Role of Nitric Oxide Signaling in Endothelial Differentiation of Embryonic Stem Cells

Article (PDF Available)inStem cells and development 19(10):1617-26 · October 2010with29 Reads
DOI: 10.1089/scd.2009.0417 · Source: PubMed
Abstract
Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guanylyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly, we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO synthase (NOS) inhibitor NG-nitroarginine methyl ester (L-NAME) for up to 11 days were not significantly different from vehicle-treated cells in EC markers. However, by 14 days, L-NAME-treated cells manifested modest reduction in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of L-NAME exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects of L-NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC, cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by L-NAME, confirming endogenous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic function.
1617
STEM CELLS AND DEVELOPMENT
Volume 19, Number 10, 2010
© Mary Ann Liebert, Inc.
DOI: 10.1089/scd.2009.0417
Signaling pathways that govern embryonic stem cell (ESCs) differentiation are not well characterized. Nitric
oxide (NO) is a potent vasodilator that modulates other signaling pathways in part by activating soluble guany-
lyl cyclase (sGC) to produce cyclic guanosine monophosphate (cGMP). Because of its importance in endothelial
cell (EC) growth in the adult, we hypothesized that NO may play a critical role in EC development. Accordingly,
we assessed the role of NO in ESC differentiation into ECs. Murine ESCs differentiated in the presence of NO syn-
thase (NOS) inhibitor NG-nitroarginine methyl ester ( -NAME) for up to 11 days were not signi cantly different
from vehicle-treated cells in EC markers. However, by 14 days, -NAME-treated cells manifested modest reduc-
tion in EC markers CD144, FLK1, and endothelial NOS. ESC-derived ECs generated in the presence of -NAME
exhibited reduced tube-like formation in Matrigel. To understand the discrepancy between early and late effects
of -NAME, we assessed the NOS machinery and observed low mRNA expression of NOS and sGC subunits
in ESCs, compared to differentiating cells after 14 days. In response to NO donors or activation of NOS or sGC,
cellular cGMP levels were undetectable in undifferentiated ESCs, at low levels on day 7, and robustly increased
in day 14 cells. Production of cGMP upon NOS activation at day 14 was inhibited by -NAME, con rming endog-
enous NO dependence. Our data suggest that NOS elements are present in ESCs but inactive until later stages of
differentiation, during which period NOS inhibition reduces expression of EC markers and impairs angiogenic
function.
Introduction
P
    as embryonic stem cells
(ESCs) have therapeutic potential for regenerative medi-
cine because they have unlimited self-renewal capacity and
can give rise to lineages comprising all 3 germ layers [ 1 , 2 ]. As
a result, ESCs are regarded as a potential unlimited source
of therapeutic cells to replace diseased tissues. With respect
to cardiovascular diseases, ESCs have been successfully dif-
ferentiated into cardiovascular lineages such as endothelial
cells (ECs), cardiomyocytes, and vascular smooth muscle
cells, and have been shown to have functional bene t in pre-
clinical models of myocardial infarction and peripheral ar-
terial disease [ 36 ].
However, the signaling pathways that regulate ESC differ-
entiation are not well characterized. Among the various im-
portant signaling molecules is nitric oxide (NO), a short-lived
free radical that is produced by nitric oxide synthase (NOS)
upon oxidation of -arginine [ 7 ]. The biological effects of NO
are in part mediated by its activation of soluble guanylate
cyclase (sGC) to produce cyclic guanosine monophosphate
(cGMP). Subsequently, cGMP modulates the activity of
cGMP-dependent protein kinases, phosphodiesterases, and
ion channels [ 8 ]. In this way, NO regulates diverse cellular
processes in the cardiovascular, central nervous system, and
immune system of the adult mammal. Several reports also
suggest a role for NO signaling in cardiac differentiation
[ 912 ]. Furthermore, defects in bicuspid aortic valves and
atrial and ventricular septa are common in endothelial NOS
(eNOS)-de cient neonates [ 13 , 14 ]. However, little is known
regarding the role of NO in ESC differentiation to ECs. In the
adult mammal, we and others have shown that NO is a po-
tent mitogen and survival factor for ECs, as well as an angio-
genic agent [ 1517 ]. Because of the important role NO plays
in maintaining EC growth in adults, we hypothesized that it
may also play a critical role in endothelial development.
Here, we report the differential expression of NO-
signaling components during endothelial differentiation and
the effect of NOS inhibition on endothelial differentiation.
Role of Nitric Oxide Signaling in Endothelial Differentiation
of Embryonic Stem Cells
Ngan F. Huang , Felix Fleissner , John Sun , and John P. Cooke
D i v i s i o n o f C a r d i o v a s c u l a r M e d i c i n e , S t a n f o r d U n i v e r s i t y , S t a n f o r d , C a l i f o r n i a .
