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DOI:10.1161/ATVBAHA.110.216044
10, 2011;
2011;31;1169-1176; originally published online Feb Arterioscler Thromb Vasc Biol
Ng, Simon Siu Man Ng, Maik Gollasch, Xiaoqiang Yao and Yu Huang
Siu Ling Wong, Chi Wai Lau, Wing Tak Wong, Aimin Xu, Chak Leung Au, Chi Fai
Cyclooxygenase-2 Expression: A Link to Vascular Inflammation
Pivotal Role of Protein Kinase C{delta} in Angiotensin II-Induced Endothelial
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Pivotal Role of Protein Kinase C

in Angiotensin IIInduced
Endothelial Cyclooxygenase-2 Expression
A Link to Vascular Inflammation
Siu Ling Wong, Chi Wai Lau, Wing Tak Wong, Aimin Xu, Chak Leung Au, Chi Fai Ng,
Simon Siu Man Ng, Maik Gollasch, Xiaoqiang Yao, Yu Huang
ObjectiveThe purpose of this study was to examine the hypothesis that angiotensin II (Ang II) induced endothelial
cyclooxygenase-2 (COX-2) expression, which in turn mediated the generation of proinflammatory cytokines.
Methods and ResultsWestern blot analysis on primary rat endothelial cells showed Ang II induced COX-2 expression,
which was abolished by cotreatment of p38 mitogen-activated protein kinase (SB 202190) and extracellular
signalregulated kinase 1/2 (PD 98059) inhibitors. Protein kinase C

(PKC

) inhibitor (rottlerin) prevented extracellular

signalregulated kinase 1/2 phosphorylation and COX-2 expression. The pivotal role of PKC

was further supported by

a similar stimulatory effect of the PKC activator on COX-2 expression, signified by Ang IIstimulated translocation of
PKC

to the plasma membrane, and confirmed by PKC

phosphorylation at Tyr311. Small interfering RNA targeting

PKC

diminished COX-2 expression, which was further abrogated by SB 202190. Human mesenteric arteries incubated
with Ang II showed increased levels of endothelial COX-2 and monocyte chemoattractant protein-1; the former was
inhibited by SB 202190 plus rottlerin, whereas the latter was prevented by COX-2 inhibitor.
ConclusionThe present study pinpoints a novel role of PKC

in Ang IIinduced endothelial COX-2 upregulation and

identifies a COX-2-dependent proatherosclerotic cytokine monocyte chemoattractant protein-1. The findings raise the
possibility of curtailing endothelial COX-2 expression as a means of limiting or preventing vascular inflammation.
(Arterioscler Thromb Vasc Biol. 2011;31:1169-1176.)
Key Words: angiotensin II

endothelium

cyclooxygenase-2

monocyte chemoattractant protein-1

protein kinase C

A
ngiotensin II (Ang II), the most prominent vasoactive
peptide in the renin-angiotensin system, causes vascular
dysfunction through an exaggerated production of reactive
oxygen species (ROS) and vascular hypercontractility through
stimulation of Ang II type 1 receptor (AT
1
R).
1
Ang II is also a
potent inducer of the expression of proinflammatory cytokines,
such as tumor necrosis factor-, and adhesion molecules such as
vascular cell adhesion molecule-1.
24
These factors are prereq-
uisite for the initiation of atherosclerosis. In addition, Ang II
activates matrix metalloproteinases, thereby promoting cell mi-
gration and adverse vascular remodeling.
5
Ang II is thus closely
associated with diseases accompanied by vascular inflammation.
Cyclooxygenase-2 (COX-2) is minimally expressed in
healthy vascular tissues, but it is highly inducible on stimulation
by growth factors, proinflammatory cytokines, and bacterial
toxins.
68
A marked upregulation of COX-2 is reported in
inflamed vascular tissues,
9
in vascular remodeling of wire-
injured mouse femoral arteries,
10
and in human atherosclerotic
plaques.
11,12
The expression of COX-2, prostanoid synthases,
and prostaglandin receptors are upregulated in blood mononu-
clear cells and plaques of patients with carotid atherosclerosis.
11
The plasma level of prostaglandin E
2
, known to activate matrix
metalloproteinases, is augmented in patients with atherosclero-
sis.
11,12
Recent studies have shown that nonsteroidal antiinflam-
matory drugs can reduce vascular inflammation
3
and that
COX-2 inhibition is beneficial in decreasing adhesion molecule
expression in cancer cell lines.
13
Both Ang II and COX-2 are associated with vascular inflam-
mation and remodeling. However, it remains to be explored
whether COX-2 could play a direct role as a downstream
effector in mediating Ang IIinduced vascular pathogenesis. The
altered endothelial cell function is the key contributor to vascular
inflammation. In the present study, we aimed to test the
hypothesis that Ang II could induce COX-2 expression in
endothelial cells, which in turn is related to generation of
proinflammatory cytokines.
Received on: September 7, 2010; final version accepted on: January 31, 2011.
From the Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences and School of Biomedical Sciences (S.L.W., C.W.L., W.T.W.,
C.L.A., X.Y., Y.H.) and Department of Surgery (C.F.N., S.S.M.N.), Chinese University of Hong Kong, Hong Kong Special Administrative Region,
China; Department of Medicine and Department of Pharmacology and Pharmacy, University of Hong Kong, Hong Kong Special Administrative Region,
China (A.X.); Medical Clinic for Nephrology and Internal Intensive Care, Charite University Medicine Berlin, Berlin, Germany (M.G.).
Correspondence to Yu Huang, PhD, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, N.T., Hong Kong Special
Administrative Region, China (E-mail yu-huang@cuhk.edu.hk); or Siu Ling Wong, PhD, School of Biomedical Sciences, Chinese University of Hong
Kong, Hong Kong Special Administrative Region, China (E-mail christine_wong@cuhk.edu.hk).
2011 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.110.216044
1169 at Chinese University of Hong Kong on April 22, 2011 atvb.ahajournals.org Downloaded from
Methods
A Supplemental Methods section is available online at
http://atvb.ahajournals.org.
Cell Culture and Treatment
Endothelial cells were freshly cultured from rat thoracic aortae.
14
The identity of the cells was confirmed by positive staining for an
endothelial cellspecific marker, platelet endothelial cell adhesion
molecule-1 (Supplemental Figure I). On confluence, cells were serum
deprived for 24 hours and incubated with Ang II (100 nmol/L) for 8
hours unless otherwise stated. When used, inhibitors were preincubated
with the cells for 30 minutes before the addition of Ang II.
Western Blot Analysis and Reverse
TranscriptionPolymerase Chain Reaction
Protein expressions and phosphorylation levels of COX-2, COX-1,
p38 mitogen-activated protein kinase (MAPK), extracellular signal
regulated kinase 1/2 (ERK1/2), and protein kinase C (PKC) isoforms
were determined by Western blotting. COX-2 mRNA stabilization
was examined by reverse transcriptionpolymerase chain reaction.
Cellular Protein Fractionation for Translocation
Study of PKC Isoforms
Endothelial cells were incubated with 100 nmol/L Ang II for 1
minute and quickly cooled on ice to quench cellular reactions.
Cytosolic and membranous proteins were extracted with the Proteo-
Extract Subcellular Proteome Extraction Kit (Calbiochem). Nuclear
proteins were extracted with custom-made high-salt buffer. Equal
amount of proteins were subjected to SDS-PAGE.
PKC

Knockdown With Small Interfering RNA

Cells were transfected with either scramble siRNA or predesigned
specific small interfering RNA (siRNA) targeting PKC

transcripts
to detect COX-2 expression after Ang II stimulation.
Suspension Antibody Array-Based
Multiplex Immunoassay
Conditioned medium from endothelial cells treated with Ang II (100
nmol/L) for 24 hours were harvested, and the levels of interleukin-6,
tumor necrosis factor- and monocyte chemoattractant protein-1
(MCP-1) were measured with MILLIPLEX MAP rodent cytokine/
chemokine panel (Millipore) using the Bio-plex Suspension Array
System (Bio-Rad), according to the manufacturers instructions.
Ang II Infusion in Rats
Sprague-Dawley rats were infused with Ang II at 0.7 mg/kg per day
or vehicle (normal saline) for 9 days using an osmotic pump. Aortae
and renal arteries were preserved for immunohistochemical staining.
Interlobal renal arteries were used for functional evaluation of
endothelium-dependent relaxations.
Tissue Culture of Human Small
Mesenteric Arteries
Human small mesenteric arteries were treated with Ang II
(1 mol/L) for 24 hours in Dulbeccos modified Eagles medium
supplemented with 10% fetal bovine serum and 1% penicillin/
streptomycin at 37C. Arteries were preserved for cryosectioning
and immunofluorescence localization of COX-2 and MCP-1.
Immunohistochemical Staining and
Immunofluorescence Localization
Localization of COX-2 in the aortae and renal arteries fromAng IIinfused
rats was determined by immunohistochemistry. Human small mesenteric
arteries harvested after a 24-hour incubation protocol were preserved for
immunofluorescence to detect COX-2 and MCP-1 expression.
Functional Examination With Myography
Endothelium-dependent relaxations of the interlobal renal arteries
from Ang IIinfused rats were determined in myographs.
ROS Detection With
Dihydroethidium Fluorescence
Production of intracellular ROS was measured with dihydroethidium
(Molecular Probes).
Data Analysis
Results represent meansSEM of 5 to 6 separate experiments. Statis-
tical significance was determined by 2-tailed Student t test or 1-way
ANOVA followed by Bonferroni post tests when more than 2 treat-
ments were compared (GraphPad Software, San Diego, CA). A proba-
bility value of less than 0.05 was regarded as statistically different.
Results
Ang II Induces COX-2 Expression in
Endothelial Cells
COX-2 expression was undetectable in the untreated quies-
cent endothelial cells and was not induced by culturing in
serum-deprived medium. Ang II at 100 nmol/L increased
COX-2 expression, reaching a maximum after an 8-hour
incubation, and this effect was concentration dependent (3 to
100 nmol/L) (Supplemental Figure IIA and IIB). Treatment
with actinomycin-D (10 mol/L), an inhibitor of RNA
synthesis, prevented Ang IIinduced COX-2 expression
(Supplemental Figure IIC) without affecting COX-1 expres-
sion (Supplemental Figure IIIC). By contrast, COX-1 was
constitutively expressed in endothelial cells, and its level was
unaffected by Ang II (Supplemental Figure IIIA and IIIB).
Ang II Upregulates COX-2 Expression via AT
1
R
Treatment with losartan (3 mol/L), an AT
1
R blocker, abolished
Ang IIinduced COX-2 expression, whereas PD 123319 (1 mol/
L), an AT
2
Rblocker, was without effect (Supplemental Figure IIC).
p38 Mitogen-Activated Protein Kinase and
ERK1/2 Jointly Mediate Ang IIInduced
COX-2 Expression
Of the 3 well-known kinase pathways, only p38 MAPKinhibitor
(SB 202190, 10 mol/L) and ERK1/2 inhibitor (PD 98059,
20 mol/L) reduced Ang IIinduced COX-2 expression, each by
50%, whereas SP 600125 (10 mol/L), a c-Jun-N-terminal
kinase inhibitor, had no effect. Cotreatment with SB 202190 and
PD 98059 produced additive inhibition (Figure 1A), suggesting a
parallel involvement of p38 MAPK and ERK1/2 in mediating Ang
IIinduced COX-2 expression. By contrast, these inhibitors did not
alter the expression of COX-1 (Supplemental Figure IIID).
When the time course of ERK1/2 and p38 MAPK activa-
tion by Ang II was examined, peak phosphorylation of both
kinases was noted 5 minutes after the addition of 100
nmol/L Ang II (Figure 1B and 1C).
PKC Activation Is Upstream of ERK1/2 in
COX-2 Induction
Treatment with GF 109203X (GFX, 2 mol/L), an inhibitor for
a broad spectrum of PKC isoforms, markedly reduced Ang
IIinduced COX-2 expression. Rottlerin (PKC

