ORIGINAL ARTICLE

L-Arginine Transport and Nitric Oxide Synthesis in Human

Endothelial Progenitor Cells
Francisca Daz-Prez, BSc,* Claudia Radojkovic, PhD,* Valeria Aguilera, BSc,* Carlos Veas, BSc,*
Marcelo Gonzlez, PhD, Liliana Lamperti, PhD,* Carlos Escudero, MD, PhD,
and Claudio Aguayo, PhD*

Abstract: Nitric oxide (NO) is an endogenous vasodilator molecule

synthetized from L-arginine by a family of nitric oxide synthases.
In differentiated human endothelial cells, it is well known that
L-arginine uptake via cationic amino acid transporters (y+/CAT) or
system y+L is required for the NO synthesis via endothelial nitric oxide
synthase, but there are no reports in human endothelial progenitor cell
(hEPC). Therefore, we isolated hEPCs from peripheral blood of healthy
donors and cultured them for either 3 (hEPC-3d) or 14 days (hEPC14d) to characterize the L-arginine transport and NO synthesis in those
cells. L-arginine transport and NO synthesis were analyzed in the
presence or absence of N-ethylmaleimide or L-nitroarginine methyl
ester, as inhibitors of y+/CAT system and nitric oxide synthases,
respectively. The results showed that L-arginine uptake is higher in
hEPC-14d than in hEPC-3d. Kinetic parameters for L-arginine transport showed the existence of at least 2 transporter systems in hEPC:
a high afnity transporter system (Km= 4.8 6 1.1 mM for hEPC-3d and
6.1 6 2.4 mM for hEPC-14d) and a medium afnity transporter system
(Km = 85.1 6 4.0 mM for hEPC-3d and 95.1 6 8 mM for hEPC-14d).
Accordingly, hEPC expressed mRNA and protein for CAT-1 (ie, system y+) and mRNA for 2 subunits of y+L system, yLAT1, and 4F2hc.
Higher L-citruline production and NO bioavailability (4-fold), and
endothelial nitric oxide synthase expression (both mRNA and protein)
were observed in hEPC-14d compared with hEPC-3d. Finally, the high
L-citruline formation observed in hEPC-14d was blocked by N-ethylmaleimide. In conclusion, this study allowed to identity a functional
L-arginine/NO pathway in two hEPC differentiation stages, which
improves the understanding of the physiology of these precursor cells.
Key Words: L-arginine, cationic amino acid transport, endothelial
progenitor cell, nitric oxide
(J Cardiovasc Pharmacol
2012;60:439449)
Received for publication May 10, 2012; accepted July 13, 2012.
From the *Department of Clinical Biochemistry and Immunology, Faculty of
Pharmacy, University of Concepcin, Concepcion, Chile; Department of
Physiology, Vascular Physiology Laboratory, Faculty of Biological Sciences,
University of Concepcin, Concepcin, Chile; and Department of Basic Sciences, Vascular Physiology Laboratory, University of Bo-Bo, Chilln, Chile.
The authors C.E. and C.A. contributed equally to this study.
Supported by Fondo Nacional de Desarrollo Cientco y Tecnolgico (FONDECYT 11070035 and 1100684), INNOVA BOBO 10CHS2671F11,
and Direccin de Investigacin, Universidad de Concepcin (DIUC 205072.031-1.0), Chile.
The authors report no conicts of interest.
Reprints: Dr Claudio Aguayo, PhD, Department of Clinical Biochemistry and
Immunology, Faculty of Pharmacy, Universidad de Concepcin, PO Box
237, Concepcin 4070415, Chile (e-mail: caguayo@udec.cl).
Copyright 2012 by Lippincott Williams & Wilkins

