Free Fatty Acid Causes Leukocyte Activation and Resultant
Endothelial Dysfunction Through Enhanced Angiotensin II
Production in Mononuclear and Polymorphonuclear Cells
Yoko Azekoshi, Takanori Yasu, Saiko Watanabe, Tatsuya Tagawa, Satomi Abe, Ken Yamakawa,
Yoshinari Uehara, Shinichi Momomura, Hidenori Urata, Shinichiro Ueda
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Abstract—Release of free fatty acid (FFA) from adipose tissue is implicated in insulin resistance and endothelial
dysfunction in patients with visceral fat obesity. We demonstrated previously that increased FFA levels cause
endothelial dysfunction that is prevented by inhibition of the renin-angiotensin system (RAS) in humans. However, the
mechanisms for FFA-mediated activation of RAS and the resultant endothelial dysfunction were not elucidated. We
investigated effects of elevated FFA on activity of circulating and vascular RAS, angiotensin II–forming activity of
leukocytes, and leukocyte activation of normotensive subjects. We showed that increased FFA levels significantly
enhanced angiotensin II–forming activity in human mononuclear (mean fold increase: 3.5 at 180 minutes; P⫽0.0016)
and polymorphonuclear (2.0; P⫽0.0012) cells, whereas parameters of the circulating and vascular RAS were not
affected. We also showed that FFA caused angiotensin II– dependent leukocyte activation, which impaired endothelial
function partly via increased myeloperoxidase release and presumably enhanced adhesion of leukocytes. We propose
that the enhanced production of angiotensin II by FFA in mononuclear and polymorphonuclear cells causes activation
of leukocytes that consequently impairs endothelial function. RAS in leukocytes may regulate the leukocyte-vasculature
interaction as the mobile RAS in humans. (Hypertension. 2010;56:136-142.)
Key Words: FFA 䡲 angiotensin 䡲 leukocyte 䡲 endothelial function
T
he levels of circulating free fatty acid (FFA), mainly
originating from lipolysis in adipose tissue, are increased
in patients with metabolic syndrome and type 2 diabetes
mellitus,1–3 reflecting resistance to the antilipolytic action of
insulin. Increased plasma FFA concentrations cause endothelial dysfunction,4 insulin resistance,5 and endothelial apoptosis.6 These observations, together with results from epidemiological studies, 7,8 suggest that FFA is involved in
atherosclerosis in subjects with insulin resistance. Recently,
we have found that FFA-induced endothelial dysfunction is
prevented by the inhibition of the renin-angiotensin (Ang)
system (RAS) in humans,9 suggesting that RAS activation by
FFA may predominantly contribute to FFA-induced endothelial dysfunction. This hypothesis appears plausible because of
the close relationship between obesity and RAS activity in
humans.10,11 In addition, RAS activation is also associated
with enhanced oxidative stress,12 which is an intermediary
mechanism by which FFA adversely alters vascular function.13 However, although the proatherogenic action of excessive Ang II has been well documented, there is little
information regarding the mechanism of RAS activation in
individuals with obesity. Indeed, only a few studies have
investigated the effects of elevated FFA on RAS activity.14
The aim of the present study was to investigate effects of
elevated FFA on RAS and to elucidate mechanisms for
FFA-induced endothelial dysfunction in humans. We also
investigated the interaction between FFA and leukocytes,
because FFA is involved in leukocyte activation through
protein kinase C redistribution15 and because leukocytes are
involved in the development of atherosclerosis. We demonstrate here that elevated FFA enhances Ang II production in
mononuclear and polymorphonuclear cells without any effect
on the circulating RAS and causes Ang-dependent leukocyte
activation, which leads to endothelial dysfunction.
Methods
Subjects
The study subjects were 49 healthy men (20 to 36 years old). None
of the subjects were taking medications on a regular basis. Subjects
had normal results from a routine physical examination and standard
Received March 10, 2010; first decision April 1, 2010; revision accepted April 28, 2010.
