Raloxifene improves vascular reactivity in pressurized septal coronary arteries of ovariectomized hamsters fed cholesterol diet.

Abstract

Although vascular effects of selective estrogen receptor modulators (SERMs) have been extensively examined in conduit arteries, whether SERMs could favorably modulate myogenic response in resistance arteries is unknown. The impact of raloxifene therapy and cholesterol diet on myogenic constriction during estrogen deficiency is unresolved. This study investigated changes in vascular reactivity and myogenic responses in female ovariectomized (Ovx) hamsters fed high-cholesterol diet (HCD) with and without chronic treatment of raloxifene. Functional studies were performed on hamster septal coronary arteries cannulated in a pressure myograph. Acetylcholine (ACh)-induced dilatation was reduced in arteries from cholesterol-fed Ovx hamsters, but not in those from cholesterol-fed hamsters, while pressure-induced myogenic constriction was unaffected. Chronic treatment with raloxifene restored ACh-induced dilatation in cholesterol-fed Ovx hamsters. U46619-induced constriction was increased in arteries from cholesterol-fed Ovx hamsters but not from cholesterol-fed control hamsters, which was normalized by chronic raloxifene treatment. The pressure-diameter relationship is presented as normalized diameter versus intraluminal pressure, while the effect of ACh or U46619 is expressed as percentage of tone at 80 mm Hg. Two-way analysis of variance (ANOVA) followed by Bonferroni post-tests were used for statistical evaluation among different treatment groups. P<0.05 was taken as statistically significant. The present results show that chronic treatment with raloxifene could benefit myogenically active coronary arteries by (i) restoring ACh-induced dilatation and (ii) reducing U46619-induced constriction without affecting pressure-induced myogenic responses in cholesterol-fed hamsters during estrogen deficiency. If such benefit can be observed in humans, raloxifene and other SERMs may be useful to preserve endothelial function and curtail vascular hypersensitivity in resistance coronary arteries in post-menopausal women with hypercholesterolemia or hyperlipidemia, a lipid condition implicated in the pathogenesis of myocardial infarction.

Full text

Page 1

Pharmacological
Research
65 (2012) 182–
188
Contents
lists
available
at SciVerse
ScienceDirect
Pharmacological
Research
journa
l h o me
pa ge:
www.elsevier.com/locate/yphr
s
Raloxifene
ovariectomized
improves
vascular
reactivity
in
pressurized
septal
coronary
arteries
of
hamsters
fed
cholesterol
diet
Yau-Chi
Xiaoqiang
Chana,∗,
Fung
Ping
Leunga,
Xiao
Yu
Tiana,
Lai
Ming
Yunga,
Chi
Wai
Laua,
Zhen
Yu
Chenb,
Yaoa,
Ismail
Laherc,
Yu
Huanga,∗
aInstitute
bSchool
cDepartment
of Vascular
Medicine,
Li Ka Shing
Institute
of
Health
Sciences,
and
School
of
Biomedical
Sciences,
Chinese
University
of
Hong
Kong,
Hong
Kong,
China
of
Life
Sciences,
Chinese
University
of
Hong
Kong,
Hong
Kong,
China
of Pharmacology
and
Therapeutics,
University
of
British
Columbia,Canada
a
r
t
i
c
l
e