INTRODUCTION
The uptake of L-arginine and other cationic amino
acids occurs through a family of membrane transporters
systems, which include at least 5 distinct groups in human1:
the Na+-independent systems (y+, y+L, b0+, b+) and the
Na+-dependent system including B0+.2 The y+ system is also
recognized as a family of cationic amino acid transporters
(CAT) with 5 members, whose transport activity maintain
the Na+ independence, they are sensitive to trans-stimulation, and its activity is altered by changes in the membrane
potencial.3,4 Likewise, system y+L comprises at least 2 distinct heteromeric amino acid transporters [the glycoprotein
4F2 heavy chain (4F2hc) combined with either amino acid
transporter (AT) carrier y+LAT1 or y+LAT2].3 In primary
cultures of differentiated human endothelium, it has been
demonstrated that L-arginine transport is mediated by
human CAT-1 (hCAT-1) and hCAT-2B,4 and system y+L
and B0+.5
Nitric oxide (NO) is an endothelial-derived vasodilator
molecule produced by the conversion of L-arginine into
L-citrulline via a family of nitric oxide synthases (NOS),
which include the Ca2+-calmodulindependent isoform present in endothelium (eNOS).6,7 Interestingly, in several cell
types, L-arginine transport has been considered to be a ratelimiting factor for NO synthesis.5,8,9 Thus, in human umbilical vein endothelial cells (HUVECs), a mature model of
endothelial cells, the eNOS activity is coupled to the L-arginine transport through hCAT-1 or hCAT-2B5 and the y+L5
system; results that were replicated also in porcine pulmonary artery endothelial cells (PAEC)10 and in endothelial
cells from human placental microvessels11 and human
saphenous veins.12 Therefore, L-arginine uptake via CATs
and y+L is required for NO synthesis in differentiated endothelial cells.
Human endothelial progenitor cells (hEPC) derived
from bone marrow are involved in vascular repair13,14
through the homing into sites of vascular injury, where they
elicit angiogenic, prosurvival, and anti-inammatory effects
and through their differentiation in mature endothelium.
Others and we have previously described that hEPC
undergo differentiation in culture. For example, on the third
day (hEPC-3d), hEPC are positive for CD34, CD133, and
Oct4 markers, which represents an immature phenotype,
whereas at day 14 (hEPC-14d), hEPC lose the expression
of those surface molecules and express membrane markers
of mature endothelium, such as vascular endothelial growth

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factor receptor 2.15,16 Accordingly, hEPC exhibited a differential expression and activity of the equilibrative and concentrative nucleoside transporters15 showing that the cell
differentiation affects the expression and function of membrane-associated transporters. Moreover, it has been
described that NO and eNOS participates in the neovascularization process via mobilization of progenitor cells from
brown marrow stem cell niches to the peripheral circulation.17 Interestingly, eNOS activity has been described in
progenitor endothelial cells,18,19 and eNOS expression is
required for mobilizing EPC from bone marrow toward
the circulation.20 Due to the dependence of L-arginine transport for NO synthesis in differentiate endothelium, we wonder whether this mechanism is present in hEPC. Therefore,
we aimed to characterize the L-arginine transport and NO
synthesis in hEPC during 2 stages of differentiation (3 and
14 days of culture).

METHODS
Isolation and Culture of hEPC
hEPC were isolated as described previously.15,16,21
Briey, fresh human peripheral venous blood was collected
from eight healthy female volunteers (25-30 y), non-smokers,
not receiving any medication and without any clinical diagnosis. This protocol was conducted according to the protocols
dened by the Declaration of Helsinki Ethic Committee, and
informed consent was signed by every volunteer participating
in this study. The total peripheral blood mononuclear cells
fraction was isolated by gradient centrifugation using Ficoll
Histopaque (Sigma Chemical, Sigma Chemical Co, St Louis,
MO), plated (1.5 106 cells/cm2) on bronectin-coated
culture dishes (Sigma), and cultured (378C, 5% CO2) up to
passage 1 in primary culture medium (PCM) composed of
endothelial growth medium (GIBCO BRL Life Technologies,
Bethesda, MD), 5 mM D-glucose, 15% fetal calf serum,
10 mg/mL of human vascular endothelial growth factor
A (VEGF-A) and 100 U/mL penicillinstreptomycin. PCM
was changed every 3 days, and hEPC were selected as cells
attached to bronectin-coated plates. In addition, experiments
were performed as described previously in hEPC cultured for
3 days (hEPC-3d) or 14 days (hEPC-14d)15,16 after cell starving (12 hours) using PCM containing 2% of serum without
growth factors.

Characterization of hEPC Differentiation

Markers by Flow Cytometry
Flow cytometry was performed on an argon laser
FACSCalibur (BD Biosciences, Pharmingen, San Diego,
CA) with an excitation wavelength at 488 nm. Detection of
CD34+/kinase insert domain receptor (KDR)+, CD34-/KDR+,
or CD34+/KDR- cell population was performed in hEPC
incubated (20 minutes, 228C) with monoclonal antibodies
against human phycoerytrin-conjugated CD34 (Becton Dickinson, San Diego, CA) and CarboxyFluorescein-conjugated
KDR (R&D System Inc, Minneapolis, MN) as previously
described.15,16

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Characterization of L-Arginine Transport