From the Department of Clinical Pharmacology and Therapeutics (Y.A., T.Y., S.W., T.T., K.Y., S.U.), University of the Ryukyus School of Medicine,
Okinawa, Japan; Department of Cardiovascular Diseases (S.A., Y.U., H.U.), Fukuoka University Chikushi Hospital, Fukuoka, Japan; Department of First
Integrated Medicine (S.M.), Saitama Medical Center, Jichi Medical University, Saitama, Japan; Current address (T.T.): Department of Nutritional
Sciences, Faculty of Health and Welfare, Seinan Jo Gakuin University, Kitakyushu-shi, Japan.
Y.A. and T.Y. are joint first authors with equal contributions to this work.
Correspondence to Shinichiro Ueda, Department of Clinical Pharmacology and Therapeutics, University of the Ryukyus School of Medicine, Okinawa
903-0215, Japan. E-mail [email protected]
© 2010 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.110.153056
136
Azekoshi et al
Endothelial Dysfunction by FFA and Angiotensin II
137
laboratory tests. All of the participants gave written informed
consent. The ethics committee of the University of the Ryukyus
approved the study protocol.
Study Protocols
Lipid/Heparin Infusion
This protocol was performed on 2 days in 8 normotensive men with
an interval of ⱖ7 days between the study days. We measured
changes in forearm blood flow (FBF) during intra-arterial infusion of
acetylcholine at 50, 100, 200, and 400 nmol/min and sodium
nitroprusside at 3, 10, and 30 nmol/min before and after a 3-hour
lipid/heparin infusion. This experiment was repeated 4 hours after a
single 50-mg oral dose of losartan. Our preliminary experiment
showed that there was no order effect of vasodilatation to acetylcholine and that losartan had no effect on endothelial function in healthy
men in the absence of lipid infusion.
Participants fasted overnight and abstained from alcohol or caffeine
for ⱖ12 hour before the study. The participants came to our
laboratory at 9:00 AM and received a continuous infusion of an
intravenous fat emulsion (Intralipid 20%, Fresenius Kabi AB) at 90
mL/h to increase their serum FFA concentrations. Heparin (Shimizu
Pharmaceutical Co, Ltd.) at 0.3 U/kg per minute was coinfused to
activate lipoprotein lipase and thereby catalyze the hydrolysis of
triglyceride.
Preparation of Palmitate and Oleate for Ex
Vivo Experiments
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Palmitate (Chem Service S-33) and oleate (Wako Pure Chemical
Industries 153-01241) solutions were prepared as described elsewhere16 with minor modifications. In brief, sodium palmitate and
oleic acid were dissolved to 50 mmol/L in 0.1 N NaOH 70% ethanol
at 70°C. FFA solutions were stored in small aliquots at ⫺80°C. FFA
preparations were thawed and complexed with FFA-free, lowendotoxin BSA (Sigma A8806 –5G) before use.
Measurement of Plasma FFA Concentration
Plasma FFA concentration was measured before and after 1- and
3-hour lipid/heparin infusions in 8 healthy men. Blood samples for
FFAs were placed in tubes containing 50 ␮L of paraoxon (diethyl
p-nitrophenyl phosphate; Sigma Chemical Company) diluted to
0.04% in diethyl ether to prevent ex vivo lipolysis.17 Samples were
centrifuged immediately at 4°C, and the plasma obtained from these
tubes was stored at ⫺80°C until assay.
Fluorogenic Enzymatic Assay for Ang
II–Forming Activity
Ang II–forming activity in mononuclear and polymorphonuclear
cells was measured according to the previously described method18
with minor alterations. A modified Ang I substrate was developed by
the addition of a fluorescent tag, N-methylanthranilic acid, at the N
terminal and 2,4-dinitrophenyl (Dnp) at the C terminal of Ang I
(Peptide Institute, Inc). Ang II–forming enzymes cleave between the
Phe and His residues, permitting the release of His-Leu-Dnp, the
quenching residue. Then, fluorescence was detected with excitation
at 355 nm and emission at 460 nm. The reaction yielded
N-methylanthranilic acid-Ang II that was measured by using the
synthesized N-methylanthranilic acid-Ang II as a standard. The final
Ang II–forming activity data were represented as the picomole yield
of N-methylanthranilic acid-Ang II per minuter per milligram of
protein. The intra-assay and interassay coefficients (n⫽12 each)
were 3.6% and 5.8% for total Ang II–forming activity.