i
n
f
o
Article
Received
Received
20
Accepted
history:
4 July
2010
in revised
form
September
2011
20 September
2011
Keywords:
Cholesterol
Raloxifene
Ovariectomy
Myogenic
Septal
Hamster
constriction
coronary
arteries
a
b
s
t
r
a
c
t
Although
ined
arteries
ing
responses
chronic
ies
from
induced
dilatation
cholesterol-fed
chronic
versus
80
tistical
present
arteries
affecting
such
lial
women
of
vascular
effects
of
selective
estrogen
receptor
modulators
(SERMs)
have
been
extensively
exam-
in
conduit
arteries,
whether
SERMs
could
favorably
modulate
myogenic
response
in
resistance
is
unknown.
The
impact
of
raloxifene
therapy
and
cholesterol
diet
on
myogenic
constriction
dur-
estrogen
deficiency
is
unresolved.
This
study
investigated
changes
in
vascular
reactivity
and
myogenic
in
female
ovariectomized
(Ovx)
hamsters
fed
high-cholesterol
diet
(HCD)
with
and
without
treatment
of
raloxifene.
Functional
studies
were
performed
on
hamster
septal
coronary
arter-
cannulated
in
a
pressure
myograph.
Acetylcholine
(ACh)-induced
dilatation
was
reduced
in
arteries
cholesterol-fed
Ovx
hamsters,
but
not
in
those
from
cholesterol-fed
hamsters,
while
pressure-
myogenic
constriction
was
unaffected.
Chronic
treatment
with
raloxifene
restored
ACh-induced
in
cholesterol-fed
Ovx
hamsters.
U46619-induced
constriction
was
increased
in
arteries
from
Ovx
hamsters
but
not
from
cholesterol-fed
control
hamsters,
which
was
normalized
by
raloxifene
treatment.
The
pressure–diameter
relationship
is
presented
as
normalized
diameter
intraluminal
pressure,
while
the
effect
of
ACh
or
U46619
is expressed
as
percentage
of
tone
at
mmHg.
Two-way
analysis
of
variance
(ANOVA)
followed
by
Bonferroni
post-tests
were
used
for
sta-
evaluation
among
different
treatment
groups.
P
<
0.05
was
taken
as
statistically
significant.
The
results
show
that
chronic
treatment
with
raloxifene
could
benefit
myogenically
active
coronary
by
(i)
restoring
ACh-induced
dilatation
and
(ii)
reducing
U46619-induced
constriction
without
pressure-induced
myogenic
responses
in
cholesterol-fed
hamsters
during
estrogen
deficiency.
If
benefit
can
be
observed
in
humans,
raloxifene
and
other
SERMs
may
be
useful
to
preserve
endothe-
function
and
curtail
vascular
hypersensitivity
in
resistance
coronary
arteries
in
post-menopausal
with
hypercholesterolemia
or
hyperlipidemia,
a
lipid
condition
implicated
in
the
pathogenesis
myocardial
infarction.
© 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
The
development
of
SERMs
allows
the
clinical
use
of
estro-
gen
replacement
estrogen-agonist/antagonist
exhibit
nervous
uterus.
substitutes
that
are
devoid
of side
effects
of
hormone
therapy.
Due
to their
tissue-specific
and
mixed
properties,
SERMs
such
as
raloxifene
estrogenic
activities
in bone,
cardiovascular
and
central
systems,
while
avoiding
harmful
effects
in breast
and
Raloxifene
exerts
estrogen-like
cardioprotective
actions
∗Corresponding
Hong
E-mail
yu-huang@cuhk.edu.hk
authors
at:
School
of
Biomedical
Sciences,
Chinese
University
of
Kong,
Shatin,
NT,
Hong
Kong,
China.
Tel.:
+852
39436787;
fax:
+852
26035022.
addresses:
yc
chan@alumni.cuhk.net
(Y.-C.
Chan),
(Y.
Huang).
and
nitric
sheep
arteries
[3]. Chronic
risks
and
recently
centrations
mechanism
lial
resistance
significant
the
to
improves
coronary
blood
flow
partly
by
releasing
endothelial
oxide
(NO)
[1,2]
in animal
studies.
Ovariectomized
(Ovx)
taking
raloxifene
have
greater
diameters
in their
coronary
compared
with
those
receiving
estrogen
or no treatment
treatment
with
raloxifene
may
reduce
cardiovascular
in women
[4]
and
prevents
endothelial
dysfunction
in young
aging
Ovx
rats
by
increasing
NO
bioavailability
[5,6].
We
have
reported
that
raloxifene
at
therapeutically
relevant
con-
inhibits
myogenic
constriction
by
an
NO-dependent
that
causally
involves
[Ca2+]ielevation
in endothe-
cells
and
subsequent
eNOS
activation
and
raloxifene
dilates
arteries
more
effectively
in female
rats,
indicating
a
gender-related
action
in microcirculation
[7].
Although
outcomes
of the
RUTH
trial
showed
that
raloxifene
failed
reduce
the
overall
risk
of
coronary
heart
disease
(CHD)
in
1043-6618/$
doi:10.1016/j.phrs.2011.09.010
– see
front
matter ©