HUANG ET AL. 1618
Immuno uorescence staining
Samples were  xed in 4% paraformaldehyde and immu-
no uorescently stained, as described previously [ 21 ], for the
antibodies sGCα
3
(Sigma, St. Louis, MO), sGCβ
3
(Sigma),
eNOS, or CD144 (BD Biosciences, San Diego, CA). Total
nuclei were stained by Hoechst 33342 (Invitrogen, Carlsbad,
CA) dye before imaging on a  uorescent microscope (Nikon,
Burlingame, CA), and images were captured with a SPOT
RT color camera (Diagnostic Instruments, Sterling Heights,
MI).
cGMP activity
To investigate the contribution of endogenous or exog-
enous NO release at various time points of differentiation,
cells were incubated for 30 min with Dulbecco’s modi-
ed Eagle’s medium (DMEM) containing calcium iono-
phore A23187 (2 × 10
5
M; Sigma), BAY-41-2272 (BAY41,
10
5
M; EMD Chemicals, Gibbstown, NJ), (±)- S -Nitroso- N -
acetylpenicillamine (SNAP, 10
4
M, EMD Chemicals), or
DMSO vehicle control. In addition, to assess the effect of
-NAME treatment on cGMP activity, after 14 days of dif-
ferentiation in the presence or absence of -NAME (10
5
M),
cells were incubated with DMEM containing one of the fol-
lowing treatments: (1) A23187 (2 × 10
5
M), (2) BAY41 (10
5
M), (3) SNAP (10
4
M), (4) BAY41+SNAP, (5) BAY41+A23187,
or (6) DMSO vehicle control (Basal). All media were further
supplemented with the cGMP phosphodiesterase inhibitor
isobutylmethylxanthine (IBMX, 10
3
M; Sigma). Af ter 30 min,
the cells were lysed in 0.1 N HCl and assayed for cGMP ac-
tivity as described by the manufacturer (Cayman Chemical,
Ann Arbor, MI). For each sample, cGMP activity was nor-
malized to the abundance of total protein, as assessed by
BCA protein quanti cation assay ( n = 3).
Fluorescently activated cell sorting (FACS)
and Matrigel tube-like formation
After 3 weeks of differentiation in -NAME (10
4
M or 10
5
M), ESC-derived endothelial cells (ESC-ECs) were puri ed
by FACS. Cells were dissociated using dissociation buffer
(Invitrogen, Carlsbad, CA), blocked in 10% normal mouse
serum, and then incubated with anti-CD144 antibody (BD
Biosciences, San Diego, CA), followed by allophycocyanin
(APC)-conjugated secondary antibody. As a negative con-
trol, immunoglobulin G isotype was used in replacement
of primary antibody. The cells were puri ed using FACS
Vantage (BD Biosciences). To perform the Matrigel tube-like
formation assay, puri ed ESC-ECs derived from -NAME
treatment were seeded on growth factor-reduced Matrigel at
a density of 1 × 10
5
cells/well in differentiation media with-
out -NAME. After 24 h, the cells were  xed in paraformal-
dehyde (4%). Phase-contrast images were taken under 10×
objectives, and the number of branch points was quanti ed
by NIH ImageJ software version 1.41o ( n = 4) [ 22 ].
Statistical analysis
All data are expressed as mean ± standard deviation.
Statistical analysis was performed by the Student’s t -test
for comparison of 2 groups or one-way analysis of variance
(ANOVA) with Holm’s adjustment for multiple comparisons.
Materials and Methods
ESC culture and differentiation
Murine ESCs (D3; ATCC, Manassas, VA) were routinely
cultured on mitotically inactivated mouse embryonic  bro-
blasts (MEF) in growth media containing leukemia inhib-
itory factor as described [ 18 ]. For differentiation studies,
ESCs were dissociated by trypsin and then pre-plated onto
gelatin-coated dishes for 60 min in growth media at 37°C
to deplete residual MEFs. Next, 10
6
ESCs were transferred
to ultra-low nonadhesive dishes to initiate embryoid body
(EB) formation in an endothelial differentiation media
that consisted of α-minimum Eagle’s media, 10% fetal bo-
vine serum (FBS), 1% penicillin/streptomycin, and 0.05 mM
β-mercaptoethanol (Sigma, St. Louis, MO) [ 12 ]. After 4 days
of suspension culture, the EBs were reattached onto 0.2%
gelatin-coated dishes and cultured in differentiation media.
Where speci ed, cells were treated with differentiation
media supplemented by NOS inhibitor NG-nitroarginine
methyl ester ( -NAME, 10
5
M or 10
4
M; Sigma) or dimethyl
sulfoxide (DMSO) as vehicle control. The media was changed
daily, and at indicated time points, samples were assayed for
gene expression, protein expression, Matrigel tube-like for-
mation assay, or cGMP activity.
Quantitative polymerase chain reaction
Samples were lysed in Trizol (Invitrogen, Carlsbad, CA),
and total RNA was isolated using the RNEasy Kit (Qiagen,
Valencia, CA). Using the Superscript II reverse transcriptase
(Invitrogen),  rst strand cDNA was synthesized accord-
ing to the manufacturer’s instruction. Primers for Taqman
qPCR assays consisted of FLK1, CD144 (VE-cadherin), von
Willebrand factor (VWF), eNOS, soluble guanylyl cyclase
(sGC) subunits α
2
, sGC
α
3
(formerly named sGC
α
1
), sGC
β
2
,
sGCβ
3
(formerly named sGC
β
1
), dimethylarginine dimeth-
ylaminohydrolase (DDAH) 1, and DDAH2 (all from Applied
Biosystems, Foster City, CA). The 18S primers were obtained
from Shetzline et al. [ 19 ]. qPCRs were performed on a 7300
Real-Time PCR system (Applied Biosystems) for 40 cycles.