Assessment of Leukocyte Adhesiveness and
Deformability by Ex Vivo Microchannel
Capillary Model
We used a microchannel flow analyzer (HR200, Kowa Co, Ltd) as an
ex vivo capillary model to assess adhesiveness and deformability of
leukocytes in whole blood.19 –21 Within 10 minutes after blood
collection into a heparinized tube, 0.1 mL of blood was drawn
through the narrow microchannels (7854-parallel, 7-␮m equivalent
diameter, 20-␮m long channels) under a constant suction of 20
cm H2O. The microscopic motion images of blood passing through
the microditches were monitored and stored on a computer system.
In offline analysis, an investigator blinded to treatments randomly
selected 5 different still images when 0.08 to 0.10 mL of blood flew
out and then counted the number of adherent or plugging leukocytes
on the microchannel terrace. Adherent leukocytes were defined as
static leukocytes with clear surface borders on still images.
Effect of a Single Dose of Losartan on FFA-Induced
Endothelial Dysfunction
Effects of Increased FFA on Circulating RAS
Lipid with heparin was infused for 3 hours, and blood samples were
collected before the infusion and 1 and 3 hours after the commencement of the infusion in 15 healthy men. Plasma renin activity, serum
Ang-converting enzyme activity, plasma concentration of Ang I and
II, and aldosterone were measured.
Effects of Increased FFA on Forearm Vascular
Responses to Ang I and II
To assess the effects of increased FFA levels on vascular RAS (ie,
Ang-converting enzyme activity in vascular endothelial cells and the
sensitivity of vascular smooth muscle cells to Ang II), we measured
changes in FBF by strain gauge plethysmography during intra-arterial infusions of Ang I and II at 0.5, 1.0, 5.0, and 10.0 pmol/min with
or without systemic lipid/heparin infusion in 10 normotensive men.
Effects of Increased FFA on Ang II–Forming Activity
This protocol was performed on 2 days with an interval of ⱖ7 days
between them. Fifteen normotensive subjects received a 3-hour
infusion of lipid/heparin or saline/heparin as a control at the same
rate in an open-label, randomized, crossover design. Blood samples
for the measurement of Ang II–forming activity in mononuclear and
polymorphonuclear cells were collected in tubes containing EDTA2Na before and 60 and 180 minutes after the start of the infusion.
Ang II–forming activity in leukocytes was also measured after
incubation of fresh whole blood drawn from 5 healthy men with 0.2
or 0.4 mmol/L of oleate/palmitate mixture (1:1 ratio) or vehicle (2%
BSA) at 37°C for 30 minutes.
Effects of Increased Serum FFA on Leukocyte Activity
This protocol was performed on 3 days in 8 normotensive men with
ⱖ7 days in a washout period between study days. First, blood
samples were collected to assess leukocyte activity using the
microchannel flow analyzer and determining the plasma levels of
myeloperoxidase (MPO) before and 60 and 120 minutes after
starting a systemic infusion of saline/heparin (study day 1) and
lipid/heparin (study day 2 or 3). Immediately after blood sampling at
120 minutes, bolus heparin (70 U/kg of body weight) was injected to
release vessel wall–immobilized MPO into the circulating
blood.22–24 Each subject received 160-mg oral doses of valsartan
(Novartis Pharmaceutical Co, Ltd) or placebo at 28 and 4 hours
before the experiment in a double-blind crossover design on study
day 2 or 3. The order of the pretreatment was randomized. As a
parameter of newly released MPO from activated leukocytes by FFA
provocation, we calculate ␦MPO⫽(serum MPO after bolus injection
of heparin during FFA provocation)⫺(serum MPO after bolus
injection of heparin during normal saline infusion). Leukocyte
adhesiveness was also assessed after incubation of fresh whole blood
from 5 healthy men with 0.2 or 0.4 mmol/L of oleate/palmitate
mixture (1:1 ratio) or vehicle (2% BSA) at 37°C for 30 minutes. This
experiment was repeated after the oral administration of valsartan in
the same subjects.
