?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
The phosphorylation state of eNOS
modulates vascular reactivity and outcome
of cerebral ischemia in vivo
Dmitriy N. Atochin,1 Annie Wang,1 Victor W.T. Liu,1 Jeffrey D. Critchlow,1 Ana Paula V. Dantas,2
Robin Looft-Wilson,3 Takahisa Murata,3 Salvatore Salomone,4 Hwa Kyoung Shin,4 Cenk Ayata,4
Michael A. Moskowitz,4 Thomas Michel,2 William C. Sessa,3 and Paul L. Huang1
1Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Boston, Massachusetts, USA. 2Cardiovascular Division,
Brigham and Women’s Hospital, Boston, Massachusetts, USA. 3Department of Pharmacology and Molecular Medicine, Yale University School of Medicine,
New Haven, Connecticut, USA. 4Stroke and Neurovascular Regulation Laboratory, Massachusetts General Hospital, Boston, Massachusetts, USA.
eNOS plays important roles in vascular function, including reg-
ulation of vascular tone, angiogenesis, vascular signaling and
remodeling, platelet aggregation, and leukocyte-endothelial inter-
actions (1–4). eNOS activity is dependent on calcium-calmodulin
activation. However, eNOS activity is also regulated at other lev-
els, including availability of its substrate l-arginine or cofactors
(NADPH, flavin adenine dinucleotide [FAD], flavin mononucleo-
tide [FMN], and tetrahydrobiopterin [BH4]), protein-protein inter-
actions with hsp90 and caveolins, posttranslational fatty acylation
(myristoylation and palmitoylation), S-nitrosylation, and phos-
phorylation (5–7). Among these, eNOS phosphorylation appears
to be an important means of regulating eNOS activity (8, 9).
eNOS phosphorylation at serine residue 1179 increases eNOS
enzymatic activity and NO production. S1179 phosphorylation is
increased by estrogens (10) and statins (11), accounting in part for
the beneficial effects of these agents on vascular function. Shear
stress and VEGF also increase S1179 phosphorylation (12). Leptin
and the protective hormone adiponectin (13, 14) increase eNOS
phosphorylation while IL-6, TNF-α, and resistin decrease it (15,
16). eNOS phosphorylation provides a potential mechanistic link
between metabolic abnormalities from diabetes or obesity and
cardiovascular risk. We hypothesize that a defect in eNOS phos-
phorylation may account for endothelial dysfunction observed in
hypertension, hyperlipidemia, diabetes, and metabolic syndrome.
To date, the effects of eNOS phosphorylation on vascular function
and disease pathogenesis have not been demonstrated in vivo.
The importance of eNOS phosphorylation at the S1179 residue
has been studied using mutant forms of eNOS. S1179A eNOS
contains an alanine, which cannot be phosphorylated, in place of
the serine. S1179D eNOS contains an aspartate with a carboxyl
side group that mimics a negatively charged phosphate group
in place of the serine. Purified S1179D eNOS enzyme has high-
er catalytic activity than WT eNOS due to both increased basal
activity and calmodulin regulation of the reductase domain of
eNOS (17). When these mutants are expressed in COS cells (8, 9)
or endothelial cells (12), both S1179A and S1179D mutants are
active. Thus, phosphorylation at S1179 is not a prerequisite for
enzymatic activity, and the S1179A mutant is not a dominant-
negative or null mutation. However, S1179A eNOS does not show
Akt-dependent NO release while S1179D shows increased NO
release even in the absence of Akt kinase (8, 9).
The molecular mechanism for the increased NOS activity in
S1179D eNOS (and eNOS phosphorylated at S1179) appears to be
enhanced electron flux through the reductase domain and reduced
calmodulin dissociation (17). Elegant crystallographic stud-
ies show that the carboxyl tail of NOS normally retards electron
transfer from the reductase domain by locking the FMN-binding
domain into an electron-accepting position (18). Phosphoryla-
tion of S1179 results in a negative charge (as does substitution by
aspartate in the S1179D mutation), inducing a conformational
change that shifts the entire FMN domain to allow enhanced
electron transfer through the reductase domain, activating the
enzyme. As a result, S1179D eNOS produces NO at resting levels
of intracellular calcium. Physiologically, shear stress, VEGF, and
insulin activate eNOS because they promote eNOS S1179 phos-
phorylation. This allows greater eNOS activation to take place at
any calcium concentration without substantially changing the
affinity of calcium-activated calmodulin for eNOS.