The data were assessed by the ∆∆Ct method [ 20 ], normal-
ized to 18S housekeeping gene, and then expressed as rela-
tive fold changes ( n = 3).
Immunoblotting
At indicated time points, samples were lysed in RIPA
buffer (Pierce, Rockford, IL) containing protease and phos-
phatase inhibitors and quanti ed for total protein quantity by
the bicinchoninic (acid) BCA protein assay (Pierce). Equally
loaded protein samples were resolved by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
then transferred onto nitrocellulose membranes (Invitrogen,
Carlsbad, CA). The membranes were blocked in 5% bovine
serum albumin (BSA) before incubating with the following
antibodies: FLK1 (Cell Signaling Technology, Danvers, MA),
eNOS (BD Biosciences, San Diego, CA), or α-tubulin (Cell
Signaling Technology). Horse radish peroxidase (HRP)-
conjugated secondary antibodies were applied, and the pro-
teins were visualized by an ECL Detection Kit (Amersham,
Piscataway, NJ). Quanti cation of protein quantities were
carried out by normalizing to α-tubulin and are expressed
in relative fold change ( n = 3).
NITRIC OXIDE SIGNALING IN EMBRYONIC STEM CELLS
1619
or 10
4
M) for 14 days ( P < 0.01) ( Fig. 1C ). These observations
at the mRNA level were further substantiated by immunob-
lots after 14 days of differentiation, demonstrating a signif-
icant inhibition of eNOS and FLK1 at the protein level with
 -NAME (10
4
M; Fig. 1D–1F ).
Effect of L -NAME on Matrigel tube-like formation
by ESC-ECs
To further explore the inhibitory effects of -NAME on
endothelial differentiation, we questioned whether chronic
treatment of -NAME could impair endothelial function in
the ESC-ECs. After 3 weeks of culture in the presence of
 -NAME (10
5
M or 10
4
M) or DMSO vehicle control, the
ESC-ECs were puri ed by the positive expression of CD144.
As shown in Figure 2B , the percentage of CD144
+
cells in
the group treated with -NAME (10
5
M) was ~20%, and
not signi cantly different from the percentage of cells in
other treatment groups (data not shown). After puri cation
based on positive expression of CD144, the cells were further
Repeated measurements of samples over time were ana-
lyzed by repeated measures ANOVA with Holms adjust-
ment. Statistical signi cance was accepted at P < 0.05.
Results
Effect of L -NAME on endothelial differentiation
of ESCs
To investigate the role of -NAME on endothelial dif-
ferentiation of ESCs, we treated differentiating ESCs with
-NAME for 14 days and carried out qPCR of endothelial
genes. After 14 days of differentiation in the presence of
-NAME, the mature endothelial marker CD144 was signif-
icantly inhibited by 34% in the presence of -NAME (10
5
M), when compared to the control group ( P < 0.01) ( Fig. 1A ).
 -NAME (10
5
M or 10
4
M) treatment also signi cantly
down-regulated eNOS expression by 50% or 52%, respec-
tively ( P < 0.01) ( Fig. 1B ). Similarly, the endothelial progen-
itor marker, FLK1, was signi cantly down-regulated by 34%
or 33%, respectively, after treatment with -NAME (10
5
M
1. 6
A
B
C
D
E
F
CD144
eNOS
FLK1
eNOS
α-tubulin
α-tubulin
FLK1
**
Control
LN 10
–5
M
LN 10
–4
M
Control
LN 10
–5
M
LN 10
–4
M
Con LN 10
–5
M LN 10
–4
M
Con LN 10
–5
M LN 10
–4
M
Control
**
**
LN 10
–5
M
LN 10
–4
M
Con
LN 10
–5
M
LN 10
–4
M
1. 2
0.8
0.4
0
ESC 4 7 11 14
Treatment Time (days)
ESC 4 7 11 14
Treatment Time (days)
ESC 4 7 11 14
Treatment Time (days)
Normalized Fold Change
1. 6
1. 2
0.8
0.4
0
1. 2
eNOS
*
FLK1
*
**
**
0.8
1
0.4
0.6
0
0.2
Normalized Fold Change
Normalized Fold Change
1. 2
0.8
1
0.4
0.6
0
0.2
Normalized Fold Change
1. 6
1. 2
0.8
0.4
0
Normalized Fold Change
F I G . 1 . Effect of NG-nitroarginine methyl ester ( -NAME) on endothelial differentiation of embryonic stem cells (ESCs). ( A )
Gene expression time course of endothelial differentiation in the absence (Con) or presence of -NAME (LN, 10
5
M or 10
4
M) for CD144, ( B ) endothelial nitric oxide synthase (eNOS), and ( C ) FLK1. Data normalized to 18S and expressed as relative
fold change. ( D ) Immunoblots for endothelial markers 14 days after differentiation in the presence of -NAME. Quanti cation
of immunoblots for ( E ) eNOS and ( F ) FLK1 ( n = 3). Statistically signi cant comparisons (* P < 0.05; ** P < 0.01).