L-arginine transport was measured as described.22,23
Briey, hEPC-3d or hEPC-14d were washed with Krebs solution [(mM): NaCl 131, KCl 5.6, NaHCO3 25, NaH2PO4 1,
HEPES 20, CaCl2 2.5, MgCl2 1 (pH 7.4)] 30 minutes before
transport assays. L-arginine transport (1 minute, 378C) was
initiated by the addition of HEPES-buffered Krebs solution
containing unlabelled L-arginine (0.1500 mM) and L-[3H]arginine (4 mCi/mL) and was stopped by the addition of cold
phosphate buffer.
Kinetic parameters of arginine inux were determined
by nonlinear regression analysis as described.15,23 Saturable
L-arginine transport kinetic parameters Vmax and Km were
calculated from data where the nonsaturable linear uptake
of L-arginine was subtracted from transport data and tted
to the MichaelisMenten hyperbola assuming a single saturable transport system.
The relative contribution of culture day on saturable
L-arginine transport kinetic parameters [maximal velocity
(Vmax) and apparent Km] was estimated from the maximal
transport capacity (Vmax/Km) values for L-arginine transport by:
I
hEPC3d=hEPC14d F

hEPC3d Km

hEPC14d Vmax
hEPC3d Vmax hEPC14d Km

where hEPC3dVmax and hEPC3dKm are the kinetics parameters

for L-arginine transport in hEPC-3d, and hEPC14dVmax and
hEPC14dKm are the kinetics parameters of L-arginine transport
in hEPC cultured for 14 days.
In parallel experiments, we determined the dependence
of extracellular Na+ for the L-arginine uptake. For this purpose,
L-arginine transport was measured in a Krebs solution in which
NaCl was replaced by an equimolar concentration of choline
chloride (Na+-free Krebs), as previously described.6,15,22
To assess the implication of the 2 components detected for
L-arginine transport, L-arginine uptake was evaluated using 2
concentrations of unlabelled L-arginine (5.0 mM or 100 mM)
in hEPC-3d or hEPC-14d pre-incubated (10 minUTES) with
Na+-Krebs or Na+-free Krebs. to investigate the participation of
y+/CAT or y+L systems, experiments were developed in presence of N-ethylmaleimide (NEM), an unspecic inhibitor of
system y+/CATs,24,25 and/or L-leucine (100 mM), a neutral
amino acid transported via y+L system in a Na+-dependent
manner. For these experiments, both NEM or L-leucine were
added together with the Na+ or Na+-free Krebs immediately
before L-[3H]-arginine transport assays as described above.

Assessment of eNOS and CAT

Protein Expression
Total eNOS and CAT protein expression was evaluated
by western blot in hEPC-3d and hEPC-14d. Total proteins
(70 mg) were separated by a polyacrylamide gel (10%) electrophoresis and transferred to Immobilon-P polyvinylidene
diuoride membranes (BioRad Laboratories, Hertfordshire,
United Kingdom). Then, membranes were probed with the
following primary antibodies: rabbit anti-hCAT-1 (1:1000)
or rabbit anti-hCAT-2 (1:250), kindly donated by Professor
Luis Sobrevia from Ponticia Universidad Catlica de Chile,
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Santiago, Chile; rabbit anti-eNOS (1:1000) or rabbit anti-b actin

(1:2000) from Santa Cruz Biotechnology, Santa Cruz, CA.
Membranes were rinsed with Tris buffer saline 0.05% Tween
and then incubated (1 hour) with a horseradish peroxidase
conjugated goat anti-rabbit antibody. Proteins were detected
by the enhanced chemiluminescence method, quantied by
densitometry and compared with b-actin expression (control).

Assessment of mRNA Levels for eNOS

and CAT
Total RNA was isolated from hEPC-3d or hEPC-14d
using the Trizol Reagent (Invitrogen, Carlsbad, CA) according
to the manufacturer instructions. One microgram of total RNA
was reverse transcribed into cDNA using Oligo (dT18), random
(10-mer) primers and avian Moloney murine leukaemia virus
reverse transcriptase (Promega, USA) for 1 hour at 378C.
Nonquantitative polymerase chain reaction (PCR) was performed in 20 mL containing 2 mL PCR buffer, 1.5 mM
Mg+2, 100 mM dNTPs, 1.0 U Taq DNA polymerase (Invitrogen), and sequence-specic oligonucleotide primers for
human CAT1 (sense:50 -GAGTTAGATCCAGCAGACCA-30 ,
antisense: 50 -TGTTCACAATTAGCCCAGAG-30 , 148 bp),
CAT-2A (sense: 50 -TATCCCGATTTTTTTGCTGT-30 , antisense: 50 -TGCAGTCAACGTGGCAGCAACT-30 , 690 pb),
CAT-2B (sense: 50 -TCCCAATGCCTCGTGTAAACT-30 ,
antisense 50 -GCACCCGATAAAGTAGCAA-30 , 115 pb),
y+LAT1 (sense: 5`-GTTTGTCTCAGGCAAGGCTC-3`, antisense: 50 -GGAACAAGGAAAGGAGGGAG-3`, 270 bp)
y+LAT2 (sense: 50 -AGACATCTTCCAGCTCATTAACTAC
TACAG-3`, antisense: 50 -CTTTTCAACTTCCTTAGCTCTA
GCCAGTA-3` 481 pb), 4F2-hc (sense: 50 -CTTTCTACTT
CATGGGTGTTTACC-3`, antisense:50 -ATCCTGAGTCTCC
TATAGCTTACCAA-3`, 332 pb), and eNOS (Sense: 50 -CC
AGCTAGCCAAAGTCACCAT-30 , antisense: 50 -GTCTCG
GAGCCATACAGGATT-30 , 350 pb). In addition, mRNA
levels of b-actin (sense: 5`-AACCGCGAGAAGATGACC
CAGATCATGTTT-3`, antisense: 5`-AGCAGCCGTGGC
CATCTCTTGCTCGAAGTC-30 , 350 bp) was used as an
internal reference. Samples were incubated for 4 minutes at
958C, followed by 25 cycles of 30 seconds at 958C, then
30 seconds at 578C (for CATs) or 548C (for 4F2hc, eNOS
and b-actin) or 588C (for LAT1 and LAT2), followed by
30 seconds at 728C, and nally 7 minutes at 728C. reverse
transcriptasepolymerase chain reaction (RT-PCR) products
were sequenced as described.5