inhibitor,
10 mol/L) but not Go 6976 (PKC
/
inhibitor, 1 mol/L)
1170 Arterioscler Thromb Vasc Biol May 2011
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inhibited COX-2 expression (Figure 1D). These inhibitors did
not affect COX-1 expression (Supplemental Figure IIIE).
Next, the sequence of events involving PKC, ERK1/2, and
p38 MAPK on Ang II stimulation was determined. PKC
inhibition would be expected to prevent the activation of
ERK1/2 and p38 MAPK if PKC is the upstream regulator of the 2
latter pathways. ERK1/2 phosphorylation was inhibited by GFX
and rottlerin but not Go 6976, with PD 98059 serving as a positive
control for ERK1/2 inhibition (Figure 1E). By contrast, p38 MAPK
phosphorylation was prevented by losartan but unaltered by GFX,
Go 6976, and rottlerin (Figure 1F), indicating that unlike ERK1/2,
activation of p38 MAPK was independent of PKC

regulation.
Reverse transcriptionpolymerase chain reaction on cells preex-
posed to Ang II for 4 hours and then treated with actinomycin-D
revealed the participation of these kinases in maintaining COX-2
mRNA stability. Posttreatments with either SB 202190, PD 98059,
or rottlerin partially reduced COX-2 mRNA levels, whereas the
combined treatments of SB 202190PD 98059 and SB
202190rottlerin did not result in synergistic reduction in the levels
of transcribed COX-2 mRNA(Supplemental Figure IID), implying
that unlike induction, these kinases may act in series to maintain
COX-2 mRNA stability posttranscriptionally.
PKC

Plays a Key Role With Little Involvement

of Other PKC Isoforms
Studies with the pharmacological inhibitor of PKC

, rottlerin,
implied the participation of PKC

in Ang IIinduced COX-2

expression. To confirmthe involvement of PKC

, a time-dependent
phosphorylation of PKC

at the activation sites was determined.

PKC

was phosphorylated at Tyr311 in less than 1 minute after the

addition of Ang II (Figure 2A). In contrast, residues Thr505 and
Tyr332 were found to be nonphosphorylated throughout the 1-hour
time course studied (Supplemental Figure IV).
Cytoplasm to membrane translocation of various PKC iso-
forms was examined by fractionating the cytosolic and mem-
branous portion of total protein from endothelial cells. This
method was validated by a clear detection of large amount of
cytosolic GAPDH in the cytosol but a minimal amount in the
membranous fraction, and vice versa for membrane-bound
NOX-2 subunit of NADPH oxidase (Supplemental Figure VA
and VB). The results showed that PKC

and PKC

were
activated on 1-minute stimulation with Ang II, which was
signified by their increased levels in membranous fractions. By
contrast, , , and isoforms were not increased in the
membranous portions of Ang IIstimulated cells compared with
that of control. PKC

was minimally expressed in endothelial

cells (Figure 2B, Supplemental Figure V). PKC

translocation
was abolished by losartan (Figure 2C). SB 202190 and PD
98059 failed to inhibit Ang IIinduced PKC

translocation
(Supplemental Figure VE), again confirming the notion that
ERK1/2 can only be a downstream target of PKC

, but not vice

versa, whereas p38 MAPK and PKC

are independent of each

other. Notably, PKC

translocation from cytosol to nucleus was

Figure 1. Ang IIinduced COX-2 expression is mediated through ERK1/2, p38 MAPK, and PKC

. A, COX-2 expression was inhibited by

SB 202190 (SB, 10 mol/L) or PD 98059 (PD, 20 mol/L) but not SP 600125 (SP, 10 mol/L). Cotreatment with SB and PD further
reduced the COX-2 expression. B and C, Time course for phosphorylation of ERK1/2 and p38 MAPK in response to Ang II (100 nmol/L). D,
COX-2 expression was inhibited by GF 109203X (GFX, 2 mol/L) or rottlerin (10 mol/L) but not Go 6976 (1 mol/L). E, ERK1/2 phosphoryla-
tion was inhibited by GFX, rottlerin, or PD 98059 (PD) but not Go 6976. F, Phosphorylation of p38 MAPK was inhibited by losartan (3 mol/L)
but not PKC inhibitors. *P0.05, **P0.01, ***P0.001 vs control; ###P0.001 vs the Ang IItreated group.
Wong et al Ang II and Endothelial COX-2 Upregulation 1171
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not detected shortly after Ang II exposure (Supplemental Figure
VIA; fractionation method was validated by strong positive
signals of GAPDH in the cytosolic portion and histone H3 in the
nuclear portion, as shown in Supplemental Figure VIB and
VIC), indicating that the action of PKC

mainly resided in the

cytosol at its peak activation state. Cotreatment with SB 202190
and rottlerin produced additive effects in suppressing the Ang
IIinduced COX-2 expression, without affecting COX-1 expres-
sion (Supplemental Figure VIIA and VIIB).
Transfection with siRNA targeting PKC

(siPKC

) further
supports the key role of PKC

. Western blot analysis con-

firmed that siPKC

successfully knocked down PKC

without
affecting PKC

(Figure 2D; Supplemental Figure VIIIA and

VIIIB). In siPKC

-transfected cells, Ang IIinduced COX-2

expression was reduced by 50%, and the remaining portion
could almost be abolished by cotreatment with SB 202190,
again indicating the operation of dual signal transduction
pathways involving PKC

and p38 MAPK. By contrast,

scramble siRNA had no effect on Ang IIinduced COX-2
expression compared with the nontransfected endothelial
cells (Figure 2D, Supplemental Figure VIIIC and VIIID).
As shown in the study of PKC translocation, PKC

was also
activated by Ang II (Figure 2B, Supplemental Figure VF).
However, the PKC

inhibitor peptide V1-2 (10 mol/L) did not

prevent Ang IIinduced COX-2 expression (Supplemental Fig-
ure VIIC). siPKC

did not suppress PKC

levels (Figure 2D,

Supplemental Figure VIIIB) but abolished COX-2 expression in
combination with the p38 MAPK inhibitor SB 202190 (Figure
2D, Supplemental Figure VIIID), thus suggesting that PKC

is
unlikely to be one of the mediators of COX-2 expression.
The inducible role of PKC

in COX-2 expression was also

pinpointed using an exogenous PKC activator, phorbol 12-my-
ristate 13-acetate (PMA). PMA at 1 mol/L time-dependently
increased COX-2 expression, whereas its negative analog, 4-
PMA, had no effect (Figure 2E, Supplemental Figure VIIIE).
Such increase in COX-2 expression was again sensitive to
inhibition by GFX and rottlerin but not Go 6976 (Figure 2E,
Supplemental Figure VIIIF).
Ang IIInduced Release of MCP-1 Is
COX-2 Dependent
The level of MCP-1 in the conditioned medium from endo-
thelial cells treated with 100 nmol/L Ang II for 24 hours
increased (Figure 3), whereas the release of interleukin-6 and
tumor necrosis factor- fell below detectable levels (data not
shown). The MCP-1 release was inhibited by losartan and 2
structurally different COX-2 inhibitors, celecoxib and NS 398
(both at 3 mol/L) (Figure 3).
Renovascular Dysfunction and Elevated
Endothelial COX-2 Expression in Ang
IIInfused Rats
Systolic blood pressure of rats rose from 104.34.6 to
174.09.6 mm Hg during a 9-day period of Ang II infusion,
and this increase was prevented by concomitant oral treat-
ment with losartan (Figure 4A). Endothelium-dependent re-
laxations were impaired in interlobal renal arteries from the
Ang IIinfused rats, and this impairment was abolished by
losartan treatment (Figure 4B). The attenuated relaxations
were restored by acute treatment with the COX-2 inhibitor
Figure 2. PKC

is activated by Ang II
stimulation, acting as a major mediator of
COX-2 expression. A, PKC

was phos-
phorylated at Tyr311 within 1 minute of
Ang II addition. B, Representative immu-
noblots of translocation of various PKC
isoforms stimulated by Ang II (100
nmol/L) and PMA (1 mol/L). C indicates
cytosol; M, membrane. C, Ang II stimu-
lated PKC

translocation to the mem-

brane, which was prevented by losartan
(3 mol/L). Cyto indicates cytosol; mb,
membrane. D, siRNA targeting PKC