HUANG ET AL. 1620
with sGCα
3
and sGCβ
3
at day 14 of differentiation. However,
sGCα
3
and sGCβ
3
were also expressed in other cell types be-
sides ESC-ECs, which is consistent with literature indicating
that sGCs are expressed in numerous mammalian tissues
[ 23 ]. These results indicated that NO-signaling components
are present in ESCs, but are up-regulated during the course
of ESC differentiation. The up-regulation of NO-signaling
components with differentiation time is consistent with the
observed effect of -NAME on EC markers at later stages of
differentiation.
Production of cGMP activity during
endothelial differentiation
To determine whether the differential gene expression
of NO-signaling components re ected the ability of the
cells to produce cGMP in response to NO stimulation, we
stimulated ESCs or differentiating cells (7 or 14 days) with
A23187, BAY41, SNAP, or DMSO vehicle control. The calcium
ionophore A23187 increases intracellular calcium levels to
increase NOS activity, and thereby increases endogenous
NO activation of sGC. SNAP is an exogenous NO donor and
mimics endogenous NO. In contrast, BAY41 activates sGC in
an NO-independent manner and can potentiate the effect of
NO to increase cGMP levels [ 8 ]. As shown in Figure 6A6D ,
ESCs did not produce detectable cGMP after the addition of
any of these agents. Basal levels of cGMP were detectable
by 7 days, and by day 14 had increased to 4.4 ± 0.1 pmol/
mg protein ( Fig. 6A ). A response to A23187 was also ob-
served at day 7 and was greater at day 14 (8.7 ± 1.3 pmol/
mg protein ( P < 0.05, Fig. 6B ). Similarly, SNAP and BAY41
were potent stimulators of sGC, with a greater response ob-
served after 14 days of differentiation (362.8 ± 19.31 pmol/
mg and 205.0 ± 15.5 pmol/mg, respectively, P < 0.05, Fig. 6C
and 6D ). Together, these data indicate that even though com-
ponents of the NOS pathway are detectable, ESCs are not
capable of generating cGMP via this pathway. Furthermore,
as ESCs differentiate, the elements of the NOS pathway are
characterized by immuno uorescence staining of CD144
and eNOS to con rm their endothelial phenotype ( Fig. 2C
and 2D ). We noted that cells previously exposed to -NAME
had alterations in function, even after -NAME withdrawal.
Although the percentages of CD144-expressing cells were
similar for all groups (20%), the cells treated for 21 days with
 -NAME(10
5
M dose only) appeared to have impairment
in function, with reduced capacity (by 25%) to form tube-
like structures in Matrigel, even in the absence of -NAME
( Fig. 3 , P < 0.05). This result demonstrated that chronic treat-
ment of -NAME during endothelial differentiation of ESCs
could impair an important endothelial function.
Temporal expression of sGC and DDAH during
endothelial differentiation
To explore the mechanism of by which -NAME affected
late but not early expression of EC markers, we surveyed
NO-signaling components during the course of endothelial
differentiation for up to 2 weeks. Using qPCR, we examined
the mRNA expression of sGC subunits (α
2
, α
3
, β
2
, and β
3
) as
well as DDAH1 and DDAH2. The mRNA abundance was
normalized to 18S housekeeping gene and expressed as fold
changes relative to the undifferentiated ESC group. As shown
in Figure 4A–4D , mRNA transcripts could be detected in
ESCs for all sGC subunits, although the relative abundance
of sGC
α
3
was particularly low. During the course of differ-
entiation, the mRNA levels increased signi cantly for each
subunit, namely a 100-fold increase in sGC
α
3
and a 10-fold
increase for sGC subunits α
2
, β
2
, and β
3
. The gene expression
pattern was similar for DDAH1 and DDAH2, showing a sig-
ni cant increase with differentiation time ( Fig. 4E and 4F , P
< 0.05). The time course of gene expression was further sup-
ported by immuno uorescence staining showing that the
increased intensity of sGCα
3
and sGCβ
3
expression as ESCs
differentiated through day 14 ( Fig. 5A and 5B ). The increased
expression of sGCα
3
and sGCβ
3
was associated in part by the
emergence of ESC-ECs, as CD144- expressing cells co-localized
10
4
10
3
10
2
CD144+
CD144+
10
1
10
0
200 400 600 800 1K
FSC
200 400 600 800 1K
FSC
20%
10
4
10
3
10
2
10
1
10
0
A
CD
B
F I G . 2 . P u r i cation and characterization of
embryonic stem cell-derived endothelial cells
(ESC-ECs). The ESCs were differentiated for
3 weeks and then puri ed by  uorescently
activated cell sorting (FACS) based on positive
expression of the mature endothelial marker,
CD144. Shown are the ( A ) isotype negative
control and ( B ) CD144
+
population. After pu-
ri cation, the CD144
+
cells were characterized
by immuno uorescence staining for ( C ) CD144
or ( D ) endothelial nitric oxide synthase (eNOS)
to con rm their endothelial phenotype. Scale
bar = 25 µm. Color images available online at
www.liebertonline.com/scd.