To define the role of eNOS phosphorylation at S1179 in vascular
function and disease pathogenesis in vivo, we generated transgenic
mice that express either S1179A or S1179D eNOS. By breeding
these animals with eNOS KO mice, we obtained mice that only
express the S1179A and S1179D mutant forms of eNOS. We deter-
Nonstandard?abbreviations?used: BH4, tetrahydrobiopterin; FAD, flavin adenine
dinucleotide; FMN, flavin mononucleotide; l-NAME, Nω-nitro-l-arginine methyl ester;
MCA, middle cerebral artery; PSS, physiologic saline; SNP, sodium nitroprusside.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 117:1961–1967 (2007). doi:10.1172/JCI29877.
1962?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
mined the effects of modulation of the S1179 phosphorylation site
on vascular reactivity, cerebral blood flow, and outcome in a middle
cerebral artery (MCA) occlusion model of stroke. Our results indi-
cate that modulation of the S1179 phosphorylation site affects
endothelium-dependent vasodilation and cerebral blood flow and
that these effects determine outcome of stroke in vivo.
Generation of S1179A and S1179D transgenic mice. We generated trans-
genic mice that express either S1179A or S1179D mutant forms of
eNOS, using the construct shown in Figure 1A. We performed site-
directed mutagenesis on bovine eNOS cDNA to generate S1179A
and S1179D mutations. The cDNAs were cloned into a vector con-
taining an HA tag to facilitate detection of transgene expression.
Expression of the eNOS cDNAs was driven using a 1.6-kb frag-
ment of the human eNOS promoter, which is endothelium spe-
cific and contains the binding sites for Sp1, GATA-2, AP-1, NF-1,
and the shear stress responsive element (19, 20). We established
multiple independent founder lines for each mutant and selected
2 lines that showed equivalent levels of expression.
To obtain mice that express the mutant forms of eNOS but not
endogenous WT eNOS, we bred the transgenic mice with eNOS
KO mice (21) and selected offspring that carried the transgenes
and were heterozygous for eNOS gene deletion. We then mated
these animals with each other to obtain animals that carried the
transgenes and were homozygous for eNOS gene deletion. Thus,
we obtained S1179D and S1179A transgenic mice on an eNOS
null background (S1179D/eNOS KO mice and S1179A/eNOS KO
mice). Figure 1B confirms the expression of the S1179A or S1179D
transgene in the absence of the native eNOS gene.
The eNOS KO mice had been backcrossed to the C57BL/6 back-
ground, while the S1179A and S1179D transgenic mice were cre-
ated on a C57BL/6-C3H F1 background. We tested the vascular
responses of eNOS KO littermates from these matings that did
not contain the S1179A or S1179D transgenes. These littermate
control eNOS KO mice had vascular responses to ACh, sodium
nitroprusside (SNP), and phenylephrine that were indistinguish-
able from de novo eNOS KO mice on a C57BL/6 background.
Expression and subcellular localization of S1179D and S1179A forms of
eNOS. Western blot analysis confirmed that the expression levels
of the mutant eNOS protein in S1179D and S1179A transgenic
mice were equivalent but less than those seen in WT mice (Figure
1C). Consistent with this, the mean arterial blood pressure read-
ings of S1179D/eNOS KO mice (124 ± 10 mmHg) and S1179A/
eNOS KO mice (129 ± 12 mmHg) were not significantly different
from those of eNOS KO mice (120 ± 10 mmHg) while WT mice
showed a mean arterial blood pressure of 90 ± 10 mmHg (n = 3–5
per group). Thus, we succeeded in reconstituting the eNOS KO
mice with equivalent but diminished levels of either S1179D or
S1179A forms of eNOS.
Figure 2 shows luminal en face vessel preparations stained by
immunofluorescence. Expression of eNOS in the endothelial cells
of WT mice is localized in a perinuclear Golgi pattern with lesser
amounts of eNOS distributed throughout the cells. Expression
of the mutant eNOS transgenes via immunostaining for the HA
tag demonstrated that both S1179A and S1179D transgenes were
expressed in a distribution similar to that observed for eNOS in
WT cells, confirming proper subcellular localization of the pro-
teins. Furthermore, staining for eNOS itself showed proper sub-
cellular localization of the mutant forms of eNOS in the S1179D/
eNOS KO and S1179A/eNOS KO mice (Figure 3). The presence
of the HA tag did not alter the subcellular localization of eNOS,
nor did it affect the relationship between eNOS and caveolin-1,
as seen in Figure 3.
Vascular reactivity in S1179A and S1179D mice. To determine how
S1179 transgenes affect vascular reactivity, we mounted the carot-
id arteries onto a pressurized myograph, constricted the vessels
with phenylephrine, and measured vascular relaxation in response
to ACh. As seen in Figure 4A, S1179D/eNOS KO mice showed
increased relaxation compared with S1179A/eNOS KO mice.
eNOS KO mice without eNOS transgenes showed little relaxation.