2011 Elsevier Ltd. All rights reserved.

Page 2

Y.-C.
Chan
et
al.
/ Pharmacological
Research
65 (2012) 182–
188
183
postmenopausal
for
from
cholesterolaemia
Diets
total
sters
risks
documented
Evaluation)
Although
striction
could
study
tivity
cholesterol
ifene.
women
with
established
CHD
or at
increased
risk
CHD
[8],
how
SERMs
could
specifically
benefit
coronary
arteries
postmenopausal
women
with
hyperlipidemia
and/or
hyper-
is still
unclear
and
it does
deserve
investigation.
rich
in fat
and
cholesterol
are
routinely
used
to increase
the
lipids
and
low-density
lipoprotein
cholesterol
(LDL-C)
in ham-
and
rabbits.
High
plasma
LDL-C
is
associated
with
increased
of
CHD,
myocardial
infarction
and
stroke
[9].
In women
with
CHD
in the
MORE
(Multiple
Outcomes
of Raloxifene
study,
raloxifene
reduces
LDL-C
[10].
estrogen
has
been
shown
to reduce
myogenic
con-
in resistance
arteries,
it is unclear
whether
raloxifene
also
favorably
modulate
myogenic
responses.
Therefore,
this
was
designed
to study
changes
in septal
coronary
artery
reac-
and
myogenic
responses
of
female
Ovx
hamsters
fed
high
diet
with
and
without
chronic
treatment
with
ralox-
2.
Materials
and
methods
This
study
was
approved
by the
Animal
Research
Ethics
Com-
mittee
The
US
1996).
of
the
Chinese
University
of
Hong
Kong
and
conformed
with
Guide
for
the
Care
and
Use
of Laboratory
Animals
published
by
the
National
Institute
of
Health
(NIH
Publication
No.85-23,
revised
2.1.
Animals
Twenty-nine
supplied
versity
diet
diet
diet
plus
Hamsters
controlled
light
before
Body
twice
the
female
Syrian
golden
hamsters
(∼12-month
old),
by the
Laboratory
Animal
Service
Center
at
Chinese
Uni-
of
Hong
Kong,
were
randomly
divided
into
5 groups:
normal
(ND,
n = 7),
high
cholesterol
diet
(HCD,
n = 5),
high
cholesterol
with
raloxifene
treatment
(HCD
+
Rf,
n
=
5),
high
cholesterol
plus
ovariectomy
(HCD
+ Ovx,
n = 6),
and
high
cholesterol
diet
ovariectomy
with
raloxifene
treatment
(HCD
+ Ovx
+ Rf,
n
= 6).
had
free
access
to either
ND or
HCD
and
maintained
under
humidity
(∼48%)
and
temperature
(20◦C),
and
a
12-h
cycle
for
15
weeks.
For
OVX
rats,
ovariectomy
was
performed
HCD
feeding.
Table
1 shows
the
composition
of
HCD
and
ND.
weights
and
food
consumption
were
measured
weekly
and
a week,
respectively,
while
wet
weight
of
uteri
was
taken
at
time
of
sacrifice.
2.2.
Raloxifene
administration
HCD
+ Rf
and
HCD
+ Ovx
+ Rf
were
subjected
to raloxifene
treatment.
of ketamine
a raloxifene-filled
subcutaneously
Hamsters
were
anesthetized
with
a 3-ml
mixture
and
xylaxine
(1.5–1,
v/v)
per
kg body
weight
and
Silastic®tubing
(Dow
Corning)
was
inserted
in the
back
of
each
hamster
[11]. The
average
Table
Composition
1
of
normal
and
high
cholesterol
diets.
Normal
diet

High
cholesterol
diet
Ingredient
Casein
Lard
Starch
Sucrose
Mineral
Vitamin
DL-Methionine
Pure
Gelatin
Fibre
Total

(%)
20

(%)
20
20
38.6
10
4
2
0.1
0.1
2
3.2
100


5

53.7
10
4
2
0.1
0
2
3.2
100

mix

mix


cholesterol




daily
difference
treatment,
raloxifene
release
in each
hamster
was
calculated
by
weight
of
raloxifene
resided
in the
tubing
before
and
after
which
was