Analysis of eNOS Activity by

L-Citrulline Formation
L-[3H]-arginine conversion into L-[3H]-citruline was
used to assess the eNOS activity in hEPC. For this purpose,
hEPC-3d or hEPC-14d were incubated with Na+Krebs solution containing 100 mM L-arginine and L-[3H]arginine
(4 mCi/ml), for 30 minutes at 378C. Then, cells were lysed
and treated with a cation ion-exchange resin Dowex-50W
(50X8-200, Na+ form) to separate L-[3H]-citrulline present
in H2O eluates from L-[3H]-arginine retained in the column.5,23 To ascertain the involvement of eNOS and CAT in
the production of L-[3H]-citrulline, NG-nitro-L-arginine
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L-Arginine Transport in Progenitor Endothelial Cells

methyl ester (L-NAME, 100 mM, an inhibitor of endothelial

nitric oxide synthase22,23) or NEM (100 mM, a nonspecic
inhibitor of cationic amino acid transporters24,25) were added
to cell cultures 30 minutes before experiments.

Assessment of Intracellular NO Formation

Intracellular NO production was determined by
using the uorescent probe 4-amino-5-methylamino-20 ,
70 -diuorouorescein diacetate (DAF-FM-DA, Calbiochem)
as described previously.26,27 Briey, hEPC-3d or hPEC-14d
were seeded in 96-well black plates, incubated during
24 hours under standard conditions and then for 4 hours in
serum-depleted medium. Afterwards, the uorescent probe
DAF-FM-DA (7.5 mM) plus L-arginine (100 mM) were
added to cultures and incubated for 30 minutes. At the end
of this period, uorescence intensity was measured using
a uorimeter (Sinergy 2, Biotec). As a positive control,
1 mM histamine was added for the last 15 minutes of DAFFM-DA incubation. To assess the involvement of eNOS
activity in NO production, cells were pre-incubated with
L-NAME (100 mM) 30 minutes before histamine.

Statistical Analysis

Results were expressed as mean 6 SEM, where n

indicates the number of different experiments (34 replicates). Data were analyzed using 1-way analysis of variance
followed by Bonferroni t test. The statistical software GraphPad Prism 5.00 (GraphPad Software Inc., CA) were used. The
P , 0.05 was considered statistically signicant.

RESULTS
Characterization of hEPC in Cultures

hEPC cultured for 3 or 14 days were analyzed by ow

cytometry to detect cell surface markers associated to cell
differentiation. The fractions of CD34+KDR- and
CD34+KDR+ cells at 3 days in culture were higher (8.5-fold
6 0.4-fold and 7.0-fold 6 0.5-fold, respectively, P , 0.05)
than cells cultured for 14 days; however, a larger fraction of
CD34-KDR+ cells (5.5-fold 6 0.2-fold, P , 0.05) was identied after 14 days compared with 3 days of culture conrming our previous reports.15,16

Characterization of L-Arginine Transport

in hEPC
L-arginine transport was assessed in hEPC-3d and hEPC14d by using L-[3H]-arginine, as described in Methods.
L-arginine uptake was linear determined until 60 seconds for
both hEPC-3d (slope = 0.00039) and hEPC-14d (slope =
0.00128) and adjusted to a double Michaelis-Menten equation
(Figs. 1A, B). EadieHofstee transformation presented a descendent exponential curve phase showing a biphasic curve (Figs. 1C,
D), where at least 2 transport components can be identied:
a medium afnity component and a high afnity component
(named C1 and C2, respectively, for component 1 and 2).
The analysis of kinetic transport curves for medium
afnity (ie, component 1) transport system was saturable in
both hEPC-3d and hEPC-14d with an apparent Km = 85 mmol
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(Fig. 2A) and 95.1 mM (Fig. 2B), respectively. EadieHofstee

