiogenic potential appears
to be related to its ability to bind and activate FPR3 on the
endothelial cell surface, thus shedding a new light on the
biological function of this chemoattractant receptor.
[
48]. Finally, FPRs have been involved in the angiogenic
responses elicited in vitro and in vivo by the vitreous ꢂuid
isolated from patients aꢁected by proliferative diabetic retin-
opathy [10]. Together, these data point to a role for FPRs
in angiogenesis, even though experimental evidences are
sometimes contradictory and the mechanism of action of
the diꢁerent FPR agonists, including their receptor selectiv-
ity, will require further investigation. Relevant to this point,
d-Succ-F3 is devoid of a signiꢀcant mitogenic activity for
HUVECs. Thus, in keeping with the chemoattractant nature
of FPRs, the pro-angiogenic activity of FPR ligands appears
to depend upon their capacity to trigger a migratory response
in endothelial cells, essential for sprouting angiogenesis
[
49]. Notably, other pro-angiogenic mediators, including
angiopoietin-1 and CCL2, CCL11, and CCL15 chemokines,
are able to induce neovessel formation in diꢁerent in vivo
assays by stimulating endothelial cell migration despite their
limited ability to activate mitogenic responses in these cells
[
50–53].
Insuꢃcient vascularization limits size and complexity of
Acknowledgements This work was supported in part by Associazione
Italiana per la Ricerca sul Cancro (IG AIRC Grant n° 18493) to M.P
and by Regione Campania, Italy [Grant “Fighting Cancer resistance:
Multidisciplinary integrated Platform for a technological Innovative
Approach to Oncotherapies (Campania Oncotherapies)”; Grant “Devel-
opment of novel therapeutic approaches for treatment‐resistant neoplas-
tic diseases (SATIN)”; Grant NANOfotonica per la lotta al CANcro
the engineered tissues [41] and a number of ischemic tissue
disorders would beneꢀt from pro-angiogenic therapies [54].
Further experiments will be required to assess the thera-
peutic potential of FPR3 ligands, including pro-angiogenic
d-Succ-F3 derivatives.
(
NANOCAN)] to M.R.; S.R. was supported by an AIRC fellowship.
Retro-inversion is often used in linear peptides to obtain
topologically similar molecules with improved stability
features deriving from the presence of protease resistant
d-amino acids [22, 23]. In our speciꢀc case, FLFLF is a
palindromic sequence; thus, d-F3 and d-BOC2 peptides are
the d-enantiomers of the corresponding l counterparts and,
at the same time, they represent their “retro-inverso” vari-
ants. Taking advantage of the pseudo-symmetry occurring
in the FLFLF sequence and the possible role played by the
C-terminal carboxyl group in engaging the heparin-binding
region of angiogenic growth factors [9], we generated and
tested the Succ-d-F3 variant where symmetry is further
enhanced by the presence of a second carboxyl moiety on
the N-terminal side of the molecule. The improved symme-
try would provide the peptide with the possibility to access
its target in at least two quasi-equivalent orientations, thus
conferring a potentially increased activity to the molecule.
Comparative data obtained with the various peptides hav-
ing diꢁerent chirality and N-terminal groups suggest that
all-l and all-d variants have orthogonal activities on VEGF-
vs FPR3-mediated angiogenic responses. Indeed, the pep-
tides of the all-l series (l-BOC2, l-F3, Succ-l-F3) suppress
angiogenesis by binding and inhibiting VEGF and other
Author contributions Conceptualization: MIN, MP; Formal analysis
and investigation: MIN, SR, CT, DC, MB, SM, MC, AS, AC; Writ-
ing—original draft preparation: MIN, MR, MP; Writing—review and
editing: MP; Funding acquisition: MP, MR; Supervision: MP.
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