Generation of S1179A and S1179D eNOS transgenic mice. (A) Trans-
genic construct. A 1.6-kb fragment of the human eNOS promoter
drives the bovine eNOS cDNA carrying the S1179D or S1179A muta-
tion, an HA tag, and the SV40 polyadenylation site. (B) Genotyping of
transgenic and native eNOS genes by PCR. Primers E1 and E2 (see
Methods) span an intron, so they amplify an 800-bp product from the
endogenous WT eNOS gene but a 280-bp product from the S1179A
and S1179D transgenes. Mice bred onto the homozygous eNOS KO
background do not show the 800-bp product. (C) Western blot analysis
of heart protein (60 μg) of WT, S1179D transgenic/eNOS KO (S1179D/
eNOS KO), and S1179A transgenic/eNOS KO (S1179A/eNOS KO)
mice, using antibody directed against total eNOS.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
WT mice showed greater relaxation than S1179D/eNOS KO mice,
consistent with the fact that the expression levels of mutant eNOS
protein were less than WT expression levels. In contrast, carotid
arteries from all 4 groups responded equally well to the endothe-
lium-independent vasodilator SNP (Figure 4B).
MCA occlusion model of stroke. To determine the effect of these
eNOS S1179 mutations on the response to cerebral ischemia, we
subjected the S1179D/eNOS KO mice and S1179A/eNOS KO
mice to an MCA occlusion model of stroke. Vessel occlusion for
1 hour followed by 23 hours of reperfusion resulted in a greater
volume of cerebral infarction in eNOS KO mice compared with
WT mice (22). Expression of the mutant S1179D eNOS trans-
gene reduced infarct area while expression of S1179A eNOS did
not (Figure 5). The functional significance of these results is
confirmed by the neurologic scoring, which shows more severe
deficits in the eNOS KO and S1179A/eNOS KO mice than in the
WT and S1179D/eNOS KO mice.
Laser speckle flowmetry assessment of cerebral blood flow. To determine
whether these differences in stroke size result from differences in
cerebral blood flow, we performed laser speckle flowmetry dur-
ing distal MCA occlusion (23). This technique measures cerebral
blood flow with high spatial (12 μm/pixel) and temporal resolu-
tion (1 image/7.5 s) and provides 2D assessment of blood flow def-
icit in real time. eNOS KO mice (n = 10) developed a significantly
larger cortical area with more severe blood flow reduction (≤20%
residual blood flow calculated using a threshold-
ing paradigm) compared with WT (n = 10) mice
(Figure 6). These data show that eNOS activity is
critical to augment blood flow in focal cerebral
ischemia. Expression of the S1179A transgene
in eNOS KO mice did not improve blood flow
(n = 5) whereas expression of the S1179D eNOS
transgene (n = 5) reduced the ischemic area to a
size close to that seen in the WT mice.
It is well established that hypertension, hyper-
lipidemia, diabetes, and smoking are associated
with increased cardiovascular risk. However, the
mechanisms by which these conditions increase
atherosclerosis are not fully understood. One of
the earliest detectable events in these conditions is
endothelial dysfunction, which precedes structur-
al changes. A relative insufficiency in vascular NO
production results in a reduction in the protective
effects of NO and increases vascular wall inflammation, thrombosis,
and smooth muscle cell proliferation. We hypothesized that abnor-
malities in eNOS phosphorylation are an important common path-
way that links diverse cardiovascular risks with endothelial dysfunc-
tion to increase propensity to atherosclerotic cardiovascular disease.
For diabetes, obesity, and the metabolic syndrome, abnormalities in
adipocyte signaling or metabolism may be the primary events that
lead to downstream vascular abnormalities.
S1179 is phosphorylated not only by Akt kinase, but also by AMP
kinase (24) and protein kinases A and G (25). In particular, insulin
activates eNOS through Akt-mediated S1179 phosphorylation,
thereby increasing blood flow and glucose uptake (26). In contrast,
adiponectin, which is vasculoprotective, increases NO production
not through Akt, but rather through AMP kinase–mediated S1179
phosphorylation (14). Thus, the phosphorylation state of S1179
may be a common integration point for multiple diverse signal-
ing systems, such as insulin and adipokines. These results support
the possibility that abnormalities in eNOS phosphorylation may
underlie endothelial dysfunction from various causes.