Effects of Increased Serum FFA and a Bolus Injection of
Heparin on FBF in Response to Acetylcholine
Eight healthy men participated in this protocol, which was performed
on 3 days with an interval of ⱖ7 days among them. Endothelial
138
Hypertension
July 2010
Forearm Blood Flow
(ml/min/100ml)
35
With saline/heparin infusion
With lipid/heparin infusion
30
25
Figure 1. Dose-response curves of intraarterial infusion of acetylcholine after
saline/heparin infusion (䡺) and lipid/heparin infusion (f) with (left) and without (right)
a single dose of losartan (*P⬍0.0001).
*
20
15
10
After losartan
5
0
50
0
100
200
400
50
0
100
200
400
Dose of Acetylcholine (nmol/min)
Results
Plasma FFA concentration before and after 1-hour and 3-hour
lipid/heparin infusions were 0.54⫾0.23, 1.56⫾0.30, and
1.97⫾0.55 milliequivalents/L (n⫽8), respectively.
Statistical Analysis
Role of Ang II in FFA-Induced
Endothelial Dysfunction
Data are presented as the mean⫾SD unless otherwise indicated.
Probability values ⬍0.05 were considered to be statistically significant. Comparisons of the dose-response curves of acetylcholine,
Ang I, and Ang II were made by repeated measure of ANOVA.
Comparison of each parameter of the circulating RAS before and
after lipid/heparin infusion and comparison of Ang II–forming
activity after the lipid infusion with that after the control infusion
were also made by repeated measure of ANOVA. The number of
adhesive leukocytes and plasma concentration of MPO were analyzed by ANOVA with a post hoc multiple comparison test.
PMNC Ang II FA (fold increase)
As we reported previously,10 increased FFA levels significantly attenuated vasodilatation to acetylcholine (P⬍0.0001,
ANOVA; Figure 1). This FFA-induced endothelial dysfunction was prevented by a single 50-mg dose of losartan (Figure
1). Lipid/heparin infusion did not significantly affect blood
pressure and baseline FBF. Elevated FFA did not affect the
FBF response to sodium nitroprusside either with or without
losartan (data are not shown).
8
6
4
2
0
Baseline
60 min
B
2.0
*
1 .5
1.0
0 .5
0
Vehicle
8
6
4
2
0
180 min
FFA 0.2 mM FFA0.4 mM
Baseline
PMNC Ang II FA (fold increase)
MNC Ang II FA (fold increase)
A
MNC Ang II FA (fold increase)
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function was evaluated by the vasodilation to intra-arterial infusion
of acetylcholine after a 1-hour systemic infusion of saline/heparin as
a control (day 1) and of lipid/heparin followed by bolus heparin or
vehicle (day 2 or 3). We initiated the FBF measurements 15 minutes
after bolus heparin or vehicle. The investigators who assessed
endothelial function were blinded to treatments.
60 min
180 min
2.0
*
1 .5
1.0
0 .5
0
Vehicle
FFA 0.2 mM
FFA0.4 mM
Figure 2. Ang II–forming activity in mononuclear (left) and polymorphonuclear (right) cells after a 180-minute lipid/heparin infusion (f)
and the activity after the saline/heparin infusion (䡺; P⫽0.0016 and P⫽0.0012; A). Ang II–forming activity in mononuclear (left) and polymorphonuclear (right) cells after the incubation with 0.2 or 0.4 mmol/L of oleate/palmitate mixture (1:1 ratio) or vehicle (2% BSA) at
37°C for 30 minutes (B; n⫽5; *P⬍0.05 vs vehicle).