(siPKC

) signicantly reduced Ang IIin-

duced COX-2 expression, which was fur-
ther abolished by SB 202190. E, PMA
induced COX-2 expression, which was inhib-
ited by GFX (2 mol/L) and rottlerin (10 mol/
L). **P0.01, ***P0.001 vs control;
##P0.01 vs the Ang IItreated group.
Figure 3. MCP-1 production as a consequence of COX-2 upregula-
tion. MCP-1 release increased after a 24-hour incubation with Ang II
(100 nmol/L), which was prevented by losartan (3 mol/L) and the
COX-2 inhibitors celecoxib and NS 398 (each at 3 mol/L).
***P0.001 vs control; ##P0.01 vs the Ang IItreated group.
1172 Arterioscler Thromb Vasc Biol May 2011
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celecoxib (3 mol/L) (Figure 4C) but not by the COX-1
inhibitor sc-560 (data not shown). Immunohistochemical
staining revealed that endothelial COX-2 expression (Figure
4D and 4E, arrows) was augmented in both aortae and renal
arteries of Ang IIinfused rats, and the increased COX-2
staining was attenuated by losartan.
Ang IIInduced COX-2 Expression and
COX-2-Dependent MCP-1 Expression in Human
Mesenteric Arteries
Ang IIinduced expression of proinflammatory MCP-1 was
also demonstrated in the endothelial layer of human mesen-
teric arteries. The left columns of Figure 5A and 5B show the
green autofluorescence from elastin of the internal elastic
lamina, which delineated the artery into endothelial and
smooth muscle layers. The middle columns feature the
fluorescence emitted from Alexa Fluor 546conjugated sec-
ondary antibodies, which were tagged to primary antibodies
against either COX-2 (Figure 5A) or MCP-1 (Figure 5B),
appearing in reddish orange together with the noise from
autofluorescence. In the overlay images in the right column,
the autofluorescence and noise are yellowish green, and the
remaining reddish orange signifies the signals from COX-2 or
MCP-1. Referring to the right column of Figure 5A, endo-
thelial COX-2 expression was remarkably augmented after a
24-hour exposure to Ang II (1 mol/L); expression was
prevented by cotreatment with SB 202190 and rottlerin but
remained unaffected in solvent control exposed to dimethyl
sulfoxide. It is noteworthy that this COX-2 expression may
contribute to the MCP-1 expression, as evidenced by an increase
of endothelial MCP-1 expression in the same arteries treated
with Ang II, which was inhibited by preincubation with cele-
coxib but not the solvent dimethyl sulfoxide (Figure 5B).
ROS Do Not Mediate COX-2 Upregulation
Dihydroethidium fluorescence in endothelial cells showed
that the ROS level rose 2-fold in 30 s after Ang II
addition, which was inhibited by losartan, the NADPH
oxidase inhibitor apocynin (100 mol/L), and ROS scaven-
gers, tiron (1 mmol/L) plus diethyldithiocarbamate
(100 mol/L) (Supplemental Figures IX and XA). Inhibitors
of ROS-producing enzymes, including apocynin (100 mol/
L), oxypurinol (xanthine oxidase inhibitor, 100 mol/L),
2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine hydrochlo-
ride (inducible nitric oxide synthase inhibitor, 30 mol/L),
N
G
-nitro-L-arginine methyl ester (nonspecific nitric oxide
synthase inhibitor, 100 mol/L), the ROS scavengers tempol,
and tiron plus diethyldithiocarbamate, however, did not
decrease the Ang IIinduced COX-2 expression (Supplemen-
tal Figure XB), thus ruling out the participation from ROS.
Indeed, exogenous H
2
O
2
induced only a small increase
(3-fold) in COX-2 expression compared with the marked
stimulation (15-fold) by Ang II (Supplemental Figure XC).
Discussion
The present study provides novel insights on the molecular
mechanism and sequence of intracellular events on the
upregulation of endothelial COX-2 expression by Ang II,
pinpointing a novel role of PKC

activated at Tyr311 and

joint contributions of ERK1/2 and p38 MAPK. We also
defined the Ang IIinduced release or expression of the
Figure 4. COX-2 upregulation and vascular dysfunction in Ang
IIinfused hypertensive rats. A, Systolic blood pressure increased
markedly in Ang IIinfused rats, which was prevented by oral
administration of losartan (Losar, 10 mg/kg per day). B, Acetylcho-
line (ACh)induced endothelium-dependent relaxations were atten-
uated in the interlobal renal arteries from Ang IIinfused rats but
not in those orally administered losartan. C, Celecoxib acutely
restored the impaired endothelium-dependent relaxations. D and E,
Endothelial COX-2 expression (indicated by red arrows) was aug-
mented in the aortae (D) and renal arteries (E) of Ang IIinfused
rats (AII), and the expression was reduced in those from Ang
IIinfused rats orally treated with losartan (LAII). Similar obser-
vations were each made in arteries from 4 rats. *P0.05,
**P0.01 vs control; #P0.05, ##P0.01 vs the Ang IIinfused
group. CTL indicates control; Endo, endothelium.
Figure 5. Ang II induces COX-2 expression and COX-2-dependent
MCP-1 expression in human mesenteric arteries. Autouorescence
(Auto, left columns, in green) signied the elastin from internal elastic
lamina (IEL) in the vessel wall. It delineated the layers of endothelial
cells (EC) and vascular smooth muscle cells (VSMC). Detection for
Alexa Fluor 546 (AF546, middle columns, in reddish orange) gave the
signals from both the elastin and the antibody-uorophore complex.
Right columns feature the overlay images of the other columns, with
the elastin appearing yellow. The reddish orange color in the right col-
umn indicates the pure signal from antibodies. A, Ang II induced en-
dothelial COX-2 expression (indicated by white arrows), which was
sensitive to SB 202190 (SB) and rottlerin (ROT). B, Ang II elevated
endothelial MCP-1 expression, which was inhibited by celecoxib.
dimethyl sulfoxide did not affect the expressions of either Ang IIin-
duced COX-2 or MCP-1. When primary antibody was neglected (ie,
negative control), the signals were purely from the elastin.
Wong et al Ang II and Endothelial COX-2 Upregulation 1173
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COX-2-dependent proatherosclerotic cytokine MCP-1. The
present results were first obtained from primary culture of rat
endothelial cells, but they were also confirmed in an in vivo
animal model of Ang IIinfused hypertensive rats and ex-
tended to tissue culture of human arteries.
Ang II, a potent vasoconstrictive peptide, plays a central role
in vascular dysfunction associated with hypertension and diabe-
tes, and AT
1
R blockers represent a major class of antihyperten-
sive drugs that interrupt Ang IIinitiated intracellular signaling.
15
Although Ang II serves as a powerful ligand to trigger transcrip-
tions of proatherosclerotic cytokines,
24
the role of the highly
inducible proinflammatory enzyme COX-2 in vascular diseases
has recently gained attention, as its overexpression is also
detected in human atherosclerotic plaques.
11
However, the rela-
tionship between Ang II and COX-2 remains elusive as to
whether COX-2 acts as a direct downstream effector of Ang II
to mediate vascular inflammation, especially in endothelial cells,
which represent the first-line initiation point of atherosclerosis.
The present study demonstrated that Ang II could markedly
upregulate COX-2 expression in endothelial cells through AT
1
R
activation, agreeing with the preliminary findings of Kramer et
al using Northern blot analysis.
16
The COX-2 expression was
jointly mediated by p38 MAPK- and ERK1/2-dependent signal-
ing pathways, as the inhibitor of either kinase, SB 202190 or PD
98059, could only partially suppress the COX-2 expression,
whereas their cotreatment resulted in additive inhibition that
almost abolished COX-2 expression. It appeared that c-Jun-N-
terminal kinase was unlikely to be involved, as its inhibitor did
not alter Ang IIinduced COX-2 expression.
Because p38 MAPK and ERK1/2 are sensitive to redox
triggers,
1719
we initially suspected that Ang IIinduced oxida-
tive stress might contribute to COX-2 expression. This possibil-
ity, however, was excluded by 2 observations. First, Ang
IIstimulated ROS production was prevented by the NADPH
oxidase inhibitor apocynin and by the ROS scavenger tiron plus
diethyldithiocarbamate; however, Ang IIinduced COX-2 ex-
pression was unaltered by these inhibitors and by inhibitors
against xanthine oxidase, inducible nitric oxide synthase, and
nitric oxide synthase uncoupling. Second, H
2
O
2
, which was
reported to increase COX-2 expression in human umbilical vein
endothelial cells,
20
induced a mild increase in COX-2 expression
compared with that stimulated by Ang II. Our results differ from
a previous study showing that in human saphenous vein endo-
thelial cells, interleukin-1-induced COX-2 expression involves
both NADPH oxidase-derived superoxide anion and H
2
O
2
.
21
Indeed, our recent findings on bone morphogenic protein-4
induced COX-2 expression also showed that the kinase mediator
p38 MAPK is activated by bone morphogenic protein-4
stimulated NADPH oxidase-derived ROS.
22
Our further exper-
imentation, aimed at understanding why oxidative stress is
involved in bone morphogenic protein-4induced COX-2 in-
duction but not that stimulated by Ang II, showed that bone
morphogenic protein-4 (20 ng/mL) induced a significantly
higher ROS level compared with Ang II at 100 nmol/L (the
concentration used in the cell-based studies) or even 1 mol/L
(Supplemental Figure XI), implying that the level of oxidative
stress induced by the agonist is an important determinant in
whether the redox-sensitive kinases are turned on by the radicals
and hence whether there is a role for the ROS to mediate COX-2
upregulation. The MAPKs are not preferentially activated by the
Ang IIinduced ROS, possibly owing to the ineffective ROS
level. Alternatively, the source and production site of ROS or
type of ROS produced may play differential roles in both
induction of COX-2 expression and stimulation of COX-2
activity. These need further investigation at subcellular levels.
PKC appears to be another possible upstream target because
PKC is associated with the G-protein-coupled AT
1
R. Herein we
defined a novel role of PKC

, activated at Tyr311, to mediate

COX-2 expression in Ang IItreated rat aortic endothelial cells.
To our surprise, PKC

is not phosphorylated at the activation

loop of residue Thr505 in response to Ang II. As pointed out by
Liu et al, phosphorylation of the activation loop may not be
necessary for PKC

activation and that PKC

-mediated apopto-
sis of HEK293T cells does not require phosphorylation at the
activation loop.
23
Indeed, earlier reports also found that mutant
PKC

with activation loop dephosphorylated was still in full

activity.
2426
As to the tyrosine residues, Tyr311 but not Tyr332
was phosphorylated by Ang II, although both are concomitantly
phosphorylated in response to oxidative stress such as H
2
O
2
.
27
As explained by Rybin et al, the agonist (Ang II or PMA)-
dependent selective phosphorylation of tyrosine residues may pos-
sibly be effected via the selective Tyr311 kinase c-Abl, whereas
other kinases (sensitive to redox changes) may cause a concurrent
phosphorylation in both residues.
27
Although we cannot detail all
the phosphorylation residues that are involved in the activation of
PKC

in response to Ang II (because of a large number of possible

phosphorylation sites and the complex regulation of PKC

), our
present data suggest that at least Tyr311 is phosphorylated to
mediate Ang IIinduced PKC

activation, without the possible

involvement of residues Thr505 and Tyr332.
Pharmacologically, Ang IIinduced COX-2 expression and
PMA (PKC activator)-induced COX-2 expression were both
sensitive to inhibition by GFX (a broad spectrum of PKC
inhibitor) and rottlerin (PKC

inhibitor). Of note, GFX and

rottlerin also inhibited Ang IIinduced ERK1/2 phosphorylation,
indicating that PKC

is likely the upstream activator of ERK1/2.