Here, we test the hypothesis that phosphorylation at S1179 is
an important determinant of vascular function and outcome of
atherosclerotic disease. To date, the effects of S1179A and S1179D
mutants have been characterized in cultured cells (12, 14, 26) and
using ex vivo gene transfer into isolated vessels (12, 27, 28). Both
S1179A and S1179D eNOS are enzymatically active, so S1179
Subcellular localization of WT and transgenic
eNOS. (A) En face immunostaining of the com-
mon carotid arteries using eNOS antibody shows
localization of eNOS in perinuclear Golgi that are
aligned along the long axes of the cells in the endo-
thelium. VE-cadherin (VE-cad) staining shows the
outlines of the endothelial cell membranes. (B) En
face immunostaining of the common carotid arter-
ies using anti-HA shows perinuclear staining in the
Golgi apparatus in a pattern similar to that of eNOS
immunostaining in WT mice. Expression levels of
the transgene are comparable in the S1179A/eNOS
KO mice and S1179D/eNOS KO mice. Original
1964?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
phosphorylation is not required for NO production. However,
the S1179D form of eNOS is associated with much greater NO
production due to enhanced electron flux through the reductase
domain of eNOS and reduced calmodulin dissociation (17). As
a result, S1179D eNOS produces NO even in the absence of Akt
kinase activity while S1179A fails to augment NO production in
the setting of Akt activation (8, 9).
We find that S1179A, mimicking diminished S1179 phosphory-
lation, impairs vasodilation in intact vessels while the phosphomi-
metic S1179D eNOS increases vasodilation. Furthermore, modu-
lation of the eNOS S1179 phosphorylation site affects cerebral
blood flow in vivo and influences stroke size following cerebral
ischemia. Because eNOS activity is regulated by multiple mecha-
nisms, our results are important for demonstrating the in vivo
relevance of this eNOS phosphorylation site to vascular function,
blood flow, and outcome in a mouse model of human disease.
Although our results show that vascular reactivity and changes in
blood flow are affected by eNOS phosphorylation at S1179, they do
not preclude additional effects. Specifically, eNOS has
been demonstrated to affect vascular smooth muscle
proliferation, leukocyte-endothelial interactions, and
platelet aggregation and adhesion. In addition, it is
possible that eNOS phosphorylation and the result-
ing increase in NO production may also influence
insulin sensitivity, as has been reported in eNOS
homozygous and heterozygous KO mice (29, 30).
En face imaging of the HA tag shows that the
human eNOS promoter (20) leads to expression in
endothelial cells with preserved subcellular localiza-
tion. Furthermore, Western blots show that expres-
sion levels of the S1179A and S1179D transgenes
are equivalent, though diminished, compared with
that of eNOS in WT mice. These considerations are
important because differences in expression levels
or localization could affect the outcome of vascular
function studies or in vivo disease models. We com-
pared S1179A/eNOS KO mice with S1179D/eNOS
KO mice to directly assess the effects of phosphoryla-
tion on endothelial function, cerebral blood flow, and
outcome of MCA occlusion. Comparisons with WT
mice may be less revealing because the phosphoryla-
tion state of the S1179 residue in WT mice cannot
be controlled and because the expression levels in
the transgenic mice were less than WT eNOS levels.
In this regard, reconstitution of eNOS KO mice with
low levels of either S1179A or S1179D transgenes may
explain why we did not observe compensatory effects
of increased transgene expression that have been
reported in some (28) but not other (12, 27) systems.
eNOS KO mice have been used as a model of
endothelial dysfunction because their phenotype
reflects the effects of absence of endothelial NO pro-
duction. However, most cardiovascular risks are not
associated with total absence of vascular NO but
rather a decrease in the amount of bioavailable NO. In
particular, shear stress, insulin, IGF-1, estrogen, and
VEGF influence eNOS activity through S1179 phos-
phorylation, so the S1179A and S1179D transgenic
mice should be more pathophysiologically relevant
models of endothelial dysfunction than mice that
totally lack eNOS. Our results establish that eNOS phosphoryla-
tion is an important determinant of vascular function, blood flow,
and outcome of cerebral ischemia in intact animals. This provides
proof of concept for the modulation of the S1179 phosphorylation
state as an approach to treating and preventing cardiovascular dis-
ease, particularly as influenced by risk factors of diabetes, obesity,
metabolic syndrome, hyperlipidemia, and hypertension.
Generation of S1179D and S1179A eNOS mutant transgenic mice. To generate
S1179A and S1179D transgenic mice, we cloned a 1.6-kb fragment (from
KpnI to HindIII) containing the human eNOS promoter into pBK-CMV
(Stratagene). Bovine eNOS cDNAs with HA tags and carrying either the
S1179A or S1179D mutations were inserted into the EcoRI and BssHII
restriction sites, placing the eNOS cDNAs in the correct orientation
downstream of the human eNOS promoter. The plasmid map of the con-
structs is shown in Figure 1A. The plasmid inserts were liberated with
MluI and NheI, purified, and injected into oocytes to obtain transgenic
Interactions between caveolin-1 (Cav-1) and eNOS are unaffected in mice carrying
transgenes. En face immunostaining of the common carotid arteries using eNOS
antibody and caveolin-1 antibody shows that interactions between eNOS and caveo-
lin-1 are unaffected in S1179D/eNOS KO mice and S1179A/eNOS KO mice. Original
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
mice. Several founder lines were obtained for S1179A and S1179D trans-
genes, and 2 lines that had equivalent levels of expression were chosen.