Azekoshi et al
B
Adherent leuko
ocyte number (/P
PHF)
A
Endothelial Dysfunction by FFA and Angiotensin II
NS + heparin
10 µm
Placebo + lipid/heparin
25
*
20
15
15
10
10
5
5
*
#
#
0
0
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Plasma Myeloperoxidase level (ng/ml)
P
**
#
20
D
Valsartan + lipid/heparin
C
*
25
139
Baseline
60 min
120 min
Vehicle
FFA 0.2
mM
FFA 0.4
mM
200
Lipid/heparin or NS/heparin div
150
*
100
50
0
NS+heparin
Placebo+lipid/heparin
Valsartan+lipid/heparin
0
60 min
120 min
Time
15 m after
bolus heparin
(70 U/kg) iv
Figure 3. Representative microphotographs of adherent or plugging leukocytes in whole blood subjected to an ex vivo microchannel
flow analyzer after a 2-hour intravenous infusion of normal saline (NS)/heparin (top), placebo⫹lipid/heparin (middle), or valsartan (160
mg/d for 2 days)⫹lipid/heparin (bottom; A). Arrows denote adherent or plugging leukocytes. Scale bar, 10 ␮m. The adherent leukocyte
number after the saline/heparin infusion (䡺), the lipid/heparin infusion and pretreatment with placebo (f), or valsartan ( ; B). *P⬍0.05
vs NS/heparin; #P⬍0.05 vs lipid/heparin. The adherent leukocyte number after the incubation of fresh whole blood collected from subjects after no pretreatment (f) and valsartan ( ) with 0.2 or 0.4 mmol/L of oleate/palmitate mixture or vehicle (2% BSA) at 37°C for 30
minutes (n⫽5; C). *P⬍0.05 and **P⬍0.01 vs no pretreatment with vehicle; #P⬍0.05 vs no pretreatment with each concentration of
oleate/palmitate mixture. Serial changes in plasma MPO levels before and during intravenous infusion of saline/heparin (Œ),
placebo⫹lipid/heparin (F), and valsartan⫹lipid/heparin (䡺); n⫽8 per protocol; *P⬍0.05; D).
Increased FFA Levels Do Not Affect the Activity
of Circulating RAS
Effect of FFA Levels on Leukocyte Activation and
the Role of RAS
There was no significant effect of increased FFA levels on
any component of the circulating RAS (please see Table
S1, available in the online Data Supplement at
http://hyper.ahajournals.org).
The lipid/heparin infusion significantly increased the number of
adherent or plugging leukocytes (Figure 3A and B). Pretreatment with valsartan significantly inhibited (P⬍0.05) the FFAinduced leukocyte activation at 120 minutes (Figure 3B). The
number of adherent or plugging leukocytes increased in a
dose-dependent manner after a 30-minute incubation with the
oleate/palmitate mixture (Figure 3C). Pretreatment with valsartan significantly inhibited (P⬍0.05) leukocyte activation by the
oleate/palmitate mixture (Figure 3C). MPO concentration after
the FFA provocation and the bolus heparin was significantly
higher than that after the control infusion and the bolus heparin
(P⬍0.05 by ANOVA), and the ␦serum MPO was significantly
decreased by pretreatment with valsartan (Figure 3D).
Effect of FFA on Vascular Response to Ang I
and II
Increased FFA levels did not significantly affect the doseresponse curves of Ang I or II (Figure S1).
Effect of FFA on Ang II–Forming Activity in
Mononuclear and Polymorphonuclear Cells
Increased FFA levels significantly enhanced the total Ang
II–forming activity at each time point in mononuclear
(P⫽0.0016) and polymorphonuclear (P⫽0.0012) cells,
whereas the infusion of saline/heparin had no effect (Figure
2A). The incubation of whole blood with FFA ex vivo
significantly enhanced the Ang II–forming activity in both
cells (Figure 2B).
Effects of Liberation of MPO From Endothelial Cells
by the Bolus Heparin on Endothelial Function
The FFA-induced endothelial dysfunction was restored by the
bolus heparin injection, which was deemed to liberate MPO
from endothelial cells (Figure 4).
140
35
Hypertension
July 2010
Forearm Blood Flow
(ml/min/100ml)
30
*
25
20
15
10
NS100ml/h+heparin0.3U/kg/min
Lipid/heparin1h
Lipid/heparin1h+heparin70U/kgbolusiv
5
0
0
100
200
300
400
Dose of Acetylcholine (nmol/min)
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Figure 4. Dose-response curves of acetylcholine after a 1-hour
intravenous infusion of NS/heparin (Œ), of lipid/heparin followed
by bolus heparin (F), and after bolus saline (E; *P⬍0.05).
Discussion
FFA released from expanded adipose tissue is a key adipocytokine in the facilitation of endothelial dysfunction and
insulin resistance in individuals with visceral fat obesity.