Activation of p38 MAPK was likely independent of PKC
regulation because its phosphorylation was unaffected by PKC
inhibitors. Interestingly, posttreatments of these kinase inhibitors
along with actinomycin-D in cells preexposed to Ang II dem-
onstrated a partial reduction in Ang IIinduced COX-2 mRNA
levels, indicating that p38 MAPK, ERK1/2, and PKC

may also
help to stabilize the COX-2 mRNAtranscribed. In contrast to the
results from Western blot analysis in which cotreatments of SB
202190PD 98059 and SB 202190rottlerin produced clear
additive effect that totally abolished Ang IIinduced COX-2
expression, neither SB 202190PD 98059 nor SB 202190rot-
tlerin showed a significant synergistic suppression on the COX-2
mRNA level, implying that the kinases may function in series in
preserving mRNA stability, which is different from COX-2
induction, when they act jointly. Doller et al recently demon-
strated in rat mesangial cells that PKC

take a critical role in

shuttling the mRNA-stabilizing factor human antigen R and
thereby posttranscriptionally maintaining COX-2 expression
induced by Ang II.
28
Of note, members of MAPK family can
also interact with human antigen R to stabilize the mRNA
coding for inflammatory mediators such as tumor necrosis
factor- and COX-2.
29,30
Ang IIinduced COX-2 expression in
1174 Arterioscler Thromb Vasc Biol May 2011
at Chinese University of Hong Kong on April 22, 2011 atvb.ahajournals.org Downloaded from
vascular smooth muscles is reported to be mediated by p38 MAPK-
and ERK1/2-mediated mRNA stabilization of COX-2 mRNA.
31
Nonetheless, taken together with the results from exogenous PKC
activator PMA which can induce rottlerin-sensitive COX-2 expres-
sion, Ang IIstimulated activation of PKC

and the MAPKs

possibly cause an upregulation of COX-2 expression by both
transcription induction and mRNA stabilization.
To address the concern over the specificity of pharmacolog-
ical inhibitors used, we studied the treatment-induced transloca-
tion of PKC

from the cytoplasm to the plasma membrane and

excluded the participation of other PKC isoforms. Translocation
of soluble cytosolic PKCto the particulated membranous formis
a key feature that signifies PKC activation.
32
Comparing the
portion of membranous PKC after Ang II stimulation with that
of control, only PKC

and PKC

were found to be responsive to

Ang II. While PKC

was activated by PMA but not Ang II,

PKC

and PKC

remained unresponsive to either treatments,

and PKC

was not expressed in endothelial cells. Indeed,

although PKC

was activated by PMA, treatments with the

PKC
/
inhibitor Go 6976 failed to suppress the PMA-induced
COX-2 expression, thus contrasting a complete abrogation by
rottlerin. Likewise, Ang II activated both PKC

and PKC

, yet
the role of PKC

in mediating COX-2 expression appears to be

negligible, as indicated by the following 2 observations. First,
PKC

inhibitor V12 did not affect Ang IIinduced COX-2

expression. Second, siPKC

markedly suppressed COX-2 ex-

pression without affecting PKC

expression. If PKC

were to be
significantly involved in mediating Ang II action, its nonsup-
pressed levels in siPKC

-transfected cells with or without

concomitant exposure to the p38 MAPK inhibitor SB 202190
would have maintained the inducible response of COX-2 ex-
pression as in the control or scramble siRNA-transfected cells.
Taken together, these data indicate that even a number of PKC
isoforms might be activated by Ang II PKC

is probably the
main isoform that is essentially coupled with endothelial COX-2
expression. It is noteworthy that inhibition of p38 MAPK by SB
202190 in combination with PKC

knockdown cells prevented

the induction of COX-2 expression, thus proving a parallel
involvement of the PKC

-ERK1/2 and p38 MAPK pathways.

Such findings are in contrast to Ang IIinduced COX-2 upregula-
tion in vascular smooth muscle cells, which depends mainly on the
ERK1/2 activity without the participation of PKC or p38 MAPK.
7
Pretreatment by inhibitor of either ERK1/2 or p38 MAPK did not
affect Ang IIinduced PKC

translocation, again confirming that

PKC

is upstream of ERK1/2 activation and independent of p38

MAPK regulation. Although PKC

can also move into the nucleus

from the cytosol,
33
our present results showed that PKC

is not
translocated into the nucleus shortly (1 minute) after Ang II
exposure, in contrast to its clear and rapid particulation at the plasma
membrane. Indeed, PKC

was found to be imported into the

nucleus at least 30 minutes after Ang II stimulation.
28
Our present
data thus suggest that PKC

, shortly after Ang II stimulation, first

translocates to the membrane where it exerts its major effect on
COX-2 induction via regulation of ERK1/2 activity. COX-2
mRNA stabilization by PKC

may occur as a consecutive event

following its delayed translocation into the nucleus.
Upregulation of endothelial COX-2 by Ang II could be demon-
strated not only in cultured cells. Indeed, our study clearly showed
an augmentation of endothelial COX-2 protein expression in the
aorta and renal arteries of Ang IIinfused rats, which was inhibited
by concomitant oral treatment of losartan. Impairment of endothe-
lium-dependent relaxations was acutely restored by the COX-2
inhibitor celecoxib, indicating that COX-2-derived vasoconstrictive
prostanoids may contribute to vascular dysfunctions, as documented
in diabetic, hypertensive, and aging animal models.
3436
Tissue culture experiments on human mesenteric arteries
substantiate the findings in cultured rat endothelial cells. Ang
IIinduced endothelial COX-2 expression was prevented by
cotreatment of SB 202190 and rottlerin. More importantly,
increased COX-2 expression was accompanied by a concomi-
tant rise in MCP-1 expression, with both proteins being immu-
nolocalized to the endothelium. This MCP-1 increase was
sensitive to COX-2 inhibition by celecoxib, clearly suggesting a
causal relationship between these 2 proinflammatory and proath-
erosclerotic cytokines. Indeed, Ang IIinduced MCP-1 release into
the conditioned medium of cultured rat endothelial cells was also
prevented by the 2 specific COX-2 inhibitors celecoxib and NS398.
Taken together, endothelial COX-2-dependent release of MCP-1
may represent a new concept in our understanding of the Ang
IIinduced vascular inflammation and atherosclerotic plaque forma-
tion involving macrophages. Which of the COX-2-derived prosta-
noids may mediate MCP-1 release warrants further investigation.
Assembling the time course of maximal activation of signal-
ing proteins, and studies using pharmacological inhibitors and
siRNA, we propose the following cellular pathways of Ang
IIinduced endothelial COX-2 expression (Figure 6). Ang II
stimulates AT
1
R to activate PKC

in 1 minute and subse-

quently ERK1/2 in 5 minutes, along with maximal PKC-
independent p38 MAPK activation in 5 minutes. COX-2
expression was effected via induction and mRNA stabilization;
it was detected at 1 hour and reached the maximum at 8 hours.
MCP-1 release detected at 24 hours was COX-2 dependent. In
view of the controversy regarding the use of COX-2 inhibitors
Figure 6. Postulated cellular mechanisms on Ang IIinduced
COX-2 expression in endothelial cells and time course of
events. Ang II acts on AT
1
R, which then activates PKC

in 1
minute. Activations of PKC

-dependent ERK1/2 and PKC

-
independent p38 MAPK occur at 5 minutes. COX-2 begins to
be expressed at 1 hour via induction and mRNA stabilization,
and the expression reaches a maximum at 8 hours. COX-2-
dependent MCP-1 release was detected in 24 hours.
Wong et al Ang II and Endothelial COX-2 Upregulation 1175
at Chinese University of Hong Kong on April 22, 2011 atvb.ahajournals.org Downloaded from
and considering that PKC

is preferentially activated in hyper-

glycemia and diabetes (detailed in Significance of Study in the
online Supplement), the present study may suggest PKC

as a
more promising target for therapeutic intervention in COX-2-
mediated vascular complications.
Sources of Funding
This study was supported by the Hong Kong Research Grant Council
(Grants CUHK465308M, CUHK466110, HKU2/07C, and HKU4/
CRF10), Deutsche Forschungsgemeinschaft, Germany-Hong Kong
Joint Research Scheme, and Focused Investment Scheme of Chinese
University of Hong Kong.
Disclosures
None.
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Wong et al., 2011, Supplemental Material
P. 1
Pivoted Role of PKC

in Angiotensin II-induced Endothelial Cyclooxygenase-2 Expression

A Link to Vascular Inflammation

1
Siu Ling Wong,
1
Chi Wai Lau,
1
Wing Tak Wong,
3
Aimin Xu,
1
Chak Leung Au,
2
Chi Fai Ng,
2
Simon Siu Man Ng,
4
Maik Gollasch,
1
Xiaoqiang Yao,
1
Yu Huang

1
Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, and School of Biomedical Sciences,
2
Department of Surgery, Chinese University of Hong Kong, HKSAR, China;
3
Department of Medicine and
Department of Pharmacology and Pharmacy, University of Hong Kong, HKSAR, China;
4
Medical Clinic for
Nephrology and Internal Intensive Care, Charit University Medicine Berlin, Germany.


Supplemental Material

Methods
This study was approved by the Animal Experimentation Ethics
Committee, Chinese University of Hong Kong. Male Sprague
Dawley rats (260-280 g) were supplied by Laboratory Animal
Service Centre, Chinese University of Hong Kong. This
investigation conformed to the Guide for the Care and Use of
Laboratory Animals published by the US National Institute of
Health (NIH Publication No. 85-23, revised 1996). The use of
human samples for experimentation was approved by the Joint
Chinese University of Hong Kong-New Territories East Cluster
Clinical Research Ethics Committee. Human mesenteric
arteries distant from the tumor were obtained during surgery for
colon cancer with informed consent from patients. The mean
age of the patients was 73 years old, ranging between 62 and
88. Only arteries from patients without hypertension (having
systolic and diastolic blood pressure lower than 140 mmHg and
90 mmHg respectively) and without diabetes (having fasting
glucose level <7.0 mmol/L) were employed in the present
study.

Cell culture
Endothelial cells were freshly cultured from rat thoracic aortae
as described elsewhere.
1
The aorta was excised free, cleaned
of perivascular adventitial adipose tissue and subjected to
digestion in collagenase (Sigma, Type IA) at 37
o
C for 15 min.
Serum containing medium was added to quench the digestion
and the mixture was centrifuged at 800 g for 15 min. The cells
were re-suspended in RPMI 1640, supplemented with 10%
fetal bovine serum (FBS) plus 1% penicillin/streptomycin (P/S)
(GIBCO) and allowed to settle for 1 h upon which the medium
was changed. The identity of the endothelial cells was
confirmed by immunocytochemical staining for an endothelial
cell specific marker, PECAM-1 (Supplemental Figure I). To
avoid the influence of culture condition on endothelial cell
phenotype, only cells from passage 1 were used in the present
study.

SDS-PAGE and Western blot analysis
Cells were serum-deprived for 24 h and incubated with Ang II
(100 nmol/L) for 8 h unless otherwise stated. When used,
inhibitors were pre-incubated with the cells for 30 min before
the addition of Ang II. After the incubation, cells were lysed in
an ice-cold RIPA buffer with a cocktail of protease inhibitors
(leupetin, 1 g/mL; aprotonin, 5 g/mL; PMSF, 100 g/mL;
sodium orthovanadate, 1 mmol/L; EGTA, 1 mmol/L; EDTA, 1
mmol/L; NaF, 1 mmol/L and -glycerolphosphate, 2 mg/mL).
The lysates were centrifuged at 20,000 g for 20 min and protein
concentration was determined by the Lowry method (BioRad).
Each protein sample (20 g) was electrophoresed through the
10% SDS-polyacrylamide gels and then transferred to an
Immobilon-P polyvinylidene difluoride (PVDF) membrane
(Millipore).
2
The membranes were blocked with 5% non-fat
skimmed milk or 1% bovine serum albumin in Tween-20
phosphate buffer saline (PBST) and probed overnight at 4
o
C
with antibodies against COX-2 (Santa Cruz), COX-1 (Cayman),
p38 MARK, ERK1/2 and PKC isoforms (Cell Signaling). After
washes in PBST, the membranes were incubated with
appropriate HRP-conjugated secondary IgG (DakoCytomation)
for 1 h at room temperature. The membranes were developed
with an enhanced chemiluminescence detection system (ECL
reagents; Amersham Pharmacia Biotech, Uppsala, Sweden)
and exposed on X-ray films (Fuji). Densitometry was performed
using a documentation program (GBOX-CHEM1-HR16,
SynGene). GAPDH antibody (Ambion, Inc) was probed as a
loading control.