The S1179A and S1179D transgenic mice were bred onto an eNOS KO
background so they would not express the endogenous WT eNOS gene.
Male mice between 8 and 12 weeks of age were used for all experiments.
All experiments were approved by the Massachusetts General Hospital
Institutional Animal Care and Use Committee. Mice were euthanized
according to the recommendations of the American Veterinary Medical
Association Panel on Euthanasia.
Genotyping. Mice were genotyped using the following eNOS gene–spe-
cific primers: E1, 5′-GGGCTCCCTCCTTCCGGCTGCCACC-3′; and E2,
5′-GGATCCCTGGAAAAGGCGGTGAGG-3′. We used conditions of
denaturation at 94°C for 30 seconds, annealing at 62°C for 60 seconds,
and polymerization at 72°C for 60 seconds; 35 cycles. E1 and E2 span an
intron, so they amplify an 800-bp product (that includes the intron) from
genomic DNA and a 280-bp product (that does not include the intron)
from the transgenes. All experiments were done on mice that express the
S1179A or S1179D transgenes but not the endogenous WT eNOS gene.
eNOS KO genotype was also confirmed by Southern blotting.
Western blot analysis. Heart tissue was homogenized to obtain protein
extracts, and 60 μg of protein was subjected to electrophoresis in a 7.5%
Tris-HCl polyacrylamide gel. Proteins were transferred to membranes,
incubated with a 1:250 dilution of primary mouse monoclonal anti-eNOS
antibody (BD Biosciences), washed, incubated with 1:1,000 horseradish
peroxidase–conjugated goat anti-mouse IgG secondary antibody, and visu-
alized by chemiluminescence (Amersham Biosciences).
Blood pressure determination. Animals were anesthetized by inhalation
using a mixture of 30% O2, 70% N2O, and 1.5% isoflurane. Body tempera-
ture was maintained at 36–37°C. The common carotid artery was exposed
by dissection, and a catheter from pulled PE-10 tubing was inserted to
measure systemic blood pressure using a fluid transducer (MacLab).
En face immunostaining. The common carotid artery was harvested, fixed
with 4% paraformaldehyde in PBS at 4°C for 10 minutes, permeabilized
with 0.3% Triton X-100 at room temperature for 30 minutes, and blocked
with 3% fetal bovine serum at room temperature for 30 minutes. The sec-
Effect of S1179A and S1179D eNOS mutations on cerebral infarct size. Mice were subjected to the filament model of MCA occlusion for 1 hour,
followed by 23 hours of reperfusion. The brains were cut into 2-mm coronal sections and stained using 2,3,5-triphenyltetrazolium chloride. (A)
Rostral to caudal distribution of infarct areas (mm2) in coronal sections. Abscissa shows the distance (mm) from the rostral surface of the brain.
n = 7 mice for each group. *P < 0.05 versus corresponding sections from S1179A/eNOS KO mice; †P < 0.05 versus corresponding sections
from eNOS KO mice using 1-way ANOVA. (B) Infarct volumes were determined by integrating the infarct areas in each section over the entire
brain, using the indirect method, which corrects for edema. *P < 0.05 versus infarct volume from S1179A/eNOS and from eNOS KO mice using
ANOVA. Neurologic scores for each group of mice are as follows: eNOS KO, 2.5; S1179A/eNOS KO, 2.67; S1179D/eNOS KO, 1.57; WT, 1.86.
Kruskal-Wallis 1-way ANOVA on ranks showed a statistically significant difference between eNOS KO and S1179A/eNOS KO mice and WT and
S1179D/eNOS KO mice (P < 0.05).
Effect of S1179D and S1179A mutations on vascular reactivity. (A) Left
common carotid arteries were constricted with phenylephrine (10–5 M)
and then subjected to increasing doses of ACh from 1 × 10–9 to 1 × 10–5 M.
S1179A/eNOS KO mice, n = 8; S1179D/eNOS KO mice, n = 8; eNOS
KO mice, n = 6; and WT mice, n = 8. *P < 0.01 by 1-way ANOVA;
†P < 0.05 by 1-way ANOVA. (B) Left common carotid arteries were
treated with l-NAME (3 × 10–4 M) for 30 minutes, constricted with phen-
ylephrine (10–5 M), and then subjected to increasing doses of SNP from
1 × 10–9 to 1 × 10–5 M. Data are expressed as mean ± SEM.
