Extract
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Introduction
The endothelial cells lining the interior surface of the
blood vessels modulate the tone of the underlying smooth
muscle by releasing endothelium-derived relaxing (EDRF)
and endothelium-derived contracting (EDCF) factors.
EDCF, a hallmark of endothelial dysfunction, is a product
of cyclooxygenase (COX) and activates thromboxane-prostanoid receptors of the vascular smooth
muscle[1-8]. Endothelium-dependent contractions are augmented by
aging[9-10],
hypertension[1,3,4,9,11],
vasospasm[12], high
glucose[13], and
diabetes[8,14].
Cardiovascular diseases augment in frequency with
aging even in the absence of established risk factors,
suggesting that aging per se alters vascular
function[15]. Endothelium-dependent relaxations are impaired with advancing age
in different species, including
humans[10,16-18]. This could be due to a reduced nitric oxide bioavailability, decreased
endothelium-dependent hyperpolarizations, or augmented
endothelium-dependent contractions.
The increased production of oxygen-derived free
radicals is an important characteristic of
aging[15,19,20]. Reactive oxygen-derived free radicals not only reduce
endothelium-dependent relaxations by scavenging nitric
oxide[21], but also play a crucial role in endothelium-dependent
contractions[3,14,22,23]. Thus, superoxide anion has been proposed
as an endothelium-derived contracting factor in the canine
basilar arteries[22] and hydroxyl radicals in the aorta of
spontaneously hypertensive rats[3] or the femoral arteries of
diabetic rats[14]. In addition to their mediator role,
oxygen-derived free radicals facilitate endothelium-dependent
contractions in the aorta of spontaneously hypertensive rats
and rabbits[3,11,23,24] and the femoral arteries of diabetic
rats[14].
COX metabolizes arachidonic acid, leading to the
production of prostaglandins and thromboxane
A2. In addition, COX can produce reactive oxygen species
(ROS)[14,19,23,25]. COX-1, the constitutive isoform of the enzyme, plays a key
role in the regulation of the physiological functions
mediated by prostanoids in most cells. It can also be induced or
upregulated by shear stress[26], and its upregulation is
responsible for the release of endothelium-derived
contracting factor in the aorta of spontaneously hypertensive
rats[27] and in the femoral arteries of rats with
streptozotocin-induced type I
diabetes[8]. COX-2, the inducible isoform,
can be induced by reactive oxidative species and contributes to
endothelial dysfunction with
aging[17,28,29,30].
These findings prompted the hypothesis that reactive
oxygen-derived free radicals are involved in
endothelium-dependent contractions in aging as they are in disease.
Therefore, the present experiments were designed to study
how aging affects endothelium-dependent contractions of
the rat femoral arteries. Special attention was paid to the
contribution of oxidative stress and that of the 2 isoforms of
COX.
Materials and methods
Experimental animals All experiments were performed
on the isolated femoral arteries of Sprague-Dawley rats. Two
groups of rats of different ages (20 weeks and 1 year) were
housed in the Laboratory Animal Unit of the University of
Hong Kong (Hong Kong), fed regular chow, and given free
access to water. On the day of the experiments, they were
anesthetized with intraperitoneal pentobarbitone sodium (70
mg/kg), anticoagulated with heparin (0.5 U/kg), and
exsanguinated. The Institutional Animal Care Committee
approved all procedures and protocols.
Tissue preparation The femoral arteries were
dissected, excised, and placed into ice-cold modified Krebs-Ringer
solution of the following composition (mmol/L): NaCl 118, KCl
4.7, CaCl2 2.5, MgSO4 1.2,
NaHCO3 25.0,
KH2PO4 1.18, calcium disodium EDTA 0.026, and glucose 11.1 (control
solution). The blood vessels were cut into rings
(1.5-2 mm in length). When the arterial rings without endothelium were
needed, the arteries were perfused with 1 mL saponin
solution (1 mg/mL, diluted with Krebs_Ringer
solution)[31] before the rings were cut. The rings were suspended in organ
chambers containing control solution (37 °C) aerated with 95 %
O2 and 5 % of CO2. They were connected to a force transducer
(Powerlab Model ML785 and ML119, ADInstruments, Inc.,
Colorado Spring, CO, USA). Changes in isometric tension
were recorded. The rings were stretched progressively to
their optimal resting tension (1.0 g; determined in
preliminary experiments) and were allowed to equilibrate for 90 min
before experimentation. Changes in tension were expressed
as a percentage of the reference contraction to 60 mmol/L
KCl (isotonic solution), obtained at the beginning of the
experiment.
The incubation time with drugs was 30 min, and the
concentration-response curves were obtained in a cumulative
way. To study the endothelium-dependent relaxations, the
preparations were exposed to phenylephrine (0.1_1 µmol/L;
in order to obtain 50%-70% of the response to KCl).
Sodium nitroprusside (10 µmol/L) was added at the end of the
experiments, and the relaxations were expressed as a
percentage of the maximal relaxation induced by the
nitrovasodilator. To study the endothelium-dependent
contractions, all experiments were performed in the presence
of Nω-nitro-L-arginine methyl ester hydrochloride
(L-NAME; 0.3 mmol/L)[4,32,33].