analyses were linear in both differentiation stages (r2 = 0.94
for hEPC-3d and 0.97 for hEPC-14d, P , 0.05). Moreover,
Vmax and transport capacity (Vmax/Km) on hEPC-14d were
signicantly higher than hEPC-3d (Table 1).
L-arginine transport curve for high afnity transport
system (ie, component 2) in hEPC-3d and hEPC tted to
a saturable curve with an apparent Km = 4.8 mM (Fig. 2C) and
6.1 mM (Fig. 2D), respectively. As for component 1, Eadie
Hofstee analyses for component 2 was linear in both stages of
differentiation (r2 = 0.95 for hEPC-3d and 0.97 for hEPC14d, P , 0.05). In addition, Vmax and transport capacity

(Vmax/Km) for component 2 on hEPC-14d was about 300-fold

higher than hEPC-3d (Table 1).
To further characterize components 1 and 2, L-arginine
uptake was quantied using 2 different concentrations (100 mM
and 5.0 mM) of this amino acid in hEPC pre-incubated with
NEM (for inhibiting y+/CAT-dependent transport) and/or
L-leucine (for inhibiting y+L system, see Methods). NEM
reduced (80% or 70%) the L-arginine uptake at 100 mM but
did not change the L-arginine transport at 5.0 mM in both
hEPC-3d (Fig. 3A) and hEPC-14d (Fig. 3B), respectively. On
the other hand, when hEPC were pre-incubated with L-leucine,
the L-arginine uptake was inhibited only at 5.0 mM in both

FIGURE 1. L-arginine transport in endothelial progenitor cells (hEPC). [3H]L-arginine transport (4 mCi/mL) was evaluated in the
presence of increasing concentrations of L-arginine (0500 mM) in hEPC-3d (A) and hEPC-14d (B). In C and D, Eadiee Hofstee
transformation of transport data from A and B, respectively. C1 and C2 are referred to component 1 and component 2,
respectively. V/S is L-arginine transport rates. The results are representative of 4 independent experiments each 1 in triplicate.
Values are mean 6 S.E.M.

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L-Arginine Transport in Progenitor Endothelial Cells

FIGURE 2. Kinetic analysis of L-arginine transport for medium and high affinity components. hEPC cultured for 3 days (A, C) or
14 days (B, D) were used for L-arginine transport assays. [3H] L-arginine transport (4 mCi/mL) was evaluated with increasing
L-arginine concentrations: 0500 mM for medium affinity (A, B) and 040 mM for high affinity components (C, D). Results are
representative of 4 independent experiments each 1 in triplicate. Values are mean 6 S.E.M.

hEPC-3d (80%) and hEPC-14d (60%). Furthermore, the

inhibitory effect of NEM and L-leucine were not cumulative
when those inhibitors were co-incubated in any of the experimental conditions. In addition, L-arginine transport at 5 and
100 mM was independent of extracellular Na+; however,
inhibition of 5 mM L-arginine transport by L-leucine was
Na+ dependent in hEPC at both differentiation stages (Figs.
4A, B).

Identification of y+/CAT and y+L Systems

in hEPC
To characterize the presence of the components of y+/
CAT and y+L systems involved in the L-arginine transport in
hEPC at both differentiation stages, we conducted analysis
of RT-PCR and western blot for some isoforms belong to the
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respective family of transporters. Thus for y+/CAT system

results showed that hEPC-3d and hEPC-14d express mRNA
for hCAT-1 and hCAT-2B, but not hCAT-2A; whereas for
y+L system it was found the presence of 4F2hc at both
stages, but only y+LAT1 was detected in hEPC-3d, whereas
y+LAT2 cannot be detected in neither hEPC-3d nor hEPC14d (Fig. 5A). Concerning hCAT-1, hCAT-2B, and 4F2hc
expression, densitometry analysis did not show statistical
difference when hEPC-3d and hEPC-14d were compared
(Fig. 5B).
In addition, western-blot analysis showed that protein
abundance of hCAT-1 was similar in hEPC-3d and hEPC-14d
(Fig. 5C), but it was signicantly lower than HUVECs
(Fig. 5C, D). On the other hand, it was not feasible to detect
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TABLE 1. Kinetic Parameters for L-Arginine Transport in hEPC

hEPC-3d
y+ (component 1)
y+L (component 2)
hEPC-14d
y+ (component 1)
y+L (component 2)

Km
(mM)

Vmax
[pmol (mg Protein)-1min-1]

Transport Capacity
(Vmax/Km)
[pmol (mg Protein)-1min)-1M)]

I/hEPC3d/hEPC14dF

85.1 6 4.0
4.8 6 1.1

0.12 6 0.001
0.004 6 0.0003

0.0014 6 0.005
0.0008 6 0.0002

95.1 6 8
6.1 6 2.4

13.5 6 1.1*
1.6 6 0.17*

0.14 6 0.04*
0.26 6 0.07*

100 6 20*
300 6 63*

L-arginine saturable transport was determined as described in Methods. Values are means 6 Standard Error (n = 12).
*P , 0.05 v/s valores correspondientes en clulas hEPC-3d. I/hEPC3d/hEPC14dF is the relative contribution of differentiation to changes in maximal transport capacity (Vmax/Km) of
L-arginine transport (see Methods).

hCAT-2 protein abundance in hEPC, but it was present in the

positive control (HUVEC, Fig. 5C).