Reverse-transcription polymerase chain reaction (RT-PCR)
The potential roles of p38 MAPK, ERK1/2 and PKC

on COX-2
mRNA stabilization were examined with RT-PCR. Primary rat
aortic endothelial cells were first exposed to Ang II (100 nmol/L)
for 4 h, after which actinomycin-D (10 mol/L) and the kinase
inhibitors (SB 202190, PD 98059 and rottlerin) were introduced
for another 4 h. The cells were lysed and the mRNA was
extracted with Trizol reagent (Invitrogen) according to the
manufacturers instruction. The extracted mRNA was reverse
transcribed using the iScriptTM cDNA synthesis kit (BioRad),
and PCR was performed with Taq DNA polymerase (Invitrogen,
Carlsbad, CA, USA) with thermal cycles of 3-min 94
o
C, 29
cycles of 45-s 94
o
C, 30-s 56
o
C, 1.5-min 72
o
C, finally followed
by 10-min 72
o
C using the forward and reverse primers of
sequences 5-TGGTGCCGGGTCTGATGATG-3 and
5-GCAATGCGGTTCTGATACTG-3 respectively.
3
The PCR
products were then resolved by electrophoresis in a 1.5%
agarose gel with ethidium bromide staining. Densitometry was
determined in the gel documentation system
(GBOX-CHEM1-HR16, SynGene) and normalized to the
internal control of GAPDH.

Cellular protein fractionation for translocation study of
PKC isoforms
Endothelial cells were incubated with 100 nmol/L Ang II for 1
min and quickly cooled on ice to quench cellular reactions.
Cytosolic and membranous proteins were extracted with
ProteoExtract Subcellular Proteome Extraction Kit according
to manufacturers instructions (Calbiochem). Cytosolic and
nuclear proteins were extracted with tailor-made Buffers A (KCl,
10 mmol/L; DTT, 1 mmol/L; PMSF, 500 mol/L; EDTA, 100
mol/L; EGTA, 100 mol/L; HEPES, 10 mmol/L, pH 7.9) and C
(NaCl, 400 mmol/L; DTT, 1 mmol/L; PMSF, 1 mmol/L; EDTA, 1
mmol/L; EGTA, 1 mmol/L, HEPES, 20 mmol/L, pH 7.9). Briefly,
cells harvested were first solubilized in Buffer A with vortexing.
After incubating on ice for 15 min, 10% NP40 was added and
the mixture was centrifuged at 12 000 rpm for 5 min at 4
o
C.
The supernatant was saved as the cytosolic portion and the
pellet was resuspended in Buffer C with vigorous vortexing.
The suspension was centrifuge at 12 000 rpm for 5 min at 4
o
C
after 15-min incubation on ice. The supernatant collected
thereafter was labeled as nuclear fraction. Method validation of
the extraction protocols were performed by cross-probing with
markers for cytosolic, membranous and nuclear proteins (i.e.
GAPDH, NOX-2 and histone H3, respectively).

PKC

knockdown with small interfering RNA (siRNA)

When primary rat endothelial cells reached 80% confluence,
the cells were transfected with siRNA by electroporation using
Amaxa Basic Nucleofector Kit for primary mammalian
endothelial cells (Lonza, Germany). Briefly, 2.5 x10
6
cells/mL
were trypsinized and washed two times with PBS, and
at Chinese University of Hong Kong on April 22, 2011 atvb.ahajournals.org Downloaded from
Wong et al., 2011, Supplemental Material
P. 2
resuspended in 100 L basic nucleofector solution and
transferred to a cuvette containing either 30 pmol scramble
siRNA or pre-designed specific siRNA targeting PKC

transcripts (Ambion). The cells were electroporated with the
Amaxa Nucleofactor
TM
apparatus, re-plated in 6-well plates
containing pre-warmed complete RPMI medium and left
undisturbed for 24 h. The cells were then serum-deprived for
24 h before an 8-h incubation with Ang II (100 nmol/L).
Inhibitors, when use, were incubated for 30 min prior to Ang II
addition.

Angiotensin II infusion in rats
Sprague-Dawley rats weighing ~250 g were anesthetized with
37.5% ketamine plus 25% xylazine in normal saline. Osmotic
minipump (ALZET, model 2ML2, Alza Pharmaceutical, Palo
Alto, CA, USA) filled with either Ang II or the vehicle (normal
saline) was implanted subcutaneously in the dorsal region. Ang
II was infused at 0.7 mg/kg/day for 9 days. Blood pressure was
regularly monitored throughout the infusion period with a
tail-cuff electrosphygmomanometer system, after which aortae
and renal arteries were excised, dissected free from
perivascular connective tissue and preserved in 4%
paraformaldehyde for immunohistchemical staining. Interlobal
renal arteries were used for functional evaluation of
endothelium-dependent relaxations.

Immunohistochemical staining
Localization of COX-2 in the aortae and renal arteries from Ang
II-infused rats were determined by immunohistochemistry. The
tissues were fixed overnight in 4% paraformaldehyde,
processed for embedding in wax and then cut into 5
m-sections. Following re-hydration and treatment with 1.4%
hydrogen peroxide in absolute methanol for 30 min at room
temperature to inhibit endogenous peroxidase activity, antigen
retrieval was performed by boiling the sections in 0.01 mol/L
sodium citrate buffer (pH 6) for 30-60 s. After rinsing in PBS,
the sections were blocked with 5% donkey serum and
incubated overnight with COX-2 antibody diluted in PBS
supplemented with 2% BSA in a humidified chamber at 4
o
C.
The sections were then incubated with biotinylated secondary
antibodies for 1 h at room temperature, followed by 1 h
incubation with peroxidase-conjugated streptavidin. DAB
(Vector Laboratories, California, USA) was used for colour
development according to the manufacturers instruction.
Counter-staining of nuclei and cytoplasm was performed with
haematoxylin and eosin respectively. Negative control was
performed in the absence of primary antibody. Images were
viewed and captured under Leica DMRBE microscope coupled
to SPOT-RT cooled CCD color digital camera using the
objective PL FLUOTAR 20x/0.50 and SPOT Advanced
software (Version 3.5.5).

Immunofluorescence localization
Human small mesenteric arteries harvested after a 24-h
incubation protocol were embedded in OCT compound (Sakura
Finetek, the Netherlands) in aluminium cryomolds, snap frozen
in isopentane pre-cooled in liquid nitrogen and cut into 10 m
thick cryostat sections. The thawed sections were air-dried,
post-fixed in 4% paraformaldehyde for 30 min, and then briefly
treated with 0.05% Triton X in PBS. The sections were blocked
with 5% donkey serum for 1 h at room temperature. Primary
antibodies against COX-2 or MCP-1 (Santa Cruz) were
incubated overnight at 4
o
C. The sections were then labeled
with Alexa Fluor 546 donkey anti-goat IgG (Invitrogen,
Molecular Probes, California, USA) for 1 h at room temperature.
The sections were cover-slipped in anti-fade mounting medium
(Vector Laboratories) and viewed under fluorescence
microscope (Nikon Eclipse Ti-U) with mercury lamp Nikon
Intensilight C-HGFI using the objective Nikon S Fluor 20x/0.75.
Images were acquired with SPOT RT3 cooled 2 MP CCD
scientific color digital camera and SPOT advanced software
(Version 4.6).

Functional examination with myography
Endothelium-dependent relaxations of the interlobal renal
arteries were determined in myographs. After subjecting the
rats to a 9 day-infusion of Ang II, interlobal renal arteries were
dissected and suspended in the myograph (Danish Myo
Technology, Aarhus, Denmark) with stainless steel wires. Each
chamber was filled with 5 mL Krebs-Henseleit solution
containing (mmol/L): NaCl 119, NaHCO
3
25, MgCl
2
1, KCl 4.7,
KH
2
PO
4
1.2, CaCl
2
2.5, and D-glucose 11.1, and aerated

with
95% O
2
-5% CO
2
at 37 C to give a pH of ~7.4. The arterial rings
were stretched to a pre-determined optimal resting tension of 2
mN and allowed to stabilize

at this basal tone for 60 min before
experiments began. Acetylcholine-induced
endothelium-dependent relaxations were evaluated in arteries
from control rats and Ang II-infused rats with or without
concomitant oral administration of losartan (10 mg/kg/day). The
acute effects of celecoxib (3 mol/L) and sc-560 (0.3 mol/L)
were tested on the relaxations in renal arteries from Ang
II-infused rats.

Reactive oxygen species (ROS) detection with
dihydroethidium (DHE) fluorescence
Production of intracellular ROS was measured with DHE
(Molecular Probes), which binds to DNA upon oxidation to emit
fluorescence. Following a 30-s treatment of Ang II, primary rat
endothelial cells seeded on cover-slips were incubated in 5
mol/L DHE for 20 min at 37
o
C. After a rinse in PBS,
fluorescence was observed under the confocal microscopy
system Olympus Fluoview FV1000 (515-nm excitation; 585-nm
long pass filter) using the objective UMPlanFI 10x/0.30. DHE
fluorescence intensity was analyzed by Olympus Fluoview
software (version 1.5; FV10-ASW1.5). Similar experiments
were performed to compare the difference in ROS levels
between 15-min exposure of Ang II and bone morphogenic
protein-4 (BMP4). Data were expressed in fold change
compared with untreated control.

Chemicals
Angiotensin II, losartan, PD 123319, actinomycin-D, SB
202190, PD 98059, SP 600125, L-NAME, AMT, GF109203X,
Go 6976, NS 398 and phorbol 12-myristate 13-acetate were
purchased from Tocris (Avonmouth, UK). Rottlerin was from
Enzo Life Sciences (New York, USA) and V1-2 from AnaSpec
(California, USA). BMP4, oxypurinol, tiron, tempol, DETCA and
4-phorbol 12-myristate 13-acetate were purchased from
Sigma-Aldrich Chemical (St Louis, MO, USA). Apocynin were
from Calbiochem, EMD Biosciences (La Jolla, CA, USA).
Celecoxib was from Pfizer and sc-560 was a kind gift from
Institut de Recherches Servier (Suresnes, France). Angiotensin
II, PD 123319, N
G
-nitro-L-arginine methyl ester, AMT, tiron,
tempol, DETCA and V1-2 were dissolved in distilled water and
the others in dimethyl sulphoxide (DMSO, Sigma). DMSO
served as solvent control.