Western blotting The arteries were dissected and cleaned
of connective tissue. To remove the remaining blood cells,
the arteries were rinsed 3 times in Krebs-Ringer solution.
The blood vessels were homogenized at 4 ºC in lysis buffer
(10 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA,
25 mmol/L sodium pyrophosphate, 1 mmol/L β-glyco-phosphate,
1 mmol/L sodium orthovanadate, 2.1 µmol/L
leupeptin, 1 mg/mL aprotinin, 1 mmol/L phenyl-methylsulfonyl fluoride, and 1%
Triton X-100) and incubated on ice for 10 min. The samples
were then centrifuged at 3000×g for 10 min at 4 ºC and the
supernatant was collected. Protein concentrations were
determined using the Bradford assay. The lysates were used
immediately or stored at _80 ºC. In all the immunoblot
experiments, equal amounts of protein were loaded in each
lane of the SDS_PAGE gels. The molecular masses of the
immunoreactive bands were determined by loading a biotinylated molecular mass standard (Bio-Rad Laboratories,
Hercules, CA, USA). After electrophoresis, the proteins were
transferred to nitrocellulose membranes
(Bio-Rad Laboratories, USA) and blocked with 5% non-fat, dry milk in
Tris buffered saline (TBS; 20 mmol/L Tris-HCl, and 140
mmol/L NaCl, pH 7.4) for 1 h. The membranes were then incubated
with the suggested dilution of corresponding primary
antibodies (COX-1, 1:300; COX-2, 1:500) overnight at room
temperature, followed by 3×10 min washes in 0.1% Tween
20-TBS (T-TBS). The blots were incubated with secondary
antibodies at 1:1000 dilutions for 2 h at room temperature,
and then washed 3 times for 10 min in T-TBS. The
membranes were then incubated with an enhanced
chemiluminescence (ECL) reagent (Amersham Biosciences,
Buck-inghamshire, UK) for 1 min and exposed to X-ray films (Fuji
Super RX medical X-ray films; Fuji Photo Film, Dusseldorf,
Germany) for the recommended optimal times, depending on
the signal strength. The software for the electrophoresis
analysis (Multi-analyst, Bio-Rad Laboratories) was used for
the densitometric measurement of the immunoreactive bands.
The presence of the protein was normalized to β-actin. All
protein presences were expressed as the percentage of the
control.
Fluorescence studies ROS were measured with
2',7'-dichlorodihydrofluorescein diacetate (DCF). Non-ionized
DCF is permeable of membrane and therefore diffuses readily
into cells. The fluorescence experiments were performed in a
dark room. The rings of the femoral arteries were loaded with
DCF solution (10 µmol/L) for 15 min followed by incubation
with the tested agents for 30 min. After rinsing 3 times in
control solution, the rings were opened longitudinally and
placed with the endothelial layer down on a microscopic
stage. ROS were measured using a confocal laser-scanning
microscope. The intensity of the production was recorded
after excitation at 488 nm and emission at 510 nm using the
appropriated software (Laser Sharp 2000, Bio-Rad Cell
Science Division Hemel Hempstead, UK). The production of
ROS measured from 9 randomly-related fields were averaged
in the endothelium and the vascular smooth muscle of each
preparation. To correct for non-specific changes in
fluo-rescence, the background signal was subtracted from the
measurements and only the changes induced by A23187 are
reported and discussed[14].
Enzymatic activities The arteries were dissected and
cleaned of connective tissue. The preparations were
homogenized at 4 ºC in lysis buffer (50 mmol/L potassium
phosphate containing 1 mmol/L EDTA, pH 7.0). The samples were
centrifuged at 10 000×g for 10 min at 4 ºC, and the
supernatant was collected. Protein concentrations were determined
using the Bradford assay. The lysates were used
immediately or stored at _80 ºC. The level of glutathione and
catalase activity was measured following the instructions of the
manufacturers.
Reagents Acetylcholine, anti-β-actin monoclonal
antibody, calcimycin (A23187) DCF, indomethacin,
L-NAME, saponin, and sodium nitroprusside were purchased from
Sigma (St Louis, MO, USA). NS-398
(N[-2-(cyclohexyloxy)-4-nitrophenyl]-methanesulphonamide), valeryl salicylate
(2-[(1-oxopenytyl)oxy]-benzoic acid), glutathione detection kits,
catalase detection kits, COX-1 monoclonal antibodies, and
COX-2 polyclonal antibodies were purchased from Cayman
(Ann Arbor, MI, USA). Heparin was purchased from LEO
Pharma (Ballerup, Denmark). Secondary antibodies [ECL
antirabbit immunoglobulin G (IgG) and ECL antimouse IgG]
were obtained from Amersham Biosciences (Bucking-hamshire, UK). Terutroban, S18886,
(3-[(6-amino-(4-chlorobenzensulphonyl)-2-methyl-5,6,7,8-tetrahydro-
napht]-1-yl) propionic acid) was a kind gift of the Institut
de Recherche Servier (Paris, France). Drug concentrations
are given as final molar concentrations in the bath solution.