Characterization of Functional Expression of

eNOS in hEPC
We characterize the presence of eNOS (mRNA and
protein) in both hEPC-3d and hEPC-14d. Thus, in hEPC-14d,

it was detected a higher mRNA for eNOS (Fig. 6A) and

eNOS protein abundance (Fig. 6B) than hEPC-3d, but
they were signicantly reduced compared with HUVEC
(ie, positive control).
Basal L-citruline formation, an indirect determination
of NO synthesis, was higher in hEPC-14d compared with
hEPC-3d. In addition, L-NAME (ie, NOS inhibitor) produced

FIGURE
3. Characterization
of
L-arginine transport mediated by
system y+ in hEPC. hEPC-3d (A)
and hEPC-14d (B) were used for
L-arginine transport assays. hEPC
were preincubated (+) with NEM
(100 mM, 30 minutes) alone or with
L-leucine (1 mM, 10 minutes). Then
L-arginine transport assays were
performed at 5 mM () or 100 mM
(n) (4 mCi/mL) as described in
Methods. Values correspond to
mean 6 S.E.M (n = 3), *P , 0.05 in
hEPC-14d v/s without NEM and
L-leucine. **P , 0.04 in hEPC-3d v/s
without NEM and L-leucine.

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L-Arginine Transport in Progenitor Endothelial Cells

FIGURE
4. Characterization
of
L-arginine transport assays mediated
by system y+L in hEPC. hEPC were
cultured for 3 days [hEPC-3d, (A)] or
for 14 days [hEPC-14d, (B)]. For
L-arginine transport assays, hEPC
were exposed to Krebs solution with
sodium (+), or to a Krebs solution
where Na+ was replaced by
N-methylglucamine-HCl (-). In parallel experiments, transport assay
was also performed in cells
preincubated in Krebs (30 minutes)
with (+) or without (-) L-Leucine
(1 mM). Then L-arginine transport
assays were performed at 5 mM ()
or 100 mM (n) (4 mCi/mL)
as described in Methods. Values
represents mean 6 S.E.M (n = 3),
*P , 0.05 v/s values with Na+ and
without L-leucine in hEPC-3d and
hEP-14d.

a signicant decrease in L-citrulline formation only in hEPC14d (Fig. 7A). In parallel experiments, NO formation
assessed by the probe DAF-FM-DA conrms that only
hEPC-14d was able to synthesize NO in response to histamine (Fig. 7B), an effect also reverted by L-NAME.

Association of y+/CAT-Mediated L-Arginine

Transport With NOS Activity
We further investigate whether L-citrulline formation in
hEPC-14d require L-arginine uptake via y+/CAT. We found that
in presence of NEM (ie, y+/CAT inhibitor), L-citruline formation
was signicantly reduced (about 65%) compared with control
without this inhibitor (0,0028 6 0,0005 vs. 0,0078 6 0,0007
dpm/(mg protein)/30 minutes, respectively, P , 0.05), reaching
similar levels observed after incubation with the NOS inhibitor,
L-NAME [0.0030 6 0.0004 dpm/(mg protein)/30 minutes].

DISCUSSION
This study demonstrated that hEPC are able to transport
L-arginine and synthetize NO. Accordingly, at least 2
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transport systems are involved in L-arginine uptake, which

would correspond to y+ (ie, hCAT-1/hCAT2B) and y+L
(ie, y+LAT1) systems. Both transport systems exhibit higher
activity in hEPC-14d compared with hEPC-3d. Similarly,
eNOS activity and NO bioavailability were signicantly higher in hEPC-14d compared with hEPC-3d. Furthermore,
L-citruline formation was signicantly reduced by NEM in
hEPC-14d, suggesting that NO synthesis in hEPC may
require L-arginine incorporation thought y+L/CAT system.
Although, the functional coupling between L-arginine transport and NO synthesis have been reported in several cell types
including human monocytes/macrophages,28 erythrocytes29,30
an mature endothelial cells,5,11 our results represent the rst
evidence suggesting this relationship in hEPC.
Kinetic analysis showed that hEPC-3d and hEPC-14d
transport L-arginine via at least 2 transport systems: a medium
afnity (Km = 80145 mM; named component 1) and a high
afnity component (Km = 310 mM; named component 2).
Accordingly, one system could be y+ (high-capacity transporter and medium afnity) and the other could be y+L
(high-afnity transport) (Table 1). Further characterization
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FIGURE 5. Expression of the members of the amino acid transporter