Significance of Study
Clinical use of COX-2 inhibitors in suppressing inflammatory
responses remains a hot topic in recent years due to their
reported adverse cardiovascular effects. While COX-2
inhibition was shown to increase blood pressure and
thrombotic events,
4, 5
it also produces benefits in patients with
hypertension and coronary artery disease.
6, 7
These seemingly
contradictory findings may suggest COX-2 taking up a Janus
role in producing different types of metabolites probably
depending on the nature of triggers under physiological and
pathological states. While the exact role of COX-2 remains
unresolved and preferentially targeting COX-2 in particular
systems is a challenging task, the present study provides a
novel insight into how COX-2 action can be modulated by
regulating its expression. In this study, we demonstrate an
important role of PKC

in mediating Ang II-induced COX-2

expression. Intriguingly, a recent study has shown that
adiponectin, a cardioprotective adipokine, up-regulates
endothelial COX-2 expression by activating
calreticulin/CD91-PI3K/Akt and promotes revascularization
after ischemia.
8
It is noteworthy that Ang II-induced COX-2
expression does not involve PI3K pathways (data not shown),
implying that COX-2 expression can be mediated by different
agonist-dependent pathways. If PKC

is selectively targeted in
a strategic manner to suppress the up-regulation of
pro-inflammatory COX-2, the expression of protective COX-2
can remain undisturbed and thus preventing the occurrence of
the scenario in which non-selective inhibition towards these
functionally different COX-2 abrogates the beneficial effects
at Chinese University of Hong Kong on April 22, 2011 atvb.ahajournals.org Downloaded from
Wong et al., 2011, Supplemental Material
P. 3
from the protective ones. Indeed, PKC

and PKC

are
preferentially activated in response to hyperglycemia and
diabetes
9
and PKC inhibitors such as ruboxistaurin targeting
the isoform has been introduced in clinical trials.
10
Inhibiting
COX-2 expression and thus subsequently down-regulating
MCP-1 release may offer an alternative to retard the initiation of
atherosclerosis. With the advancement in the development of
phosphopeptide mimetics, specific peptide inhibitors of PKC

may uncover novel therapeutic potential to combat vascular
inflammatory disease.

References
1. Huang Y, Chan NW, Lau CW, Yao XQ, Chan FL,
Chen ZY. Involvement of endothelium/nitric oxide in
vasorelaxation induced by purified green tea
(-)epicatechin. Biochim Biophys Acta.
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2. Wong SL, Leung FP, Lau CW, Au CL, Yung LM, Yao
X, Chen ZY, Vanhoutte PM, Gollasch M, Huang Y.
Cyclooxygenase-2-derived prostaglandin F2alpha
mediates endothelium-dependent contractions in the
aortae of hamsters with increased impact during
aging. Circ Res. 2009;104(2):228-235.
3. Jones MK, Sasaki E, Halter F, Pai R, Nakamura T,
Arakawa T, Kuroki T, Tarnawski AS. HGF triggers
activation of the COX-2 gene in rat gastric epithelial
cells: action mediated through the ERK2 signaling
pathway. FASEB J. 1999;13(15):2186-2194.
4. Bresalier RS, Sandler RS, Quan H, Bolognese JA,
Oxenius B, Horgan K, Lines C, Riddell R, Morton D,
Lanas A, Konstam MA, Baron JA, Adenomatous
Polyp Prevention on Vioxx Trial I. Cardiovascular
events associated with rofecoxib in a colorectal
adenoma chemoprevention trial. N Engl J Med.
2005;352(11):1092-1102.
5. Capone ML, Tacconelli S, Francesco LD, Petrelli M,
Patrignani P. Cardiovascular effects of valdecoxib:
transducing human pharmacology results into clinical
read-outs. Expert Opin Drug Saf. 2008;7(1):29-42.
6. Widlansky ME, Price DT, Gokce N, Eberhardt RT,
Duffy SJ, Holbrook M, Maxwell C, Palmisano J,
Keaney JF, Jr., Morrow JD, Vita JA. Short- and
long-term COX-2 inhibition reverses endothelial
dysfunction in patients with hypertension.
Hypertension. 2003;42(3):310-315.
7. Chenevard R, Hurlimann D, Bechir M, Enseleit F,
Spieker L, Hermann M, Riesen W, Gay S, Gay RE,
Neidhart M, Michel B, Luscher TF, Noll G, Ruschitzka
F. Selective COX-2 inhibition improves endothelial
function in coronary artery disease. Circulation.
2003;107(3):405-409.
8. Ohashi K, Ouchi N, Sato K, Higuchi A, Ishikawa TO,
Herschman HR, Kihara S, Walsh K. Adiponectin
promotes revascularization of ischemic muscle
through a cyclooxygenase 2-dependent mechanism.
Mol Cell Biol. 2009;29(13):3487-3499.
9. Meier M, King GL. Protein kinase C activation and its
pharmacological inhibition in vascular disease. Vasc
Med. 2000;5(3):173-185.
10. Geraldes P, King GL. Activation of protein kinase C
isoforms and its impact on diabetic complications.
Circ Res. 2010;106(8):1319-1331.
















Supplemental Figure I.
Confirmation of the endothelial identity in the primary
culture of cells. Primary culture of cells from rat thoracic
aortae were confirmed to be endothelial cells, which were
positively stained with an endothelial cell-specific maker,
PECAM-1. Fibroblasts served as a negative control to the
staining.


Supplemental Figure II.
AT
1
R-mediated COX-2 expression reaches a maximum in 8
h with 100 nmol/L Ang II and COX-2 mRNA is stabilized by
p38 MAPK, ERK1/2 and PKC

. A and B, COX-2 expression in

primary rat endothelial cells increased in response to Ang II in
(A) a time-dependent (1-8 h, 100 nmol/L) and (B)
concentration-dependent (3-100 nmol/L, 8 h) manner. C, Ang II
(100 nmol/L)-induced COX-2 expression was prevented by
pre-treatment of actinomycin-D (10 mol/L) and losartan (3
mol/L), but unaffected by the presence of PD 123319 (1
mol/L) and the solvent DMSO. D, Inhibitors of p38 MAPK (SB
202190, SB, 10 mol/L), ERK1/2 (PD 98059, PD, 20 mol/L)
and PKC

(rottlerin, ROT, 10 mol/L) all partially reduced

COX-2 mRNA stability in endothelial cells pre-exposed to Ang II
(100 nmol/L) for 4 h with a subsequent treatment of
actinomycin-D (AD, 10 mol/L). Results represent means
SEM of 4-6 experiments. **P<0.01, ***P<0.001 versus control;
#
P<0.05,
##
P<0.01 and
###
P<0.001 compared to Ang II (A-C) or
Ang II + AD group (D).





PECAM-1
Endothelial
cells
Fibroblasts
Negative
Control
200 m
PECAM-1
Endothelial
cells
Fibroblasts
Negative
Control
200 m
0 3 10 30 100
0
10
20
Ang II (nmol/L)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
***
#
#
COX-2
GAPDH
0 3 10 30 100
0
10
20
Ang II (nmol/L)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
***
#
#
0 3 10 30 100
0
10
20
Ang II (nmol/L)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
***
#
#
COX-2
GAPDH
COX-2
GAPDH
1 2 4 8
0
10
20 Control
Ang II 100 nmol/L
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
**
COX-2
GAPDH
1 2 4 8
0
10
20 Control
Ang II 100 nmol/L
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
**
COX-2
GAPDH
COX-2
GAPDH
C
o
n
t
r
o
l
A
n
g

I
I
A
c
t
in
o
m
y
c
in
-
D
L
o
s
a
r
t
a
n
P
D

1
2
3
3
1
9
D
M
S
O
0
10
20
Ang II
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
### ###
COX-2
GADPH
C
o
n
t
r
o
l
A
n
g

I
I
A
c
t
in
o
m
y
c
in
-
D
L
o
s
a
r
t
a
n
P
D

1
2
3
3
1
9
D
M
S
O
0
10
20
Ang II
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
### ###
C
o
n
t
r
o
l
A
n
g

I
I
A
c
t
in
o
m
y
c
in
-
D
L
o
s
a
r
t
a
n
P
D

1
2
3
3
1
9
D
M
S
O
0
10
20
Ang II
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
### ###
COX-2
GADPH
COX-2
GADPH
A B
0
5
10
15
20
C
O
X
-
2

m
R
N
A

l
e
v
e
l
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
Ang II
AD
SB
PD
ROT
DMSO
+ + + + + + + + +
+ + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
#
##
#
##
#
**
0
5
10
15
20
C
O
X
-
2

m
R
N
A

l
e
v
e
l
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
Ang II
AD
SB
PD
ROT
DMSO
+ + + + + + + + +
+ + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
#
##
#
##
#
**
C D
0 3 10 30 100
0
10
20
Ang II (nmol/L)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
***
#
#
COX-2
GAPDH
0 3 10 30 100
0
10
20
Ang II (nmol/L)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
***
#
#
0 3 10 30 100
0
10
20
Ang II (nmol/L)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
***
#
#
COX-2
GAPDH
COX-2
GAPDH
1 2 4 8
0
10
20 Control
Ang II 100 nmol/L
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
**
COX-2
GAPDH
1 2 4 8
0
10
20 Control
Ang II 100 nmol/L
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
***
**
COX-2
GAPDH
COX-2
GAPDH
C
o
n
t
r
o
l
A
n
g

I
I
A
c
t
in
o
m
y
c
in
-
D
L
o
s
a
r
t
a
n
P
D

1
2
3
3
1
9
D
M
S
O
0
10
20
Ang II
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
### ###
COX-2
GADPH
C
o
n
t
r
o
l
A
n
g

I
I
A
c
t
in
o
m
y
c
in
-
D
L
o
s
a
r
t
a
n
P
D

1
2
3
3
1
9
D
M
S
O
0
10
20
Ang II
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
### ###
C
o
n
t
r
o
l
A
n
g

I
I
A
c
t
in
o
m
y
c
in
-
D
L
o
s
a
r
t
a
n
P
D

1
2
3
3
1
9
D
M
S
O
0
10
20
Ang II
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
### ###
COX-2
GADPH
COX-2
GADPH
A B
0
5
10
15
20
C
O
X
-
2

m
R
N
A

l
e
v
e
l
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
Ang II
AD
SB
PD
ROT
DMSO
+ + + + + + + + +
+ + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
#
##
#
##
#
**
0
5
10
15
20
C
O
X
-
2

m
R
N
A

l
e
v
e
l
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
Ang II
AD
SB
PD
ROT
DMSO
+ + + + + + + + +
+ + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
#
##
#
##
#
**
C D
at Chinese University of Hong Kong on April 22, 2011 atvb.ahajournals.org Downloaded from
Wong et al., 2011, Supplemental Material
P. 4
Supplemental Figure III.
Unaltered COX-1 expression in the presence of Ang II and
various inhibitors. Constitutive expression of COX-1
remained changed in response to (A) Ang II across time (1-8 h,
100 nmol/L), (B) different concentrations of Ang II (3-100
nmol/L, 8h), and (C-F) various inhibitors.



Supplemental Figure IV.
PKC

was not phosphorylated at Threonine 505 (T505) or

Tyrosine 332 (Y332) in response to Ang II (100 nmol/L). In
contrast, these residues were phosphorylated by H
2
O
2
(5
mmol/L) at 10 min, which served as a positive control.
