NITRIC OXIDE SIGNALING IN EMBRYONIC STEM CELLS
1621
signi cantly reduced the levels of cGMP production after
A23187 stimulation by 58% ± 14%, con rming that A23187
increased cGMP by activating endogenous NOS ( P < 0.05,
Fig. 6E ). -NAME also blunted the increase in cGMP induced
by BAY41, which is consistent with the effect of endogenous
NO to potentiate the effect of BAY41. The potentiation of the
effect of BAY41 by A23187 was also inhibited by -NAME
(by 33% ± 6%, P < 0.05), which is consistent with the effect
of A23187 to increase NO synthesis. The effect of SNAP to in-
crease cGMP levels (either alone or in the presence of BAY41)
up-regulated, and increasing levels of basal and stimulated
cGMP are observed in response to endogenous NO or exog-
enous stimulation of sGC.
We further assessed whether chronic -NAME treatment
could differentially regulate cGMP levels at late stages of
differentiation. To do so, we differentiated ESCs for 14 days
in the presence of -NAME (10
5
M) or vehicle control and
measured cGMP levels after stimulating with A23187 (to
stimulate endogenous NO synthesis) or the exogenous acti-
vators of sGC, SNAP, or BAY41. Chronic -NAME treatment
Control LN 10
–5
M
Control LN 10
–5
M
125
*
75
100
25
50
0
% Change in Branch
Points
F I G . 3 . Effect of NG-nitroarginine methyl ester ( -NAME) treatment on tube-like formation of embryonic stem cell-derived
endothelial cells (ESC-ECs) in Matrigel. Endothelial differentiation of ESCs was carried out in the presence of -NAME (LN, 10
5
M or 10
4
M) for 21 days. After puri cation by CD144
+
expression, the ESC-ECs were assessed for tube-like formation upon with-
drawal of -NAME for 24 h. Representative images of tube-like formation for cells having undergone chronic -NAME or di-
methyl sulfoxide (DMSO) vehicle control treatment are shown along with quanti cation of branch points ( n = 4). Prior exposure
to  -NAME (10
5
M dose only) inhibited tube formation. Statistically signi cant comparisons (* P < 0.05) Scale bar = 500 µm.
A
Normalized Fold Change
C
Normalized Fold Change
E
Normalized Fold Change
F
Normalized Fold Change
D
Normalized Fold Change
B
Normalized Fold Change
12
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
sGCα2
sGCα3 sGCβ3
DDAH 1 DDAH 2
sGCβ2
10
8
6
4
0
2
300
250
200
150
100
0
50
2.5
2.0
1. 5
1. 0
0.5
0.0
14
12
10
8
6
0
2
4
6
5
0
2
1
4
3
20
15
10
5
0
ESC 4071114 ESC 4071114
ESC 4071114 ESC 4071114
ESC 4071114 ESC 40711
Time (days)Time (days)
14
F I G . 4 . T i m e c o u r s e o f s o l u b l e
guanylyl cyclase (sGC) and
dimethylarginine dimethylam-
inohydrolase (DDAH) gene
expression during endothe-
lial differentiation. Embryonic
stem cells (ESCs) were cultured
in differentiation media for 14
days and assayed at speci ed
time points by quantitative
PCR. Data show n is nor mali zed
to 18S housekeeping gene and
expressed as fold changes rela-
tive to ESC group. *Statistically
signi cant compared to ESCs
group, P < 0.05 ( n = 3).
HUANG ET AL. 1622
increases VEGF expression [ 16 ]. Inhibition of NOS substan-
tially reduces growth factor-induced angiogenesis, whereas
enhancing endogenous NO synthesis promotes angiogenesis
[ 17 , 26 ]. In particular,  -NAME blocks VEGF-induced cGMP
production in human umbilical venous ECs when cultured
in collagen gels [ 27 ]. Whereas -NAME is not metabolized
by cells, asymmetrical dimethylarginine (ADMA) is an en-
dogenous NOS inhibitor that is degraded by DDAH. Plasma
ADMA is elevated in vascular diseases and associated disor-
ders, due in part to impairment in DDAH activity [ 28–30 ].
In view of the importance of NO in mammalian physiolog y
and particularly its role in adult angiogenesis, it is somewhat
surprising that embryonic lethality is not observed with ho-
mozygous eNOS de ciency. Even animals de cient for all 3
NOS isoforms may survive to adulthood (although hyperten-
sive, infertile, and with nephrogenic diabetes insipidus) [ 31 ].