systems y+/CAT and y+L in hEPC.
Analysis of members of the system
y+/CAT and y+L were assessed by
RT-PCR and western blot. A, mRNA
levels of hCAT-1, hCAT-2A, hCAT2B, y+L1, y+L2, 4F2hc were determined by RT-PCR in hEPC cultured
for 3 (hEPC-3d) or 14 days (hEPC14d). HUVEC were used as positive
control. B, Relative mRNA expression
for y+/CAT transporters compared to
b-actin. C, Representative western
blot for hCAT-1, hCAT-2 and b-actin
(internal reference) in hEPC-3d and
hEPC-14d. D, Densitometry analysis
for CAT-1 () and CAT-2 (n) protein
abundance, compared with b-actin.
Values are mean 6 S.E.M (n = 4).
*P , 0.05 v/s values for CAT-1 in
HUVEC.

of these systems (or components) allowed us to identify the

activity of hCAT-1 and y+LAT (see below). Both systems have
been described in several primary cultures22,31,32 and cell
lines33,34 of mature endothelium, but there are no studies showing the expression and activity of these transporters in hEPC.
Nevertheless, CAT-1 expression and activity has been identied in the cell line K562, a progenitor cell model for erythrocytes.28,35 Reported kinetic parameters for L-arginine transport
in those cells were Km = 38 mM and Vmax = 815 [pmol (mg
protein)/(min)]. Interestingly, those reports indicated that about

80% of L-arginine transport was associated with hCAT-1,

whereas the residual 20% correspond to another transport system not yet identied. Taken into account other report30 in
human erythroid cells, it is feasible that the residual component
could be a member of the system y+L, such as y+LAT2. Moreover, similar proportion in the y+ and y+Lmediated L-arginine
transport have been described in differentiated human endothelial cells22,31,34 or in porcine aorta endothelial cells.36,37
To identify which members of the y+ and y+L systems
are involved in L-arginine uptake in hEPC, we conducted

FIGURE 6. Expression of eNOS in

hEPC. Presence of eNOS was analysis
by RT-PCR and western blot. A) Upper
panel: representative image of semiquantitative RT-PCR for eNOS and
b-actin (housekeeper gene). Lower
panel: Relative mRNA levels of transporter detected as in upper panel. B)
Upper panel: representative image of
western blot for eNOS and b-actin
(internal reference). Lower panel:
densitometric ratios for eNOS/b-actin
protein abundance. mRNA and protein extracts from HUVEC were used
as positive controls. Values mean 6
S.E.M (n = 3), *P , 0.05 v/s hEPC-3d.

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FIGURE 7. L-citrulline formation and NO bioavailability in

hEPC. NO synthesis was estimated in hEPC cultured during 3
(hEPC-3d) and 14 days (hEPC-3d). Cells were incubated in
absence (ie, control ) or presence of L-nitroarginine methyl
ester (L-NAME, 100 mM) (n) for 30 minutes. A, L-citrulline
formation assay; or B, NO detection with the 4,5-diaminofluorescein diacetate (DAF-2DA, 1 mM, 30 minutes) probe
were performed as described in Methods. Values mean 6
S.E.M (n = 3), *P , 0.05 v/s hEPC-3d, **P , 0.04 in hEPC-14d
v/s control.

experiments using 2 different concentrations (100 and 5 mM)

of this amino acid. Regarding the system y+, the identication
of hCAT-1 were performed at 100 mM of L-arginine in presence or absence of NEM (ie, an inhibitor of y+ system).5,24,38
Our results showed a reduced L-arginine uptake in the presence of this CAT-1 inhibitor in both hEPC-3d and hEPC-14d.
In addition, RT-PCR and western blot analysis showed the
expression of hCAT-1 in both hEPC-3d and hEPC-14d conrming the presence of this transporter in those stages of
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L-Arginine Transport in Progenitor Endothelial Cells