1966?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
tions were incubated with mouse anti-eNOS antibody (BD Biosciences), rat
anti–VE-cadherin antibody (BD Biosciences), rat anti-HA antibody (Roche
Diagnostics), or rabbit anti–caveolin-1 antibody (Santa Cruz Biotechnol-
ogy Inc.) at dilutions between 1:200 and 1:400 at 4°C for 12 hours. They
were then incubated with a second antibody, either Alexa Fluor 488–con-
jugated goat anti-mouse IgG or Alexa Fluor 568–conjugated goat anti-rat
IgG (Invitrogen) at 1:200 dilution at room temperature for 1 hour. The
vessels were then visualized using a scanning microscope (Zeiss) with an
Axiovert 200M imaging system.
Vascular reactivity studies. Phenylephrine, ACh, Nω-nitro-l-arginine methyl
ester (l-NAME), and SNP were obtained from Sigma-Aldrich and dissolved
in distilled water to make stock solutions. Mice were anesthetized with
30% O2, 70% N2O, and 2% isoflurane. Carotid arteries were removed and
mounted onto glass cannulas in a pressure myograph (DMT). The vessels
were maintained at 85 mmHg pressure and 37°C. They were perfused in
physiologic saline (PSS) with the following composition: 130 mM NaCl,
4.7 mM KCl, 1.17 mM MgSO4, 14.9 mM NaHCO3, 1.6 mM CaCl2, 1.18
mM KH2PO4, 0.026 mM EDTA, and 5.5 mM glucose. PSS was aerated at
95% O2 and 5% CO2 to maintain a pH of 7.4. The vessel diameter was con-
tinuously recorded using a video system controlled by custom software
IonWizard (version 4.4; IonOptix Corp.). Vessels were equilibrated for 45
minutes and constricted with 10–5 M phenylephrine. Response curves were
then generated by stepwise application of increasing concentrations of
ACh from 10–9 to 10–5 M to the organ bath. The vessels were washed with
PSS and equilibrated for 30 minutes. Basal NO production was inhibited
by applying l-NAME (3 × 10–4 M, 30 minutes). Dose-response curves for
SNP were then generated by stepwise addition of increasing SNP concen-
trations from 10–9 to 10–5 M.
At the end of the experiments, passive diameters were determined by
applying Ca2+-free PSS containing 2 mM EGTA. ACh and SNP relaxation
responses were expressed as percentage change in diameter after phenyl-
ephrine preconstriction compared with the difference between calcium-
free diameter and diameter after phenylephrine constriction, using the
following equation: percentage dilation = 100% × [(Dx – Di)/(DCa-free – Di)],
where D is the measured arterial diameter and subscripts x, i, and Ca-free
denote arterial diameters at each dose of agonist (x), initial diameter fol-
lowing phenylephrine constriction (i), and in Ca-free buffer (Ca-free).
Filament model of MCA occlusion. For infarct volume, we used the filament
model of MCA occlusion. Mice were anesthetized with 1.5% isoflurane in
a mixture of 30% O2 and 70% N2O. Body temperature was maintained at
37°C using a thermostatic heating blanket (FHC). A flexible fiberoptic
probe (Perimed) was affixed to the skull over the brain area supplied by the
MCA (2 mm posterior and 6–7 mm lateral to bregma) for relative blood
flow measurements by laser Doppler flowmetry. Baseline cerebral blood
flow values were measured before internal carotid artery ligation and MCA
occlusion and considered to be 100% flow. MCA occlusion was caused by
inserting an 8-0 nylon filament (Doccol Corp.) covered by silicon into the
internal carotid artery and advancing it to the origin of the MCA. Ischemia
was confirmed by reduction in blood flow to less than 20% of control val-
ues, measured by laser Doppler flowmetry. Reperfusion was confirmed by
Doppler after withdrawal of the filament.
Determination of infarct size. Infarct size was determined by staining
with 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich). Brains
were cut into 2-mm–thick coronal sections using a mouse brain matrix
(RBM-200C; Activational Systems), stained with 2% TTC for 1 hour at
37°C in the dark, and photographed with a digital camera. Sections were
analyzed using an image analysis system (MCID M4; Imaging Research).
Infarct sizes were determined by the indirect method, which corrects for
edema (contralateral hemisphere volume minus volume of nonischemic
Neurologic scoring. Mice were examined for neurologic deficits 23 hours
after MCA occlusion using a 5-point scale (31). Normal motor function
was scored as 0, flexion of the contralateral torso and forearm on lifting the
animal by the tail as 1, circling to the contralateral side but normal posture
at rest as 2, leaning to the contralateral side at rest as 3, and no spontane-
ous motor activity as 4.