Our results provide a partial explanation for this phenom-
enon. We observe that early in differentiation, although the
key elements of the NOS pathway are present, they are func-
tionally inactive. Accordingly, other signaling systems for en-
dothelial growth must be primary early in development. In
the adult mammal, other signaling molecules such as prosta-
cyclin, epoxyeicosatrienoic acids, carbon monoxide, hydrogen
peroxide, adrenomedullin, or C-type natriuretic peptide pro-
vide for redundancy, and become more evident when NOS
is inhibited. It is possible that one or more of these signaling
molecules play an important role in vascular development.
Previous studies have surveyed the differential expression
of NO-signaling components during cardiac differentiation
of ESCs. When murine embryoid bodies are incubated with
the NOS antagonist N-monomethyl-L-arginine ( -NMMA)
was not affected by -NAME, consistent with the fact that
SNAP is an exogenous NO donor and not dependent upon
NOS activity. These observations con rm that by day 14,
elements of the NOS pathway are active, and can generate
cGMP in response to endogenous or exogenous NO.
Discussion
The salient  ndings of this study are that (1) ESCs pos-
sess key elements of the NO-signaling machinery, but this
pathway is functionally inactive in ESCs; (2) with differen-
tiation, there is an increase in the expression of endothelial
surface markers, eNOS, and subunits of sGC, in association
with increasing activity of the NO-signaling machinery as
assessed by cGMP production; and (3) chronic inhibition of
NOS has no effect early in differentiation, but by 14 days of
differentiation modestly inhibits the expression of endothe-
lial surface markers CD144, FLK1, and eNOS, as well as the
formation of endothelial tubes.
The NOS pathway is ubiquitous in the adult mammal,
with endothelial, neuronal, and inducible NOS isoforms play-
ing critical roles in every organ system, in particular cardio-
vascular homeostasis, thrombosis, neurotransmission, and
immunity [ 24 ]. With respect to vascular reactivity and struc-
ture, NO plays a dominant role as a vasodilation factor that
also potently inhibits vascular smooth muscle proliferation,
leukocyte in ltration, and platelet adherence and aggrega-
tion [ 25 ]. Furthermore, NO is a powerful agonist of EC prolif-
eration and migration. There is also a reciprocal reinforcing
relationship between NO and vascular endothelial growth
factor (VEGF), as VEGF stimulates NO synthesis, and NO
sGCα3
ESC d5 d8 d14
ESC d5 d8 d14
sGCα3/
Hoechst
sGCβ3
sGCα3 MergeCD144
sGCβ3 MergeCD144
sGCβ3/
Hoechst
A
B
C
F I G . 5 . I m m u n o uorescence staining
depicting soluble guanylyl cyclase (sGC)
α
3
and sGCβ
3
expression during the course
of endothelial differentiation in embryonic
stem cells. Immuno uorescence staining is
shown for ( A ) sGCα
3
and ( B ) sGCβ
3
, with cor-
responding Hoechst 33342 nuclear dye. ( C )
After 14 days of differentiation, the cells were
immuno uorescently stained for both CD144
and sGCα
3
or sGCβ
3
, with corresponding
Hoechst 33342 nuclear dye. Scale bar = 200
µm ( A and B ); 25 µm ( C ) . C o l o r i m a g e s a v a i l -
able online at www.liebertonline.com/scd.
NITRIC OXIDE SIGNALING IN EMBRYONIC STEM CELLS
1623
during cardiac differentiation of murine ESCs [ 11 ]. Mujoo et
al. also demonstrated an increase of sGC subunits during
the course of differentiation of human ESCs for 14 days, but
the authors reported a progressive decrease in sGC
β
2
mRNA
from day 0 to day 14 [ 10 ]. These observed differences may
be attributed to differences in the ESC species and strains,
or -nitroarginine, the number of EB-derived cardiomyo-
cytes is not affected, but  brillogenesis in the cardiomyo-
cytes is reduced [ 32 ]. This effect is mimicked by ODQ (an
antagonist of soluble guanylate cyclase). These observations
are consistent with those of Krumenacker et al. who showed
an increase in expression of sGC
α
1
and sGC
β
1
expression
*
*
*
2000
1000
500
400
300
200
100
10
Basal A23187 A23187+BAY41 SNAP+BAY41BAY41
Endogenous NO Exogenous NO
SNAP
0
cGMP (pmol/mg)
Con
LN 10
–5
M
E
6
Basal A23187
SNAP BAY41
*
*
*
*
*
*
*
*
5
4
3
2
1
0
500
400
300
200
100
0
12
10
8
6
4
2
0
ESC
cGMP (pmol/mg)cGMP (pmol/mg)
250
200
150
100
50
0
cGMP (pmol/mg) cGMP (pmol/mg)
day 7 day 14 ESC day 7 day 14
ESC day 7 day 14ESC day 7 day 14
AB
CD
F I G . 6 . Effect of endogenous or exogenous activators of nitric oxide (NO) on cyclic guanosine monophosphate (cGMP) ac-
tivity. At different time points of differentiation, cell lysates were assayed for cGMP production after ( A ) no treatment (basal),
( B ) A23187, ( C ) SNAP, or ( D ) BAY41 stimulation. ( E ) After 14 days of differentiation in media containing NG-nitroarginine
methyl ester ( -NAME) (10
5
M), cells were stimulated with activators of NO release, and lysates were assayed for cGMP.