Supplemental Figure V.
Translocation study (from cytosol to the membrane) of
various PKC isoforms in response to Ang II and PMA. A
and B, Validation on the extraction method of cytosolic and
membranous protein by detecting cytosolic GAPDH and
membrane-bound NOX-2. C through I, Translocation of various
PKC isoforms in response to 1-min stimulation of Ang II (100
nmol/L) or PMA (1 mol/L). Cyto, cytosol; M, membrane.
**P<0.01 and ***P<0.001 compared to the (A) cytosolic protein
or (B-H) membranous portion of control.









Supplemental Figure VI.
PKC

translocation study (from cytosol to the nucleus) in
response to Ang II. A, PKC

is not translocated into the

nucleus after 1-min Ang II stimulation. B and C, Fractionation of
cytosolic (cyto) and nuclear proteins was validated by the
cytosolic (b, GAPDH) and nuclear (c, histone H3) markers
respectively. ***P<0.001 versus cyto.

Supplemental Figure VII.
Ang II-induced COX-2 expression is mediated by p-38
MAPK and PKC

, without involvement of PKC

. A and B,
Co-treatment of SB 202190 (SB, 10 mol/L) and rottlerin (10
mol/L) abolished Ang II-induced COX-2 expression, while
these inhibitors did not affect COX-1 expression. C, V1-2 (10
mol/L) did not reduce COX-2 expression. ***P<0.001
compared to control;
#
P<0.05,
##
P<0.01 and
###
P<0.001
compared to Ang II-treated group.

Supplemental Figure VIII.
PKC

mediates COX-2 expression induced by Ang II or

exogenous PMA. A and B, Small interfering RNA targeting
PKC

(siPKC

) abolished expression of PKC

but not PKC

. C
and D, Ang II-induced COX-2 expression was significantly
reduced in siPKC

-transfected cells, treatment with SB 202190

(SB) further prevented COX-2 up-regulation by Ang II. E,
COX-2 expression peaked at 4-h incubation of PMA (1 mol/L),
while its negative analogue 4-PMA (1 mol/L) did not induce
COX-2 expression. F, PMA-induced COX-2 expression was
inhibited by GFX (2 mol/L) and rottlerin (10 mol/L), but not
Go 6976 (1 mol/L). *P<0.05, **P<0.01 and ***P<0.001
compared to respective control;
#
P<0.05,
##
P<0.01 and
###
P<0.001 compared to Ang II- or PMA-treated group.
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
COX-2
GAPDH
COX-2
GAPDH
A B C
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
COX-2
GAPDH
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
Ang II
Ang II
Ang II
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
COX-2
GAPDH
COX-2
GAPDH
A B C
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
COX-2
GAPDH
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
#
##
***
###
COX-2
GAPDH
COX-2
GAPDH
A B C
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
C
o
n
tro
l
A
n
g
II
V
1
-2

L
o
s
a
rta
n
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
##
COX-2
GAPDH
COX-2
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
C
o
n
tro
l
A
n
g
II
S
B
R
o
ttle
rin
S
B
+
R
o
ttle
rin
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
Ang II Ang II
Ang II Ang II
Ang II
0 3 10 30 100
0.0
0.5
1.0
1.5
Ang II (nmol/L)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
0 3 10 30 100
0.0
0.5
1.0
1.5
Ang II (nmol/L)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
1 2 4 8
0.0
0.5
1.0
1.5
Control
Ang II 100 nmol/L
Time (h)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
1 2 4 8
0.0
0.5
1.0
1.5
Control
Ang II 100 nmol/L
Time (h)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
C
o
n
tro
l
A
n
g
II
A
p
o
c
y
n
in
O
x
y
p
u
rin
o
l
T
e
m
p
o
l
T
iro
n
+
D
E
T
C
A
A
M
T
L
-N
A
M
E
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
tro
l
A
n
g
II
A
p
o
c
y
n
in
O
x
y
p
u
rin
o
l
T
e
m
p
o
l
T
iro
n
+
D
E
T
C
A
A
M
T
L
-N
A
M
E
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
tr
o
l
A
n
g
II
G
F
X
G
o
6
9
7
6
R
o
ttle
r
in
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
tr
o
l
A
n
g
II
G
F
X
G
o
6
9
7
6
R
o
ttle
r
in
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
Ang II
C
o
n
t
r
o
l
A
n
g
II
S
B
P
D
S
P
S
B
+
P
D
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
t
r
o
l
A
n
g
II
S
B
P
D
S
P
S
B
+
P
D
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
Ang II
C
o
n
tro
l
A
n
g
II
A
c
tin
o
m
y
c
in
-
D
L
o
s
a
r
ta
n
P
D
1
2
3
3
1
9
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
tro
l
A
n
g
II
A
c
tin
o
m
y
c
in
-
D
L
o
s
a
r
ta
n
P
D
1
2
3
3
1
9
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
Ang II
D E F
A B C
0 3 10 30 100
0.0
0.5
1.0
1.5
Ang II (nmol/L)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
0 3 10 30 100
0.0
0.5
1.0
1.5
Ang II (nmol/L)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
1 2 4 8
0.0
0.5
1.0
1.5
Control
Ang II 100 nmol/L
Time (h)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
1 2 4 8
0.0
0.5
1.0
1.5
Control
Ang II 100 nmol/L
Time (h)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
C
o
n
tro
l
A
n
g
II
A
p
o
c
y
n
in
O
x
y
p
u
rin
o
l
T
e
m
p
o
l
T
iro
n
+
D
E
T
C
A
A
M
T
L
-N
A
M
E
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
tro
l
A
n
g
II
A
p
o
c
y
n
in
O
x
y
p
u
rin
o
l
T
e
m
p
o
l
T
iro
n
+
D
E
T
C
A
A
M
T
L
-N
A
M
E
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
tr
o
l
A
n
g
II
G
F
X
G
o
6
9
7
6
R
o
ttle
r
in
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
tr
o
l
A
n
g
II
G
F
X
G
o
6
9
7
6
R
o
ttle
r
in
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
Ang II
C
o
n
t
r
o
l
A
n
g
II
S
B
P
D
S
P
S
B
+
P
D
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
t
r
o
l
A
n
g
II
S
B
P
D
S
P
S
B
+
P
D
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
Ang II
C
o
n
tro
l
A
n
g
II
A
c
tin
o
m
y
c
in
-
D
L
o
s
a
r
ta
n
P
D
1
2
3
3
1
9
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
Ang II
C
o
n
tro
l
A
n
g
II
A
c
tin
o
m
y
c
in
-
D
L
o
s
a
r
ta
n
P
D
1
2
3
3
1
9
D
M
S
O
0.0
0.5
1.0
1.5
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-1
GAPDH
COX-1
GAPDH
Ang II
D E F
A B C
Ang II
0 1 2 3 4 5 10 15 30 45 60 10 (min)
H
2
O
2
p-PKC(T505)
p-PKC(Y332)
PKC
GAPDH
Ang II
0 1 2 3 4 5 10 15 30 45 60 10 (min)
H
2
O
2
p-PKC(T505)
p-PKC(Y332)
PKC
GAPDH
Control Ang II
0
1
2
3
4
5
cyto nuclear
P
K
C

(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
PKC

Control Ang II
0
1
2
3
4
5
cyto nuclear
P
K
C

(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
PKC

GAPDH
cyto nuclear
0
2
4
6
8
G
A
P
D
H
(
O
p
t
ic
a
l
d
e
n
s
i
t
y
)
***
GAPDH
cyto nuclear
0
2
4
6
8
G
A
P
D
H
(
O
p
t
ic
a
l
d
e
n
s
i
t
y
)
***
cyto nuclear
0
2
4
6
8
H
is
t
o
n
e

H
3
(
O
p
t
ic
a
l
d
e
n
s
it
y
)
Histone
H3
***
cyto nuclear
0
2
4
6
8
H
is
t
o
n
e

H
3
(
O
p
t
ic
a
l
d
e
n
s
it
y
)
Histone
H3
cyto nuclear
0
2
4
6
8
H
is
t
o
n
e

H
3
(
O
p
t
ic
a
l
d
e
n
s
it
y
)
Histone
H3
***
A B C
Control Ang II
0
1
2
3
4
5
cyto nuclear
P
K
C

(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
PKC

Control Ang II
0
1
2
3
4
5
cyto nuclear
P
K
C

(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
PKC

GAPDH
cyto nuclear
0
2
4
6
8
G
A
P
D
H
(
O
p
t
ic
a
l
d
e
n
s
i
t
y
)
***
GAPDH
cyto nuclear
0
2
4
6
8
G
A
P
D
H
(
O
p
t
ic
a
l
d
e
n
s
i
t
y
)
***
cyto nuclear
0
2
4
6
8
H
is
t
o
n
e

H
3
(
O
p
t
ic
a
l
d
e
n
s
it
y
)
Histone
H3
***
cyto nuclear
0
2
4
6
8
H
is
t
o
n
e

H
3
(
O
p
t
ic
a
l
d
e
n
s
it
y
)
Histone
H3
cyto nuclear
0
2
4
6
8
H
is
t
o
n
e

H
3
(
O
p
t
ic
a
l
d
e
n
s
it
y
)
Histone
H3
***
A B C
C
o
n
tr
o
l
S
c
r
a
m
b
le
s
iP
K
C
0.0
0.5
1.0
Control
Ang II
C
O
X
-
2
/
G
A
P
D
H
*** ***
*
###
C
o
n
tr
o
l
S
c
r
a
m
b
le
s
iP
K
C
0.0
0.5
1.0
Control
Ang II
C
O
X
-
2
/
G
A
P
D
H
*** ***
*
###
Control Scramble siPKC
0.0
0.5
1.0
P
K
C

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
Control Scramble siPKC
0.0
0.5
1.0
P
K
C

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
C
o
n
tr
o
l
S
c
r
a
m
b
le
s
iP
K
C
+
S
B

s
iP
K
C
0.0
0.5
1.0
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
#
C
o
n
tr
o
l
S
c
r
a
m
b
le
s
iP
K
C
+
S
B

s
iP
K
C
0.0
0.5
1.0
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
#
Control Scramble siPKC

0.0
0.5
1.0
P
K
C

e
x
p
r
e
s
s
i
o
n
0 0.5 1 2 4 8 8
0
5
10
15
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
**
**
***
***
***
PMA
4-
PMA
0 0.5 1 2 4 8 8
0
5
10
15
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
**
**
***
***
***
PMA
4-
PMA
A
C
E
C
o
n
t
r
o
l
P
M
A
G
F
X
G
o

6
9
7
6
R
o
t
t
le
r
in
D
M
S
O
0
5
10
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
PMA (4 h)
***
##
##
C
o
n
t
r
o
l
P
M
A
G
F
X
G
o

6
9
7
6
R
o
t
t
le
r
in
D
M
S
O
0
5
10
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
PMA (4 h)
***
##
##
B
D
F
C
o
n
tr
o
l
S
c
r
a
m
b
le
s
iP
K
C
0.0
0.5
1.0
Control
Ang II
C
O
X
-
2
/
G
A
P
D
H
*** ***
*
###
C
o
n
tr
o
l
S
c
r
a
m
b
le
s
iP
K
C
0.0
0.5
1.0
Control
Ang II
C
O
X
-
2
/
G
A
P
D
H
*** ***
*
###
Control Scramble siPKC
0.0
0.5
1.0
P
K
C