However, the mechanism by which FFA contributes to
endothelial dysfunction and subsequent atherosclerosis in
humans is unclear. Here, we show that FFA-induced enhancement of Ang II production in mononuclear and polymorphonuclear cells is a key mechanism for leukocyte
activation associated with high FFA concentration, which
leads to endothelial dysfunction, in part through the immobilized MPO on endothelium and oxidative stress, although
the possibility of a direct effect of FFA on endothelial cell NO
synthase cannot be excluded.25 These results suggest that
FFA activates RAS in leukocytes, and the activated RAS
modulates the dysfunctional interaction between endothelium
and leukocytes, which is believed to be a crucial early step in
the development of atherosclerosis.
We demonstrated that increased FFA levels significantly
shift the dose-response curve of acetylcholine toward the
right without any changes in that of the sodium nitroprusside and that an Ang II type 1 blocker prevented such
vascular dysfunction.9 Our results strongly suggest that
activated RAS predominantly contributes to FFA-induced
endothelial dysfunction; however, the specific RAS systems that are involved must be identified. The experiments
in the present study have partly identified these RAS
systems.
First, we investigated the effects of FFA on circulating
RAS. Previous evidence has suggested an association
between obesity and activated circulating RAS in humans.10,11 Given the evidence for abnormal FFA metabolism in visceral fat obesity,26 it can be assumed that
long-term exposure to high FFA levels might affect the
circulating RAS activity. Short-term elevation of FFA for
a few hours, however, did not affect the activity of
circulating RAS in the present study. Even a longer
duration of lipid infusion may not have affected the results,
because Umpierrez et al14 have found no significant
increment in plasma renin activity and aldosterone concentration after a 48-hour lipid/heparin infusion. These
results suggest that the FFA-induced endothelial dysfunction might not be mediated by the activation of
circulating RAS.
We then hypothesized that vascular RAS activity would be
activated and modulate vascular function. Indeed, Nielsen et
al27 showed a significant correlation between the vascular
response to Ang II and the plasma palmitate concentration. In
terms of Ang II–forming activity from Ang I, Barton et al28
showed the activation of renal Ang-converting enzyme activity in obese animals. Increased FFA levels, however, did not
alter the FBF response to either Ang I or II in the present
study. This result suggests that acutely increased FFA levels
do not enhance Ang II production from Ang I by Angconverting enzyme in vascular endothelial cells and in the
circulation. The lack of an enhanced response to Ang II may
also indicate that acute short-term increases in FFA levels do
not enhance the sensitivity of the Ang II type 1 receptor in
vascular smooth muscle cells. However, the assessment of the
effect of FFA on the sensitivity of the vasculature to Ang II
seems complex because it has been suggested that some FFAs
inhibit the binding of Ang II to its receptor.29
Another local RAS that is relevant to the pathophysiology
of endothelial dysfunction is the RAS in leukocytes. We
showed that enzymes responsible for Ang II production were
functionally present in human mononuclear and polymorphonuclear cells and significantly enhanced by increased FFA
levels. Because human leukocytes contain all components of
RAS, enhanced activity of Ang II–forming enzyme is assumed to result in the enhanced production and release of
Ang II in the presence of sufficient amounts of substrates.
Therefore, one simple and plausible explanation for FFAinduced, RAS-mediated endothelial dysfunction is the enhancement of NADPH oxidase activity and reactive oxygen
species production in endothelial cells by Ang II from
leukocytes. In fact, we demonstrated previously that vitamin
C prevented FFA-induced endothelial dysfunction in humans9 and that palmitate enhanced reactive oxygen species
production and NADPH oxidase expression.13 However, the
enhanced release of Ang II from leukocytes, if any, was not
detectable as changes in the plasma concentration of Ang II,
the systemic blood pressure, or the forearm vascular tone in
the present study, which raises doubts that the increased Ang
II production in leukocytes could be functionally relevant to
vascular function.
We then assumed that FFA could affect leukocyte
functions other than Ang II–forming activity through
increased Ang II production. We clearly demonstrated that
increased FFA levels significantly promoted the adhesion
of leukocytes and enhanced MPO release presumably from
neutrophils and monocytes, which both were partially
prevented by previous administration of Ang II type 1
blocker. Furthermore, we showed that incubation of an
FFA mixture with blood directly caused leukocyte activation and that the previous administration of valsartan
significantly attenuated this reaction. These results
Azekoshi et al
Endothelial Dysfunction by FFA and Angiotensin II
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
strongly suggest that increased Ang II production in white
blood cells is relevant to leukocyte activation and not a
paraphenomenon and that valsartan inhibits FFA-induced
leukocyte activation via the leukocyte Ang II type 1
receptor. It can also be proposed, therefore, that Ang II has
an autocrine/paracrine effect in white blood cells.