Asterisk with brackets indicates statistically signi cant comparisons, and asterisk without brackets indicate statistically sig-
ni cant when compared to embryonic stem cells (ESCs) ( P < 0 . 0 5 ) .
HUANG ET AL. 1624
9 . M u j o o K , V G S h a r i n , N S B r y a n , J S K r u m e n a c k e r , C S l o a n , S P a r v e e n ,
L E N i k o n o f f , A Y K o t s a n d F M u r a d . ( 2 0 0 8 ) . R o l e o f n i t r i c o x i d e s i g -
naling components in differentiation of embryonic stem cells into
m y o c a r d i a l c e l l s . P r o c N a t l A c a d S c i U S A 1 0 5 : 1 8 9 2 4 1 8 9 2 9 .
1 0 . M u j o o K , J S K r u m e n a c k e r , Y W a d a a n d F M u r a d . ( 2 0 0 6 ) .
Differential expression of nitric oxide signaling components
in undifferentiated and differentiated human embryonic stem
c e l l s . S t e m C e l l s D e v 1 5 : 7 7 9 7 8 7 .
1 1 . K r u m e n a c k e r J S , S K a t s u k i , A K o t s a n d F M u r a d . ( 2 0 0 6 ) .
Differential expression of genes involved in cGMP-dependent
nitric oxide signaling in murine embryonic stem (ES) cells and
E S c e l l - d e r i v e d c a r d i o m y o c y t e s . N i t r i c O x i d e 1 4 : 1 1 1 .
1 2 . D a n a l a c h e B A , J P a q u i n , W D o n g h a o , R G r y g o r c z y k , J C M o o r e ,
C L M u m m e r y , J G u t k o w s k a a n d M J a n k o w s k i . ( 2 0 0 7 ) . N i t r i c
oxide signaling in oxytocin-mediated cardiomyogenesis . Stem
C e l l s 2 5 : 6 7 9 6 8 8 .
1 3 . L e e T C , Y D Z h a o , D W C o u r t m a n a n d D J S t e w a r t . ( 2 0 0 0 ) .
Abnormal aortic valve development in mice lacking endothe-
l i a l n i t r i c o x i d e s y n t h a s e . C i r c u l a t i o n 1 0 1 : 2 3 4 5 2 3 4 8 .
1 4 . F e n g Q , W S o n g , X L u , J A H a m i l t o n , M L e i , T P e n g a n d S P
Y e e . ( 2 0 0 2 ) . D e v e l o p m e n t o f h e a r t f a i l u r e a n d c o n g e n i t a l s e p -
tal defects in mice lacking endothelial nitric oxide synthase .
C i r c u l a t i o n 1 0 6 : 8 7 3 8 7 9 .
1 5 . N i e b a u e r J , J D u l a k , J R C h a n , P S T s a o a n d J P C o o k e . ( 1 9 9 9 ) . G e ne
transfer of nitric oxide synthase: effects on endothelial biology .
J A m C o l l C a r d i o l 3 4 : 1 2 0 1 1 2 0 7 .
1 6 . D u l a k J , A J ó z k o w i c z , A D e m b i n s k a - K i e c , I G u e v a r a , A
Z d z i e n i c k a , D Z m u d z i n s k a - G r o c h o t , I F l o r e k , A W ó j t o w i c z , A
S z u b a a n d J P C o o k e . ( 2 0 0 0 ) . N i t r i c o x i d e i n d u c e s t h e s y n t h e s i s
of vascular endothelial growth factor by rat vascular smooth
m u s c l e c e l l s . A r t e r i o s c l e r T h r o m b V a s c B i o l 2 0 : 6 5 9 6 6 6 .
1 7. J a c o b i J , K S y d o w , G v o n D e g e n f e l d , Y Z h a n g , H D a y o u b , B
W a n g , A J P a t t e r s o n , M K i m o t o , H M B l a u a n d J P C o o k e . ( 2 0 0 5 ) .
Overexpression of dimethylarginine dimethylaminohydro-
lase reduces tissue asymmetric dimethylarginine levels and
e n h a n c e s a n g i o g e n e s i s . C i r c u l a t i o n 1 1 1 : 1 4 3 1 1 4 3 8 .
1 8 . C a o F , K E v a n d e r B o g t , A S a d r z a d e h , X X i e , A Y S h e i k h , H
W a n g , A J C o n n o l l y , R C R o b b i n s a n d J C W u . ( 2 0 0 7 ) . S p a t i a l a n d
temporal kinetics of teratoma formation from murine embry-
onic stem cell transplantation . Stem Cells Dev 16 : 883 891 .
1 9 . S h e t z l i n e S E , R R a l l a p a l l i , K J D o w d , S Z o u , Y N a k a t a , C R S w i d e r ,
A K a l o t a , J K C h o i a n d A M G e w i r t z . ( 2 0 0 4 ) . N e u r o m e d i n U : a
Myb-regulated autocrine growth factor for human myeloid leu-
k e m i a s . B l o o d 1 0 4 : 1 8 3 3 1 8 4 0 .