0.0986
?g/kg
body
weight/day.
2.3.
Ovariectomy
For
HCD
+ Ovx
and
HCD
+ Ovx
+ Rf
groups,
ovariectomy
was
per-
formed
underwent
a
after
before
HCD
feeding.
During
ovariectomy,
the
hamsters
anesthesia
followed
by aseptic
bilateral
ovariectomy
via
mid-abdominal
route
[6].
Raloxifene
treatment
started
2 weeks
ovariectomy
in HCD
+ Ovx
+ Rf
group.
2.4.
Coronary
artery
preparation
After
15-week
treatment
with
of
raloxifene,
hamsters
were
euthanized
in
outer
nective
which
nology,
various
between
chamber
with
a temperature
cannula
solution
intraluminal
lated.
(model
nulae
seated
luminal
conditions.
(Zeiss
ize
Changes
using
Denmark).
by
CO2inhalation.
The
hearts
were
removed
and
placed
ice-cold
Krebs
solution.
Septal
coronary
arteries
(∼250
?m
in
diameter)
were
dissected
and
cleaned
of
surrounding
con-
tissues.
Each
artery
was
cut
into
2–3
mm
long
ring
segments
were
mounted
on
a pressure
myograph
(Danish
Myo
Tech-
Denmark).
Changes
in vessel
diameter
were
measured
at
intraluminal
pressures.
Briefly,
each
artery
was
cannulated
two
cannulae
(outer
diameter
of ∼100
?m)
in a
10-ml
filled
with
Krebs
solution
which
was
continuously
gassed
95%
O2–5% CO2and
maintained
at
37◦C (pH
7.4)
monitored
by
probe.
One
end
of the
artery
was
first
secured
on
a
with
two
fine
nylon
sutures
and
then
perfused
with
Krebs
at a pressure
difference
<20
mmHg
to remove
residual
blood.
The
other
end
of
the
artery
was
then
cannu-
Glass
cannulae
were
prepared
using
a micropipette
puller
P-87,
Shutter
Instrument
Company,
Novato,
CA).
Both
can-
were
connected
to separate
Krebs
solution-containing
bottles
on
a
pressure
regulator
that
controlled
flow
rate
and
intra-
pressure.
Myogenic
constriction
developed
under
no-flow
A video
camera
attached
to a light-inverted
microscope
Axiovert
40 Microscope,
Model
110P)
was
used
to visual-
the
artery
and
data
were
recorded
and
stored
on
a computer.
in outer
diameter
and
luminal
pressure
were
determined
Myo-View
software
(version
1.1
P,
Photonics
Engineering,
2.5.
Experimental
protocols
2.5.1.
Development
After
was
20
pressure
up
solution
ment.
response
diameter
of myogenic
constriction
mounting
in pressure
myograph,
intraluminal
pressure
increased
from
20 to 100
mmHg
at
room
temperature
in
mmHg
increment
each
5 min.
After
this
procedure,
intraluminal
was
maintained
at
80 mmHg
and
superfusate
was
warmed
gradually
to 37◦C using
a
built-in
heating
device.
The
chamber
was
kept
at 37◦C for
the
remaining
part
of
the
experi-
When
the
superfusate
temperature
reached
37◦C,
myogenic
developed
sharply,
as
recorded
by a decrease
in the
vessel
that
stabilized
after
10–20
min.
2.5.2.
Pressure–diameter
To
ment
obtained
20
experiment,
EGTA-containing
repeated
each
relationships
compare
difference
in myogenic
responses
in various
treat-
groups,
the
pressure–diameter
relationships
of arteries
were
by
stepwise
increment
of
intraluminal
pressure
from
to 120
mmHg
in Krebs
solution
at 37◦C.
At the
end
of
the
arteries
were
superfused
with
a Ca2+-free
and
2
mM
Krebs
solution
and
pressure–diameter
curve
was
again
to obtain
the
maximum
passive
vasodilatation
at
pressure
step
(Fig.
1).