Distal MCA occlusion. In order to perform laser speckle flowmetry through-
out the entire ischemic period starting 5 minutes before its onset, we chose
to occlude the MCA distally using a microvascular clip. Distal MCA occlu-
sion can be performed during laser speckle flowmetry while the animal is on
the stereotaxic frame and allows the imaging to continue uninterrupted. In
contrast, filament occlusion of MCA requires the mouse to be in the supine
position during insertion of the filament. Mice were anesthetized as above,
intubated, paralyzed (pancuronium bromide, 0.4 mg/kg i.v. given every 45
Effect of S1179A and S1179D eNOS mutations on cerebral blood flow.
(A) Laser speckle contrast images in mice subjected to MCA occlu-
sion. Superimposed areas (blue) indicate regions with ≤30% resid-
ual blood flow. Imaging field dimensions are 6 × 8 mm. Thresholded
images were recorded after 60 minutes of ischemia. (B) Composite
graph showing the area of cortex with ≤20% (black) or 21–30% (blue)
residual blood flow compared with preischemic baseline. *P < 0.05,
comparing the area with ≤20% of residual blood flow of eNOS KO
and S1179A/eNOS KO mice versus WT and S1179D/eNOS KO mice.
CBF, cerebral blood flow.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 117 Number 7 July 2007
minutes), mechanically ventilated (model SAR-830 ventilator, CWE), and
placed in a stereotaxic frame (Kopf). Arterial blood pressure, blood gases,
and pH were monitored every 30 minutes. The temporalis muscle was sepa-
rated from the temporal bone and removed. A burr hole (2 mm diameter)
was drilled under saline cooling in the temporal bone overlying the MCA
just above the zygomatic arch. The dura was kept intact, and the MCA was
occluded using a microvascular clip (Zen clip; Oswa).
Laser speckle imaging of cerebral blood flow. For continuous monitoring of
cerebral blood flow and ischemic area, a laser diode (780 nm) with a pen-
etration depth of 500 μm was used to illuminate the intact skull surface.
Raw speckle images were acquired with a video camera (Cohu) and used
to compute speckle contrast, a measure of speckle visibility related to the
velocity of the scattering particles and therefore cerebral blood flow. Ten
consecutive raw speckle images were acquired at 15 Hz and processed
to speckle contrast using a sliding grid of 7 × 7 pixels. Contrast images
were converted to correlation time values, and relative cerebral blood flow
images were calculated. Perfusion images were obtained every 7.5 seconds
starting before ischemia and continuing throughout the experiment. The
areas of severe (0%–20% residual blood flow, representing core ischemic
areas) and moderate (21%–30% residual blood flow, representing penum-
bra) blood flow deficits were quantified (mm2) by using a thresholding
paradigm as previously described (23).
Statistics. All results are expressed as mean ± SD. Responses to dilators
were calculated as percentage reversal of induced tone or constriction.
Statistical analysis was performed using 1-way ANOVA and post hoc test
for infarct size. The area of cerebral blood flow deficit was analyzed using
2-way ANOVA for repeated measures. Statistical analysis for neurological
deficit was performed using Kruskal-Wallis 1-way analysis of variance on
ranks. Differences of P < 0.05 were considered significant.
This work was supported by US Public Health Service grants
NS10828, NS033335, HL057818, and HL048426 (to P.L. Huang).
Received for publication July 27, 2006, and accepted in revised
form April 10, 2007.
Address correspondence to: Paul L. Huang, Cardiovascular Research
Center, Massachusetts General Hospital, 149 Thirteenth Street,
Room 4101, Charlestown, Massachusetts 02129, USA. Phone: (617)
724-9849; Fax: (617) 726-5806; E-mail: email@example.com.
Dmitriy N. Atochin and Annie Wang contributed equally to this
1. Bredt, D.S., and Snyder, S.H. 1994. Nitric oxide:
a physiologic messenger molecule. Annu. Rev. Bio-
2. Moncada, S., and Higgs, A. 1993. The L-arginine-
nitric oxide pathway. N. Engl. J. Med. 329:2002–2012.
3. Dudzinski, D.M., Igarashi, J., Greif, D., and Michel,
T. 2006. The regulation and pharmacology of
endothelial nitric oxide synthase. Annu. Rev. Phar-
macol. Toxicol. 46:235–276.
4. Moncada, S. 2006. Adventures in vascular biology:
a tale of two mediators. Philos. Trans. R. Soc. Lond. B
Biol. Sci. 361:735–759.
5. Alderton, W.K., Cooper, C.E., and Knowles, R.G.
2001. Nitric oxide synthases: structure, function
and inhibition. Biochem. J. 357:593–615.
6. Huang, P.L. 2005. Unraveling the links between
diabetes, obesity, and cardiovascular disease. Circ.
7. Shaul, P.W. 2002. Regulation of endothelial nitric
oxide synthase: location, location, location. Annu.
Rev. Physiol. 64:749–774.
8. Dimmeler, S., et al. 1999. Activation of nitric oxide
synthase in endothelial cells by Akt-dependent
phosphorylation. Nature. 399:601–605.