In terms of the integration of leukocytes, RAS, and
endothelial function, MPO may be one of the key players.
We showed that the liberation of MPO from endothelial
cells by bolus injection of heparin restored the FFAinduced endothelial dysfunction. These results, together
with clear experimental evidence that MPO impairs NO
availability in an H2O2-dependent manner,23 suggest that
RAS-mediated leukocyte activation by FFA may facilitate
endothelial dysfunction partly through the release of MPO
and possibly enhanced leukocyte adhesion. It is reasonably
assumed that the release of cytokines and reactive oxygen
species from activated T lymphocytes, which has been
shown to be Ang dependent,30 may also adversely modulate vascular function.31
The extrapolation of our results to patients with visceral fat
obesity is not necessarily straightforward. Individuals with
visceral fat obesity have chronically elevated FFA levels.
However, we studied healthy subjects after an acute (not
chronic) elevation of FFA. Further investigations in patients
with chronically elevated concentrations of FFA are apparently warranted.
In conclusion, increased FFA levels caused by lipid/
heparin infusion in humans, which mimic the lipid profile of
individuals with visceral fat obesity and insulin resistance,
significantly enhanced Ang II–forming activity in mononuclear and polymorphonuclear cells. This enhancement was
implicated in FFA-induced endothelial dysfunction as a
mobile RAS presumably through leukocyte activation.
Perspectives
Our present results may provide a better understanding of the
underlying mechanisms for the development of atherosclerosis in subjects with visceral fat obesity and in patients with
established diabetes mellitus whose FFA levels are assumed
to be high. In particular, we propose that leukocyte RAS
activation plays a pivotal role in the development of endothelial dysfunction in subjects with high FFA levels as the
mobile RAS in humans, which may be a future therapeutic
target.
Acknowledgments
We thank Mayumi Kobayashi and Yoko Tagomori for their technical
assistance in the assay for Ang II–forming activity in leukocytes.
Sources of Funding
This work was supported in part by Grants-in-Aid from the Ministry
of Education, Science, and Culture of Japan (16590439 and
13670734 to S.U., 21590916 to H.U., and 20590542 to K.Y.).
Disclosures
None.
141
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Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Free Fatty Acid Causes Leukocyte Activation and Resultant Endothelial Dysfunction
Through Enhanced Angiotensin II Production in Mononuclear and Polymorphonuclear
Cells
Yoko Azekoshi, Takanori Yasu, Saiko Watanabe, Tatsuya Tagawa, Satomi Abe, Ken
Yamakawa, Yoshinari Uehara, Shinichi Momomura, Hidenori Urata and Shinichiro Ueda
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Hypertension. 2010;56:136-142; originally published online June 7, 2010;
doi: 10.1161/HYPERTENSIONAHA.110.153056
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Online Supplement
FFA causes leukocyte activation and resultant endothelial dysfunction through
enhanced angiotensin II production in mononuclear and polymorphonuclear cells
Yoko Azekoshi MD1**, Takanori Yasu MD1**, Saiko Watanabe MD1, Tatsuya Tagawa
MD1*, Satomi Abe BS2, Ken Yamakawa MD1, Yoshinari Uehara MD2, Shinichi
Momomura MD3, Hidenori Urata MD2, Shinichiro Ueda MD1
1
Department of Clinical Pharmacology & Therapeutics, University of the Ryukyus
School of Medicine, Okinawa, Japan, 2Department of Cardiovascular Diseases,
Fukuoka University Chikushi Hospital, Fukuoka, Japan, 3Department of First Integrated
Medicine, Saitama Medical Center, Jichi Medical University, Saitama, Japan.