2 0 . L i v a k K J a n d T D S c h m i t t g e n . ( 2 0 0 1 ) . A n a l y s i s o f r e l a t i v e g e n e
expression data using real-time quantitative PCR and the
2 ( - D e l t a D e l t a C ( T ) ) M e t h o d . M e t h o d s 2 5 : 4 0 2 4 0 8 .
2 1 . H u a n g N F , S P a t e l , R G T h a k a r , J W u , B S H s i a o , B C h u , R J L e e
a n d S L i . ( 2 0 0 6 ) . M y o t u b e a s s e m b l y o n n a n o brous and micro-
p a t t e r n e d p o l y m e r s . N a n o L e t t 6 : 5 3 7 5 4 2 .
2 2 . C a i a d o F , C R e a l , T C a r v a l h o a n d S D i a s . ( 2 0 0 8 ) . N o t c h p a t h w a y
modulation on bone marrow-derived vascular precursor cells
regulates their angiogenic and wound healing potential . PLoS
O N E 3 : e 3 7 5 2 .
2 3 . B u d w o r t h J , S M e i l l e r a i s , I C h a r l e s a n d K P o w e l l . ( 1 9 9 9 ) . T i s s u e
distribution of the human soluble guanylate cyclases . Biochem
B i o p h y s R e s C o m m u n 2 6 3 : 6 9 6 7 0 1 .
2 4 . B r y a n N S , K B i a n a n d F M u r a d . ( 2 0 0 9 ) . D i s c o v e r y o f t h e n i t r i c
oxide signaling pathway and targets for drug development .
F r o n t B i o s c i 1 4 : 1 1 8 .
2 5 . C o o k e J P . ( 2 0 0 3 ) . F l o w , N O , a n d a t h e r o g e n e s i s . P r o c N a t l A c a d
S c i U S A 1 0 0 : 7 6 8 7 7 0 .
2 6 . J a n g J J , H K H o , H H K w a n , L F F a j a r d o a n d J P C o o k e . ( 2 0 0 0 ) .
Angiogenesis is impaired by hypercholesterolemia: role of
a s y m m e t r i c d i m e t h y l a r g i n i n e . C i r c u l a t i o n 1 0 2 : 1 4 1 4 1 4 1 9 .
2 7 . P a p a p e t r o p o u l o s A , G G a r c í a - C a r d e ñ a , J A M a d r i a n d W C S e s s a .
( 1997 ). Nitric oxide production contributes to the angiogenic
properties of vascular endothelial growth factor in human en-
d o t h e l i a l c e l l s . J C l i n I n v e s t 1 0 0 : 3 1 3 1 3 1 3 9 .
as well as varying induction media formulations. In a re-
cent study of human and mouse ESCs, NO donors and sGC
activators could increase the mRNA expression of cardiac-
speci c genes myosin light chain and Nkx2.5, whereas NOS
inhibitors decreased their mRNA expression [ 9 ].
A limitation of the current study is the inherent heteroge-
neity of the population of differentiating cells, which includes
ESC-ECs. Nevertheless, this mixed population can respond to
pharmacological agents and provide useful biochemical and
signaling cues to better understand the role of NO in ESC
differentiation.
In summary, we have demonstrated that NO-signaling
elements are present in ESCs, although the NOS pathway
is functionally inactive until later in differentiation.
Pharmacological inhibition of NOS has no observed effect
early in differentiation on endothelial gene expression and
surface markers, but later in differentiation causes a modest
reduction of these genes and markers, as well as a modest
impairment of endothelial tube-like formation. Our studies
indicate that later in differentiation NO plays a role in endo-
thelial development and function, but other signaling path-
ways must play a greater role early in development.
Acknowledgments
This study was supported by grants from the National
Institutes of Health (RO1 HL-75774, R01 CA098303, R21
HL085743, 1K12 HL087746), the California Tobacco Related
Disease Research Program of the University of California
(1514RT-0169), the American Heart Association (0970036N) and
the California Institute for Regenerative Medicine (RS1-00183),
and the Stanford Cardiovascular Institute. N.F.H. was sup-
ported by a fellowship from the American Heart Association.
Author Disclosure Statement
No competing  nancial interests exist.
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Received for publication October 22, 2009
Accepted after revision January 9, 2010
Prepublished on Liebert Instant Online January 11, 2010
Address correspondence to:
Dr. John P. Cooke
Division of Cardiovascular Medicine
Stanford University
300 Pasteur Drive
Stanford, CA 94305-5406
E-mail : john.cooke@stanford.edu
    • "Nitric oxide (NO) has been extensively studied for its role in modulating SC cell behavior to regulate aqueous outflow [3,103,139]. Because of the important role that NO plays in facilitating SC cell functions and endothelial junctional integrity, this warrants exploring the role of NO in SC development [140]. Furthermore, given the vascular origin of the SC and its lymphatic-like development and characteristics, it is important to consider soluble factors involved in vascular endothelial and lymphatic cell differentiation. "
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