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
Control Scramble siPKC
0.0
0.5
1.0
P
K
C

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
C
o
n
tr
o
l
S
c
r
a
m
b
le
s
iP
K
C
+
S
B

s
iP
K
C
0.0
0.5
1.0
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
#
C
o
n
tr
o
l
S
c
r
a
m
b
le
s
iP
K
C
+
S
B

s
iP
K
C
0.0
0.5
1.0
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
***
#
Control Scramble siPKC

0.0
0.5
1.0
P
K
C

e
x
p
r
e
s
s
i
o
n
0 0.5 1 2 4 8 8
0
5
10
15
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
**
**
***
***
***
PMA
4-
PMA
0 0.5 1 2 4 8 8
0
5
10
15
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
**
**
***
***
***
PMA
4-
PMA
A
C
E
C
o
n
t
r
o
l
P
M
A
G
F
X
G
o

6
9
7
6
R
o
t
t
le
r
in
D
M
S
O
0
5
10
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
PMA (4 h)
***
##
##
C
o
n
t
r
o
l
P
M
A
G
F
X
G
o

6
9
7
6
R
o
t
t
le
r
in
D
M
S
O
0
5
10
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
PMA (4 h)
***
##
##
B
D
F
cyto mb
0
2
4
6
N
O
X
-
2
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
**
NOX-2
cyto mb
0
2
4
6
N
O
X
-
2
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
**
cyto mb
0
2
4
6
N
O
X
-
2
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
**
NOX-2
cyto mb
0
5
10
G
A
P
D
H
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
***
GAPDH
cyto mb
0
5
10
G
A
P
D
H
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
***
cyto mb
0
5
10
G
A
P
D
H
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
***
GAPDH
C
o
n
tr
o
l
A
n
g
II
S
B
P
D
D
M
S
O
0
2
4
6
cyto mb
Ang II
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

**
C
o
n
tr
o
l
A
n
g
II
S
B
P
D
D
M
S
O
0
2
4
6
cyto mb
Ang II
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

**
Control Ang II PMA
0
2
4
6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

** **
Control Ang II PMA
0
2
4
6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

** **
Control Ang II PMA
0
2
4
6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
2
4
6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0.0
0.2
0.4
0.6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0.0
0.2
0.4
0.6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
4 cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
4 cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
4 cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

** **
Control Ang II PMA
0
1
2
3
4 cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

** **
A B C
D E F
G H I
cyto mb
0
2
4
6
N
O
X
-
2
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
**
NOX-2
cyto mb
0
2
4
6
N
O
X
-
2
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
**
cyto mb
0
2
4
6
N
O
X
-
2
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
**
NOX-2
cyto mb
0
5
10
G
A
P
D
H
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
***
GAPDH
cyto mb
0
5
10
G
A
P
D
H
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
***
cyto mb
0
5
10
G
A
P
D
H
(
O
p
t
i
c
a
l

d
e
n
s
i
t
y
)
***
GAPDH
C
o
n
tr
o
l
A
n
g
II
S
B
P
D
D
M
S
O
0
2
4
6
cyto mb
Ang II
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

**
C
o
n
tr
o
l
A
n
g
II
S
B
P
D
D
M
S
O
0
2
4
6
cyto mb
Ang II
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

**
Control Ang II PMA
0
2
4
6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

** **
Control Ang II PMA
0
2
4
6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

** **
Control Ang II PMA
0
2
4
6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
2
4
6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0.0
0.2
0.4
0.6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0.0
0.2
0.4
0.6
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
4 cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
4 cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

Control Ang II PMA

0
1
2
3
4 cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

** **
Control Ang II PMA
0
1
2
3
4 cyto mb
P
K
C

(
O
p
t
i
c
a
l

d
e
n
i
s
t
y
)
PKC

** **
A B C
D E F
G H I
at Chinese University of Hong Kong on April 22, 2011 atvb.ahajournals.org Downloaded from
Wong et al., 2011, Supplemental Material
P. 5




Supplemental Figure IX.
Representative images of DHE fluorescence. The left panel
of each pair of images showed the DHE fluorescence signal (in
red) on the production of reactive oxygen species, while the
right panel showed the brightfield images of the endothelial
cells.



Supplemental Figure X.
Reactive oxygen species are not involved in Ang
II-induced COX-2 expression. A, DHE fluorescence showed
that production of reactive oxygen species in the endothelial
cells increased about 2-fold in 30 s after Ang II (100 nmol/L)
addition, which were inhibited by losartan (3 mol/L), apocynin
(100 mol/L) and tiron (1 mmol/L) plus DETCA (100 mol/L). B,
COX-2 expression was not inhibited in the presence of various
ROS inhibitors. C and D, H
2
O
2
only induced a very low level of
COX-2 expression compared to Ang II, and COX-1 expression
was not altered with either H
2
O
2
or Ang II treatment. *P<0.05
and ***P<0.001 versus control;
#
P<0.05 compared with Ang
II-treated group.












A













B


















Supplemental Figure XI.
Different levels of oxidative stress were induced by Ang II
and BMP4 in the endothelial cells. A, Representative images
of DHE fluorescence in rat aortic endothelial cells in response
to Ang II (0.1 and 1 mol/L) and BMP4 (20 ng/mL). B, BMP4
induced a higher level of reactive oxygen species compared to
Ang II. *P<0.05 versus control;
#
P<0.05 compared with Ang
II-treated group.
0 1 2 4 8 8
0
10
20
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
H
2
O
2
*
***
Ang II
0 1 2 4 8 8
0
10
20
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
H
2
O
2
*
***
Ang II
C
o
n
t
r
o
l
A
n
g

I
I
L
o
s
a
r
t
a
n
A
p
o
c
y
n
in
T
ir
o
n

+

D
E
T
C
A
D
M
S
O
0
1
2
3
R
e
l
a
t
i
v
e

f
l
u
o
r
e
s
c
e
n
c
e
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
# #
#
Ang II
C
o
n
t
r
o
l
A
n
g

I
I
L
o
s
a
r
t
a
n
A
p
o
c
y
n
in
T
ir
o
n

+

D
E
T
C
A
D
M
S
O
0
1
2
3
R
e
l
a
t
i
v
e

f
l
u
o
r
e
s
c
e
n
c
e
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
# #
#
C
o
n
t
r
o
l
A
n
g

I
I
L
o
s
a
r
t
a
n
A
p
o
c
y
n
in
T
ir
o
n

+

D
E
T
C
A
D
M
S
O
0
1
2
3
R
e
l
a
t
i
v
e

f
l
u
o
r
e
s
c
e
n
c
e
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
# #
#
Ang II
0 1 2 4 8 8
0.0
0.5
1.0
Time (h)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
H
2
O
2
Ang II
COX-1
GAPDH
0 1 2 4 8 8
0.0
0.5
1.0
Time (h)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
H
2
O
2
Ang II
COX-1
GAPDH
C
o
n
t
r
o
l
A
n
g

I
I
A
p
o
c
y
n
in
O
x
y
p
u
r
in
o
l
T
e
m
p
o
l
T
ir
o
n

+

D
E
T
C
A
A
M
T
L
-
N
A
M
E
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
***
Ang II
C
o
n
t
r
o
l
A
n
g

I
I
A
p
o
c
y
n
in
O
x
y
p
u
r
in
o
l
T
e
m
p
o
l
T
ir
o
n

+

D
E
T
C
A
A
M
T
L
-
N
A
M
E
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
COX-2
GAPDH
***
Ang II
A B
C D
0 1 2 4 8 8
0
10
20
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
H
2
O
2
*
***
Ang II
0 1 2 4 8 8
0
10
20
Time (h)
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
H
2
O
2
*
***
Ang II
C
o
n
t
r
o
l
A
n
g

I
I
L
o
s
a
r
t
a
n
A
p
o
c
y
n
in
T
ir
o
n

+

D
E
T
C
A
D
M
S
O
0
1
2
3
R
e
l
a
t
i
v
e

f
l
u
o
r
e
s
c
e
n
c
e
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
# #
#
Ang II
C
o
n
t
r
o
l
A
n
g

I
I
L
o
s
a
r
t
a
n
A
p
o
c
y
n
in
T
ir
o
n

+

D
E
T
C
A
D
M
S
O
0
1
2
3
R
e
l
a
t
i
v
e

f
l
u
o
r
e
s
c
e
n
c
e
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
# #
#
C
o
n
t
r
o
l
A
n
g

I
I
L
o
s
a
r
t
a
n
A
p
o
c
y
n
in
T
ir
o
n

+

D
E
T
C
A
D
M
S
O
0
1
2
3
R
e
l
a
t
i
v
e

f
l
u
o
r
e
s
c
e
n
c
e
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
***
# #
#
Ang II
0 1 2 4 8 8
0.0
0.5
1.0
Time (h)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
H
2
O
2
Ang II
COX-1
GAPDH
0 1 2 4 8 8
0.0
0.5
1.0
Time (h)
C
O
X
-
1

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
H
2
O
2
Ang II
COX-1
GAPDH
C
o
n
t
r
o
l
A
n
g

I
I
A
p
o
c
y
n
in
O
x
y
p
u
r
in
o
l
T
e
m
p
o
l
T
ir
o
n

+

D
E
T
C
A
A
M
T
L
-
N
A
M
E
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
***
Ang II
C
o
n
t
r
o
l
A
n
g

I
I
A
p
o
c
y
n
in
O
x
y
p
u
r
in
o
l
T
e
m
p
o
l
T
ir
o
n

+

D
E
T
C
A
A
M
T
L
-
N
A
M
E
0
10
20
C
O
X
-
2

e
x
p
r
e
s
s
i
o
n
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
COX-2
GAPDH
COX-2
GAPDH
***
Ang II
A B
C D
Ang II 0.1 mol/L
Ang II 1 mol/L
BMP4
Control
500 m
Ang II 0.1 mol/L Ang II 0.1 mol/L
Ang II 1 mol/L Ang II 1 mol/L
BMP4 BMP4 BMP4
Control
500 m
Control
500 m
Control
500 m 500 m
C
o
n
t
r
o
l
m
o
l
/
L
)

A
n
g

I
I

(
0
.
1

m
o
l
/
L
)

A
n
g

I
I

(
1

B
M
P
4

0
1
2
D
H
E

f
l
u
o
r
e
s
c
e
n
c
e
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
*
*
*
NS
#
C
o
n
t
r
o
l
m
o
l
/
L
)

A
n
g

I
I

(
0
.
1

m
o
l
/
L
)

A
n
g

I
I

(
1

B
M
P
4

0
1
2
D
H
E

f
l
u
o
r
e
s
c
e
n
c
e
(
F
o
l
d

o
f

c
o
n
t
r
o
l
)
*
*
*
NS
#
at Chinese University of Hong Kong on April 22, 2011 atvb.ahajournals.org Downloaded from