9. Fulton, D., et al. 1999. Regulation of endothelium-
derived nitric oxide production by the protein
kinase Akt. Nature. 399:597–601.
10. Hisamoto, K., et al. 2001. Estrogen induces the Akt-
dependent activation of endothelial nitric-oxide
synthase in vascular endothelial cells. J. Biol. Chem.
11. Kureishi, Y., et al. 2000. The HMG-CoA reductase
inhibitor simvastatin activates the protein kinase
Akt and promotes angiogenesis in normocholester-
olemic animals. Nat. Med. 6:1004–1010.
12. Scotland, R.S., et al. 2002. Functional reconstitu-
tion of endothelial nitric oxide synthase reveals the
importance of serine 1179 in endothelium-depen-
dent vasomotion. Circ. Res. 90:904–910.
13. Vecchione, C., et al. 2002. Leptin effect on
endothelial nitric oxide is mediated through Akt-
endothelial nitric oxide synthase phosphorylation
pathway. Diabetes. 51:168–173.
14. Chen, H., Montagnani, M., Funahashi, T., Shi-
momura, I., and Quon, M.J. 2003. Adiponectin
stimulates production of nitric oxide in vascular
endothelial cells. J. Biol. Chem. 278:45021–45026.
15. Shen, Y.H., et al. 2006. Up-regulation of PTEN
(phosphatase and tensin homolog deleted on chro-
mosome ten) mediates p38 MAPK stress signal-
induced inhibition of insulin signaling. A cross-
talk between stress signaling and insulin signaling
in resistin-treated human endothelial cells. J. Biol.
16. Kim, F., Gallis, B., and Corson, M.A. 2001. TNF-
alpha inhibits flow and insulin signaling leading
to NO production in aortic endothelial cells. Am. J.
Physiol. Cell Physiol. 280:C1057–C1065.
17. McCabe, T.J., Fulton, D., Roman, L.J., and Sessa,
W.C. 2000. Enhanced electron flux and reduced
calmodulin dissociation may explain “calcium-
independent” eNOS activation by phosphoryla-
tion. J. Biol. Chem. 275:6123–6128.
18. Garcin, E.D., et al. 2004. Structural basis for isozyme-
specific regulation of electron transfer in nitric-oxide
synthase. J. Biol. Chem. 279:37918–37927.
19. Robinson, L.J., Weremowicz, S., Morton, C.C., and
Michel, T. 1994. Isolation and chromosomal local-
ization of the human endothelial nitric oxide syn-
thase (NOS3) gene. Genomics. 19:350–357.
20. Guillot, P.V., et al. 1999. A vascular bed-specific
pathway. J. Clin. Invest. 103:799–805.
21. Huang, P.L., et al. 1995. Hypertension in mice lack-
ing the gene for endothelial nitric oxide synthase.
22. Huang, Z., et al. 1996. Enlarged infarcts in
endothelial nitric oxide synthase knockout mice
are attenuated by nitro-L-arginine. J. Cereb. Blood
Flow Metab. 16:981–987.
23. Ayata, C., et al. 2004. Laser speckle flowmetry for
the study of cerebrovascular physiology in normal
and ischemic mouse cortex. J. Cereb. Blood Flow
24. Chen, Z.P., et al. 1999. AMP-activated protein
kinase phosphorylation of endothelial NO syn-
thase. FEBS Lett. 443:285–289.
25. Butt, E., et al. 2000. Endothelial nitric-oxide syn-
thase (type III) is activated and becomes calcium
independent upon phosphorylation by cyclic
nucleotide-dependent protein kinases. J. Biol. Chem.
26. Montagnani, M., Chen, H., Barr, V.A., and Quon,
M.J. 2001. Insulin-stimulated activation of
eNOS is independent of Ca2+ but requires phos-
phorylation by Akt at Ser (1179). J. Biol. Chem.
27. Akiyama, M., et al. 2002. Expression and func-
tion of recombinant S1179D endothelial nitric
oxide synthase in canine cerebral arteries. Stroke.
28. Sorenson, J., et al. 2005. Expression and function of
recombinant S1179D endothelial NO synthase in
human pial arteries. Stroke. 36:158–160.
29. Cook, S., et al. 2004. Partial gene deletion of
endothelial nitric oxide synthase predisposes to
exaggerated high-fat diet-induced insulin resistance
and arterial hypertension. Diabetes. 53:2067–2072.
30. Cook, S., et al. 2003. Clustering of cardiovascu-
lar risk factors mimicking the human metabolic
syndrome X in eNOS null mice. Swiss Med. Wkly.
31. Huang, Z., et al. 1994. Effects of cerebral ischemia
in mice deficient in neuronal nitric oxide synthase.