*Present address: Department of Nutritional Sciences, Faculty of Health and Welfare,
Seinan Jo Gakuin University, Kitakyushu-shi, Japan
**Co-first authors with equal contributions to this work
Running title: Endothelial dysfunction by FFA and angiotensin II
Correspondence: Dr. Shinichiro Ueda
Department of Clinical Pharmacology & Therapeutics
University of the Ryukyus School of Medicine, Okinawa 903-0215, Japan
Tel: +81-98-895-1195; Fax: +81-98-895-1447
E-mail: [email protected]
Expanded Methods
Measurement of forearm blood flow (FBF) by strain gauge plethysmography during the
intra-arterial infusion of drugs
All experiments were performed in a quiet, temperature-controlled room (22°C - 24°C).
FBF was measured bilaterally by strain gauge, venous occlusion plethysmography
during the intra-arterial infusion of drugs including acetylcholine (Daiichi
Pharmaceutical Co., Ltd.), sodium nitroprusside, Ang I and Ang II (Delivert, Toa Eiyo,
Fukushima, Japan) through a 27-gauge needle inserted into the brachial artery, as
described previously.1
Measurement of circulating RAS activity and plasma myeloperoxidase (MPO) level
Plasma renin activity2 and plasma aldosterone concentrations3 were measured by
standardized radioimmunoassay. Serum ACE activity was measured by the standardized
method with artificial substrate.4 Plasma concentrations of Ang I and II were measured
by radioimmunoassay.5 Plasma levels of MPO were measured by an enzyme-linked
immunosorbent assay kit (Bio Check, Inc., CA).
Cell isolation
Mononuclear and polymorphonuclear cells were separated by gradient centrifugation
using Lymphoprep and Polymorphprep (AXS-Shield, Oslo, Norway), respectively, as
previously described.6 The mononuclear cell layers isolated by Lymphoprep contain
1%-2% polymorphonuclear cells, and the polymorphonuclear cell layers isolated by
Polymorphoprep contain 1%-4% mononuclear cells, as stated by the manufacturer. We
confirmed these purity levels in a pilot study (n=3, data not shown). Each cell fraction
was collected and rinsed with an equal volume of physiologic saline and pelleted by
centrifugation at 250 × g for 10 min at room temperature and stored at -80°C until assay.
The cell fraction was frozen on methanol/dry ice and thawed three times, then
centrifuged at 5,000 rpm for 10 min at 4°C. The pellets were resuspended in assay
buffer (10 mM Tris buffer, pH 7.4 containing 400 mM KCl and 0.01% Triton X-100)
and homogenized with a hand homogenizer on ice. The protein concentration of the
homogenate was measured by BCA Protein Assay Reagent by Pierce (Rockford, IL).
Online references
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enzyme in serum. Clin Chem. 1979;25:1259-1262.
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levels of renin-angiotensin-aldosterone, bradykinin, and catecholamines in healthy
subjects. Clin Chim Acta. 1989; 181: 197–200.
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correlation between chymase-like angiotensin II-forming activity in mononuclear cells
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Table S1. Circulating renin-angiotensin system before and after the lipid heparin/
infusion
Variables
Plasma renin activity (mg/ml/hr)
Plasma aldosterone (pg/ml)
Serum ACE activity (IU/L)
Angiotensin I (pmol/min)
Angiotensin II (pmol/min)
Baseline
2.1 ± 0.8
60min
1.5 ± 0.9
180min
1.6 ± 0.7
p
ns
105.8 ± 88.3
12.5 ± 3.1
53.6 ± 7.7
19.5 ± 4.7
87.1 ± 26.5
13.0 ± 3.1
51.1 ± 12.8
19.8 ± 5.8
89.5 ± 44.5
12.9 ± 3.1
57.0 ± 17.7
17.5 ± 4.0
ns
ns
ns
ns
0.40 ± 0.13
0.33 ± 0.11 ns
Angiotensin II/I ratio
0.37 ± 0.09
ACE, angiotensin converting enzyme;
Figure S1
Figure S1. Effect of elevated free fatty acid (FFA) on vasoconstriction to angiotensin
(ANG) I and II
Percent changes in forearm blood flow (FBF) ratio, which was calculated as the FBF of
the infused arm divided by that of the non-infused arm, during intra-arterial infusion of
ANG I (A) and ANG II (B) with saline/heparin (open circles) or lipid/heparin (closed
circles) infusion for 1 h.