differentiation. Despite functional experiments showed a higher activity of component 1 (ie, hCAT1) in hEPC-14d than
hEPC-3d (about 100-fold), the mRNA and protein levels
for hCAT-1 were similar in hEPC cultured for 3 and 14 days.
This apparent controversy could be associated with the transporter availability in the cell membrane (ie, localization of functional transporter in the cells). Information regarding hCAT-1
translocation is limited; however, previous studies38 demonstrated that external stimuli-like insulin induces a higher expression of hCAT-1 in the plasmatic membrane, which was
associated to a high L-arginine transport in HUVECs (ie, differentiated endothelium). Nevertheless, further studies should be
carried out to prove this issue in hEPC.
On the other hand, the characterization of the high
afnity system was carried out with transport experiments
performed at 5 mM of L-arginine in the presence of L-lysine
but without sodium. As reported in undifferentiated cells29,30
and in differentiated cells such as human peripheral blood
mononuclear cells39,40 or HUVEC,5 our study suggests functional presence of y+L system in both hEPC-3d and hEPC14d. Remarkably, transport activity associated to this system
was about 300 times higher in hEPC-14d compared with
3 days cultured cells. In addition, mRNA levels of 4F2hc
were the highest expressed in both stages of differentiation.
In contrast, we could not detect the mRNA for y+L1 or y+L2
in hEPC-14d. In fact, cells on the 14th day of culture lose the
expression (ie, mRNA levels) of y+L1 that was observed on
the third day of culture. Unfortunately, there are not specic
antibodies for y+L1 or y+L2 commercially available, and then
we do not know whether changes observed in mRNA levels
are correlated with protein translation. However, because
transport activity related with this high afnity system is
increased in hEPC-14d, we can speculate that either translation or protein migration toward the membrane of the y+L
member(s) might be increased in those cells.
Elevated levels of L-arginine are necessary for cell
growth and cell differentiation.29 In this regard, other reports
have shown an increase in the y+L system activity and
a decrease in the y+ system activity, as a phenomena associated with the differentiation of monocytes into macrophages28
or with the proliferation of primary culture of monocytes
isolated from mouse bone marrow.41 Therefore, it is expected
that during cell maturation of progenitor cells, L-arginine
transporters activity changes, probably according to the phenotype of the differentiated mature cell. Among other possibilities, it is feasible that high L-arginine transport in hEPC14 may correspond to the capacity of NO synthesis.
Regarding this issue, we found that eNOS activity and
NO bioavailability were higher in hEPC-14d than in hEPC3d. These results agree with previous reports20,21 where the
eNOS activity has been described in progenitor endothelial
cells. Functional signicance of eNOS expression in hEPC is
under investigation. However, eNOS-/- mice exposed to
VEGF showed no mobilization of EPC from bone marrow
toward the blood circulation,20 suggesting that eNOS is
required by EPC-mediated regeneration.
In addition, higher L-arginine transport, NO and eNOS
activity in hEPC-14d than hEPC-3d, might suggest that
L-arginine inux in involved in the generation of NO in hEPC.
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Daz-Prez et al

In this regard, several studies,8,42 including our groups

reports,38 have shown that NO synthesis depends on the
L-arginine transporters activity in mature endothelium. This
association has been reinforced by the co-localization of
hCAT-1 and eNOS in the caveolae of mature endothelial
cells.43,44 Our results showed that in hEPC-14d, L-citruline
formation was reduced in the presence of NEM at the
same level that the reduction observed after incubation
with L-NAME, suggesting that L-arginine transport though
y+/CAT system is coupled to NO synthesis by eNOS. In
addition, our results also suggest that a member of y+ system,
probably hCAT-1, would be required by eNOS activity. However, more studies are necessary for conrming the coupling
between hCAT-1 and eNOS in hEPC.
The functional implication of the variation in the
L-arginine/NO pathway in hEPC according with the stages
of differentiation has not been described. However, it is
known that hEPC at early stages (ie, 3 days) fails in the
formation of tube-like structures; whereas hEPC at late stages
(ie, 14 days) have not problem at all to form this structures on
Matrigel assays.18 On the other hand, transplantation of EPCs
overexpressing eNOS has been linked with inhibition of neointimal hyperplasia and recuperation of vascular function after
balloon injury in rats.45 These results, associated with lack of
mobilization of EPC from bone marrow toward the blood
circulation in response to VEGF observed in eNOS-/- mice43
reinforce the importance NO synthesis by hEPC in their
capacity for recovering vascular function after injury.
In conclusion, our results showed that hEPC incorporate L-arginine, which associates to the enzymatic synthesis
of NO by eNOS. Analysis of the kinetic parameters of
L-arginine transport demonstrated that hEPC express 2
systems of cationic amino acid transporters corresponding
to y+ and y+L. The L-arginine/NO pathway is highly activated
in hEPC-14d compared with hEPC-3d, suggesting that the
differentiation determine the expression and/or activity of
transport systems. These changes on the transport capacity
and NO synthesis could correspond to variations in functional
properties of hEPC, such as angiogenic capacity and mobilization. These results improve the functional characterization
of hEPC and contribute to the understanding of physiological
processes involving NO.

ACKNOWLEDGMENTS
The authors thank Dr Luis Sobrevia from Ponticia
Universidad Catlica de Chile, for donating cationic amino
acid transport antibodies.
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