Page 3

184
Y.-C.
Chan
et al.
/ Pharmacological
Research
65 (2012) 182–
188
Fig.
experimental
tion.
endothelium-dependent
intraluminal
the
1.
Tracing
of
vessel
diameter
change
with
corresponding
intraluminal
pressure.
Upper
panel:
a representative
original
trace
with
a snapshot
displaying
the
whole
protocol
performed
in an endothelium-intact
septal
coronary
artery
from
a rat
fed
normal
diet.
It took
∼20
min
for
a full
generation
of
myogenic
constric-
Thereafter,
two
sets
of
stepwise
intraluminal
pressure
increment
were
performed
to obtain
control
vessel
diameter
data.
Acetylcholine
(ACh)
was
used
to examine
dilatation
while
U46619
was
used
to trigger
concentration-dependent
constriction
after
washout
of
ACh
in myogenically
active
arteries
at 80
mmHg
pressure.
Finally,
the
vessel
was
fully
dilated
by perfusion
of
a Ca2+-free
Krebs
solution
containing
2 mM
EGTA
for
the
purpose
of
data
calculation.
Lower
panel:
intraluminal
pressure
changes
corresponding
to vessel
diameter
changes.
2.5.3.
Effects
After
of drugs
full
development
of
myogenic
constriction,
intralumi-
nal
vessel
Vasodilatation
of myogenic
was
NO,
ence
(NOS)
time
pressure
was
maintained
at 80
mmHg
and
changes
in outer
diameter
in response
to ACh
and
U46619
were
recorded.
to ACh
(3 nM–30
?M)
was
studied
in the
presence
tone
while
vasoconstriction
to U46619
(1–300
nM)
examined
after
ACh
washout.
To assess
the
role
of
endothelial
myogenic
constriction
was
compared
in the
absence
and
pres-
(30-min
incubation)
of
30
?M
L-NAME,
a nitric
oxide
synthase
inhibitor.
Drugs
were
applied
through
the
superfusate
at
the
indicated
by
arrows
(Fig.
1).
2.6.
Serum
lipid
and
lipoproteins
Serum
determined
USA).
sured
low-density
magnesium
triglycerides
(TG)
and
total
cholesterol
(TC)
levels
were
with
enzymatic
kits
(Sigma
Chemical,
St.
Louis,
MO,
High-density
lipoprotein
cholesterol
(HDL-C)
was
mea-
after
precipitation
of
low-density
lipoprotein
(LDL)
and
very
lipoprotein
(VLDL)
with
phosphotungstic
acid
and
chloride,
using
a commercial
kit
(Sigma)
[12].
2.7.
Drugs
and
solutions
Acetylcholine
L-NAME
([9,11-Dideoxy-11?,9?-epoxymethano-prostaglandin
were
purchased fromSigma.
hydroxyphenyl)benzo[b]thiophen-3-yl][4-[2-piperidinoethoxy]-
phenyl]ketone
Center
dissolved
stock
2.5
([2-(acetyloxy)-N,N,N-trimethylethanaminium]),
(Nw-nitro-L-argininemethyl

ester),

and

U46619
F2?])


Raloxifene

([6-hydroxy-2-(4-
hydrochloride)
was
a gift
from
Lilly
Corporate
(Indianapolis,
IN,
USA).
Raloxifene
and
U46619
were
in DMSO
(Sigma).
Further
dilutions
were
made
from
solutions.
Krebs
solution
contained
(mM):
119
NaCl,
4.7
KCl,
CaCl2, 1 MgCl2, 25 NaHCO3, 1.2
KH2PO4, and
11
d-glucose.
Ca2+-free
exclusion
Krebs
solution
was
identical
to Krebs
solution
with
the
of
Ca2+and
the
addition
of
2 mM
EGTA.
2.8.
Data
analysis
The
pressure–diameter
relationship
is
presented
as
normal-
ized
of
absence
percentage
culated
passive
particular
complete
are
analysis
were
were
diameter
(DN) versus
intraluminal
pressure.
DNis
the
ratio
vessel
diameter
at a
particular
pressure
in the
presence
and
of
Ca2+. The
effect
of
ACh
or U46619
is expressed
as
of
tone
(% tone)
at
80
mmHg.
Percentage
of tone
was
cal-
as
(Dmax−
Dp)/(Dmax− D80) × 100%,
where
Dmax=
maximal
diameter
in the
absence
of
Ca2+; Dp=
diameter
at the
concentration
of
ACh
or U46619;
D80= diameter
after
development
of
myogenic
response
at
80
mmHg.
Results
means
±
SEM
of
separate
arteries
from
n animals.
Two-way
of
variance
(ANOVA)
followed
by
Bonferroni
post-tests
used
for
statistical
evaluation
when
more
than
two
groups
compared.
P < 0.05
was
taken
as
statistically
significant.
3.
Results
3.1.
Animal
parameters
Hamsters
sacrifice
them
sue,
(Fig.
in all
groups
had
similar
body
weight
at
the
time
of
(Fig.
2a).
Daily
food
consumption
did
not
differ
among
(data
not
shown).
Ovariectomy
caused
atrophy
of uterine
tis-
which
was
unaffected
by
chronic
treatment
with
raloxifene
2b).
3.2.
Serum
cholesterol,
triglyceride
and
LDL
Serum
vated
cholesterol,
triglyceride
and
LDL
were
significantly
ele-
in HCD-fed
groups.
Ovariectomy
did
not
cause
further

Page 4

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/ Pharmacological
Research
65 (2012) 182–
188
185
0
100
200
Body Weight (g)
*
NS NS
ND
HCD+Ovx
HCD+Ovx+Rf
HCD
HCD+Rf
0.0
0.4
0.8
1.2
Uterine Weight (g)
***
NS
NS
a
b
Fig.
diet;
significant.
2.
Body
weight
(a)
and
wet
uterine
weight
(b)
at the
time
of
sacrifice.
ND,
normal
HCD,
high-cholesterol
diet;
Ovx,
ovariectomy;
Rf,
raloxifene
treatment.
NS,
not
Results
are
means
± SEM
of
5–6 hamsters
(*P < 0.05;***P < 0.001).
increases
ifene
in serum
levels
of
cholesterol,
triglyceride
and
LDL;
ralox-
treatment
did
not
affect
these
parameters
(Fig.
3a–c).
3.3.
High
cholesterol
diet
and
myogenic
response
Pressure-induced
and
U46619-induced
ferent
constriction
was
similar
in arteries
from
ND
HCD
hamsters
(Fig.
4a).
ACh-induced
dilatation
(Fig.
4b)
and
constriction
(Fig.
4c)
in HCD
group
were
not
dif-
from
those
in ND
group.
3.4.
hamsters
Ovariectomy
and
myogenic
response
in high
cholesterol-fed
Ovariectomy
arteries
opment
in
In
constriction
did
not
modify
pressure-induced
constriction
of
from
HCD
hamsters
(Fig.
4d).
After
steady-state
devel-
of
myogenic
tone,
ACh
induced
paradoxical
constriction
HCD
+
Ovx
hamsters
but
dilatation
in HCD
hamsters
(Fig.
4e).
addition,
ovariectomy
significantly
enhanced
U46619-induced
in HCD
hamsters
(P < 0.05,
Fig.
4f).
3.5.
Ovariectomy
and
myogenic
response
in normal-fed
hamsters
Upon
ovariectomy,
septal
coronary
arteries
from
Ovx
hamsters
fed
as
tomy
constriction
and
normal
diet
exhibited
the
same
diameter–pressure
relationship
those
fed
with
a high-cholesterol
diet
(Fig.
5a).
However,
ovariec-
neither
altered
ACh-induced
dilatations
nor
U46619-induced
in the
vessels
from
normal
diet-fed
hamsters
(Fig.
5b
c).
3.6.
cholesterol-fed
Effect
of
raloxifene
on vascular
reactivity
in high
OVX
hamsters
Chronic
constriction
U46619
diet
raloxifene
treatment
did
not
modify
pressure-induced
(Fig.
6a),
dilatation
to ACh
(Fig.
6b),
or constriction
to
in sham-operated
control
hamsters
fed
high-cholesterol
(Fig.
6c).
0
100
200
300
400
Cholesterol (µg/ml)
ND
HCD+Ovx
HCD+Ovx+Rf
HCD
HCD+Rf
0
100
200
LDL (µg/ml)
**
***
a
b
c
*
0
100
200
300
Triglyceride (µg/ml)
Fig.
time
raloxifene
***P < 0.001
3. Serum
levels
of
cholesterol
(a),
triglyceride
(b)
and
LDL
(c)
of
hamsters
at
the
of
sacrifice.
ND,
normal
diet;
HCD,
high
cholesterol
diet;
Ovx,
ovariectomy;
Rf,
treatment.
Results
are
means
± SEM
of
5–6
hamsters
(*P < 0.05;**P < 0.01;
between
ND
and
other
groups).
Likewise,
induced
a
significantly
hamsters
striction
chronic
raloxifene
treatment
did
not
affect
pressure-
constriction
in coronary
arteries
from
Ovx
hamsters
fed
high-cholesterol
diet
(Fig.
6d).
However,
raloxifene
therapy
restored
ACh-induced
dilatations
in HCD
+ Ovx
(P <
0.05,
Fig.
6e)
and
also
abolished
the
augmented
con-
to U46619
in the
same
group
of hamsters
(P <
0.05,
Fig.
6f).
3.7.
Effect
of
L-NAME
on myogenic
tone
L-NAME
affect
from
(30
?M)
added
during
experiment
was
proved
not
to
pressure-induced
constriction
in septal
coronary
arteries
all
treatment
groups
(data
not
shown).
4.
Discussion
The
present
study
shows
that
in myogenically
active
sep-
tal
vascular
in
deficiency.
induced
pressure-induced
therapy
coronary
arteries
chronic
treatment
with
raloxifene
benefits
function
without
significantly
altering
lipid
profile
high-cholesterol-fed
OVX
female
hamsters
during
estrogen
The
main
findings
include
(i)
ovariectomy
elicited
ACh-
constriction
in cholesterol-fed
hamsters
without
affecting
myogenic
constriction,
(ii)
chronic
raloxifene
restored
ACh-induced
dilatation
only
in cholesterol-fed

Page 5

186
Y.-C.
Chan
et al.
/ Pharmacological
Research
65 (2012) 182–
188
0 4080

120
0.6
0.8
1.0
ND
HCD
Intraluminal pressure
(mmHg)
Normalized
diameter
-9-8
ACh (log M)
-7 -6 -5-4
0
50
100
150
ND
HCD
% Myogenic tone
-9 -8-7-6
100
150
200
ND
HCD
U46619 (log M)
% Myogenic tone
0
Intraluminal pressure
(mmHg)
40 80120
0.6
0.8
1.0
HCD+Ovx
HCD
Normalized
diameter
-9 -8
ACh (log M)
-7-6 -5-4
0
100
200
300
HCD+Ovx
HCD
% Myogenic tone
-9 -8 -7-6
100
300
500
700
HCD+Ovx
HCD
U46619 (log M)
% Myogenic tone
a
b
c
d
e
f
*
*
Fig.
myogenically
tion
of
high-cholesterol
high-cholesterol
coronary
response
myogenic
means
4.
Effect
of
high-cholesterol
diet
on
vessels
(a)
pressure–diameter
curves
in
active
septal
coronary
arteries,
(b)
concentration-dependent
dilata-
to acetylcholine
(ACh),
(c)
U46619-induced
constriction
after
full
development
myogenic
constriction
in arteries
from
hamsters
fed
with
normal
diet
(ND)
and
diet
(HCD).
Effect
of
ovariectomy
on
vessels
from
hamsters
fed
diet,
(d)
pressure–diameter
curves
in myogenically
active
septal
arteries
in HCD
and
HCD
+ Ovx
hamsters,
(e)
concentration-dependent
to ACh
and
(f)
U46619-induced
constriction
after
full
development
of
constriction
in arteries
from
HCD
and
HCD
+ Ovx
hamsters.
Results
are
± SEM
of
5–7 hamsters
(*P < 0.05
between
curves).
Ovx
cholesterol-fed
The
a resistance
intraluminal
key
detected
ies
only
on
example,
myogenic
elevated
tive
myogenic
vascular
on
A
ies
endothelium-dependent
terol
uterine
peripheral
high
hamsters,
and
(iii)
the
increased
constriction
to U46619
in
Ovx
hamsters
was
prevented
by
raloxifene.
myogenic
response
describes
the
extent
of
constriction
of
blood
vessel
in response
to a stepwise
increment
of
pressure.
Myogenic
regulation
of vascular
tone
is a
intrinsic
regulator
of
blood
flow
in cardiac
microcirculation.
We
no
difference
in myogenic
constriction
in coronary
arter-
from
hamsters
fed
normal
or cholesterol
(0.1%)
diet.
To date,
a few
studies
have
examined
the
impact
of
cholesterol
diet
vascular
myogenic
responses
and
reported
different
results.
For
the
basilar
arteries
from
cholesterol
(2%)-fed
rabbits
lost
constriction
[13]
while
hyperlipidemia
in pregnant
rats
myogenic
constriction
in mesenteric
arteries
due
to selec-
attenuation
of
an NO-mediated
vasodilator
component
of
the
response
[14], thus
indicating
a significant
difference
in
beds
and
species
regarding
the
impact
of
high
cholesterol
myogenic
tone.
previous
study
reported
that
mesenteric
resistance
arter-
of
cholesterol-fed
hamsters
exhibited
a moderately
reduced
relaxation
to acetylcholine
[15]. Choles-
diet-related
endothelial
dysfunction
is also
described
in rat
arteries
[16], porcine
coronary
arteries
[17], and
human
arteries
[18]. However,
the
present
study
shows
that
cholesterol
diet
only
led
to insignificant
increase
in myogenic
a
b
c
040 80 120
0.6
0.8
1.0
ND
ND+Ovx
HCD+Ovx
Intravascular Pressure
(mmHg)
Normalized
Diameter
-9 -8-7 -6 -5-4
0
100
200
300
ND
ND+Ovx
HCD+Ovx
ACh (log M)
% Myogenic tone
-9-8-7-6
0
200
400
600
ND
ND+Ovx
HCD+Ovx
U46619 (log M)
% Myogenic tone
*
*
Fig.
cholesterol
arteries,
U46619-induced
in
(HCD
5. Effect
of
ovariectomy
on
vessels
from
hamsters
fed
normal
and
high-
diet
(a)
pressure–diameter
curves
in myogenically
active
septal
coronary
(b)
concentration-dependent
dilatation
to acetylcholine
(ACh)
and
(c)
constriction
after
full
development
of
myogenic
constriction
arteries
from
Ovx
hamsters
fed
normal
(ND
+ Ovx)
and
high-cholesterol
diet
+ Ovx).
Results
are
means
± SEM
of
6 hamsters
(*P < 0.05
between
curves).
constriction
vasodilatation
in species,
ditions,
vessels.
duit
with
duction
also
endothelial
Ovariectomy
tion
these
choline
Chronic
response
and
insignificant
decrease
in acetylcholine-induced
in septal
coronary
arteries,
suggesting
a difference
in isometric
(other
studies)
and
isobaric
(our
study)
con-
or in conduit
(other
studies)
and
resistance
(our
study)
The
attenuated
endothelium-dependent
relaxation
in con-
arteries
from
hypercholesterolaemic
hamsters
is associated
a
diminished
bioavailability
of
NO
[19,20]. Increased
pro-
of
oxidized-LDL
not
only
reduced
the
NO
function
but
exacerbated
oxidative
stress
to cause
further
impairment
of
function
[21,22].
severely
impaired
acetylcholine-induced
dilata-
in cholesterol-fed
hamsters,
and
septal
coronary
arteries
from
animals
paradoxically
constricted
in response
to acetyl-
(≥100
nM),
suggesting
a complete
loss
of NO
function.
treatment
with
raloxifene
prevented
the
constrictor
to acetylcholine,
and
restored
vasodilatation.
Local

Page 6

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/ Pharmacological
Research
65 (2012) 182–
188
187
0
Intraluminal pressure
(mmHg)
4080

120
0.6
0.8
1.0
HCD
HCD+Rf
Normalized
diameter
-9 -8-7-6 -5 -4
0
50
100
150
HCD
HCD+Rf
ACh (log M)
% Myogenic tone
-9-8 -7-6
50
100
150
200
250
HCD
HCD+Rf
U46619 (log M)
% Myogenic tone
-9-8 -7-6-5-4
0
100
200
300
HCD+Ovx
HCD+Ovx+Rf
ACh (log M)
% Myogenic tone
-9 -8 -7-6
0
200
400
600
HCD+Ovx
HCD+Ovx+Rf
U46619 (log M)
% Myogenic tone
0
Intraluminal pressure
(mmHg)
4080120
0.6
0.8
1.0
HCD+Ovx
HCD+Ovx+Rf
Normalized
diameter
*
*
a
b
c
d
e
f
Fig.
high-cholesterol
tal
(ACh)
genic
and
ment
pressure–diameter
concentration-dependent
constriction
hamsters
ifene
curves).
6. Effect
of
chronic
raloxifene
treatment
on
vessels
from
hamsters
fed
diet
(a)
pressure–diameter
curves
in myogenically
active
sep-
coronary
arteries,
(b)
concentration-dependent
dilatation
to acetylcholine
and
(c)
U46619-induced
constriction
after
full
development
of
myo-
constriction
in
arteries
from
hamsters
fed
cholesterol
diet
without
(HCD)
with
raloxifene
treatment
(HCD
+ Rf).
Effect
of
chronic
raloxifene
treat-
on
vessels
from
ovariectomized
hamsters
fed
high-cholesterol
diet,
(d)
curves
in myogenically
active
septal
coronary
arteries,
(e)
dilatation
to acetylcholine
(ACh)
and
(f)
U46619-induced
after
full
development
of
myogenic
constriction
in arteries
from
Ovx
fed
high-cholesterol
diet
without
(HCD
+ Ovx)
and
with
treatment
of
ralox-
(HCD
+ Ovx
+ Rf).
Results
are
means
± SEM
of
5–6
hamsters
(*P < 0.05
between
vascular
derived
Estrogen
endothelial
oxidative-stress-related
our
raloxifene
mediated
acetylcholine
Our
endothelial
acetylcholine
sters.
receptor-mediated
the
apy.
of
tal
30
trast,
arteries
spontaneously
tone
is regulated
by a balance
between
endothelium-
relaxing
(EDRF)
and
contracting
factors
(EDCF)
[23].
deficiency
disturbs
this
balance
as
ovariectomy
reduces
function
by reducing
NO
availability
or enhancing
vasoconstriction
[6].
In agreement
with
findings,
Wong
et
al.
reported
that
chronic
therapy
with
in aging
Ovx
female
rats
increased
endothelial
NO-
relaxation
and
suppressed
Ovx-related
contraction
to
in large
conduit
arteries
[6].
results
indicate
that
raloxifene
treatment
improves
function
by increasing
vascular
responsiveness
to
during
estrogen
deficiency
in cholesterol-fed
ham-
However,
it cannot
be
discounted
that
a
decreased
EDCF
release/function
may
also
contribute
to
improved
endothelial
function
resulting
from
raloxifene
ther-
It is unlikely
that
raloxifene
therapy
enhances
the
basal
release
NO
in resistance
arteries
since
myogenic
constriction
of
sep-
coronary
arteries
was
similar
in the
absence
and
presence
of
?M
L-NAME
in all
treatment
groups
(data
not
shown).
By
con-
raloxifene
did
prevent
the
loss
of
basal
NO
release
in conduit
from
aged
OVX
rats
[6]
and
in pulmonary
arteries
from
hypertensive
rats
[24].
Taken
together,
it
is
likely
that
menting
production
lowing
Coronary
in
this
constrictions
vented
shows
U46619-induced
cholesterol-fed
increased
of
hamsters,
dilatations
high-cholesterol
observed
In
chronic
coronary
dilatation
out
which
benefit
may
vascular
menopausal
a
infarction.
raloxifene
treatment
improves
endothelial
function
by aug-
the
function
and
release
of
EDRFs
or/and
inhibiting
the
and
function
of
EDCFs
in septal
coronary
arteries
fol-
ovariectomy.
artery
constriction
to U46619
was
markedly
enhanced
cholesterol-fed
OVX
hamsters.
Mericli
et
al.
reported
that
thromboxane
A2 analogue
caused
greater
coronary
artery
in Ovx
rats
and
that
estrogen
treatment
pre-
hypersensitivity
to U46619
[25]. The
present
study
clearly
that
chronic
raloxifene
therapy
normalized
the
augmented
constriction
in septal
coronary
arteries
from
Ovx
hamsters.
Likewise,
raloxifene
prevented
the
contraction
of
aortas
in aging
Ovx
rats
[6].
The
absence
changes
in myogenic
responses
in the
vessels
from
normal-fed
together
with
their
unaltered
responses
to ACh-induced
and
U46619-induced
constrictions,
indicates
solely
the
diet
and
ovariectomy
contributed
to the
above
phenotypes.
conclusion,
the
results
of
the
present
investigation
reveal
that
treatment
with
raloxifene
could
benefit
resistance-sized
arteries
after
ovariectomy
by (i)
restoring
ACh-induced
and
(ii)
reducing
agonist-induced
constriction
with-
affecting
myogenic
constriction
in cholesterol-fed
hamsters,
is beyond
its
modulation
of plasma
lipid
profile.
If such
can
be
observed
in humans,
raloxifene
and
other
SERMs
be
useful
in preserving
endothelial
function
and
curtailing
hypersensitivity
in resistance
coronary
arteries
in post-
women
with
hypercholesterolemia
or hyperlipidemia,
lipid
condition
implicated
in the
pathogenesis
of
myocardial
Conflict
of
interests
None.
Acknowledgements
This
study
was
supported
by
Research
Grants
Council
of
Hong
Kong
(2012CB517805)
(4362/04M),
National
Basic
Research
Program
of
China
and
CUHK
Focused
Investment
Scheme.
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