Data analysis Data are presented as mean±SEM;
n refers to the number of rats. The statistical analysis was done
by one-way ANOVA followed by post-hoc comparison
using the Bonferroni test, or by two-way ANOVA, as
appropriate (Prism version 3a, GraphPad Software, San Diego,
CA, USA). Differences were considered to be statistically
significant when P was less than 0.05.
Results
General condition At the beginning of experiments, the
body weight was comparable in both groups. With aging,
the rats gradually gained body weight and the 1-year-old
rats were significantly heavier than the younger ones (20
weeks 684.5±23.6 g, 1 year 840.0±21.6 g,
P<0.05).
Endothelium-dependent relaxations Acetylcholine
induced a concentration-dependent relaxation (Figure 1A,1B).
The acetylcholine-induced relaxation was slightly, but
significantly reduced in the arteries from the 1-year-old rats.
Indomethacin (an inhibitor of COX, 5 µmol/L) prevented the
reduced response in the arteries from the 1-year-old group
(Figure 1B).
The calcium ionophore A23187 induced a
concentration-dependent relaxation at lower concentrations (0.1
nmol/L_0.1 µmol/L) and a secondary increase in tension at higher
(0.3_1 µmol/L) concentrations (Figure 1C,1D). In the arteries from
the 1-year-old rats, A23187 induced a comparable relaxation,
but an enhanced secondary contraction. The secondary
contraction was inhibited by indomethacin (Figure 1D).
Endothelium-dependent contractions In the presence of
L-NAME (an inhibitor of nitric oxide synthase, 0.3 mmol/L),
quiescent rings with endothelium of 20-week and 1-year-old
rats did not contract when exposed to increased
concentrations of acetylcholine (data not shown). Under the same
conditions, A23187 induced concentration-dependent
contractions in rings with, but not in those without endothelium.
The contractions in rings with endothelium were significantly
greater in the arteries from the 1-year_old rats than that in
those from the younger rats (Figure 2). The
endothelium-dependent contractions were abolished by indomethacin and
terutroban (a blocker of thromboxane-prostanoid receptor,
0.1 µmol/L; Figure 3A). Valeryl salicylate or NS-398
(preferential inhibitors of COX-1, 3 mmol/L and COX-2, 1
µmol/L, respectively) partially inhibited the contraction to
A23187 in rings with endothelium (Figure 3B).
DCF fluorescence study In the presence
of L-NAME (0.3 mmol/L), the calcium ionophore A23187 (0.3 µmol/L) increased
the fluorescence intensity in the endothelium of the arteries of
the 1-year_old rats, but not of those of the 20-week-old rats.
This increase was reduced significantly by indomethacin
(Figure 4, right), but not by terutroban (data not shown). A23187
did not significantly increase the fluorescence intensity in the
underlying smooth muscle in either group (data not shown).
Western blotting The protein levels of COX-1 in the
arteries with endothelium from the 1-year-old rats was
significantly higher than that in preparations from the
20-week-old animals (Figure 5A,5B), but was comparable in the
arteries without endothelium of the 2 groups(data not shown).
The protein level of COX-2 was increased in the arteries
with endothelium from the 1-year-old rats (Figure 5C,5D),
while it was comparable in the arteries without endothelium
of the 2 groups (data not shown).
Catalase and glutathione The activity of catalase was
reduced significantly in the femoral arteries with
endothelium from the 1-year-old rats (Figure 6A).The global level of
glutathione was not significantly different in the femoral
arteries with endothelium in the 2 groups (Figure 6B).
Discussion
The present experiments were designed to study the
endothelial function in the process of aging. Endothelial
dysfunction is attributed to either impaired
endothelium-dependent relaxations, augmented endothelium-dependent
contractions, or both[34]. The relaxation induced by the
receptor-mediated agonist acetylcholine was slightly, but
significantly reduced in the femoral arteries from the 1-year-old
rats. Indomethacin, an inhibitor of COX, restored the blunted
response, suggesting that an endothelium-dependent
contracting factor derived from COX exists in the femoral
arteries from 1-year-old rats. For the receptor-independent
response to the calcium ionophore
A23187[35], the concentration-dependent relaxation was comparable between the
groups, but the secondary concentration-dependent
contraction was significantly higher in the arteries from the
1-year-old rats than in that from the younger rats. The
secondary contraction was also prevented by indomethacin, which is
in agreement with the data of acetylcholine-induced relaxation.
Thus, these results confirm that endothelial dysfunction with
aging can be attributed to the occurrence of enhanced
endothelium- and COX-dependent
contractions[10,17,29].
To further study endothelium-dependent contractions in
rat femoral arteries, all of the experiments were performed
with quiescent preparations in the presence of
L-NAME, an inhibitor of nitric oxide synthase, since nitric oxide
exerts negative feedback on this
response[4,32,33]. Endothelium-dependent contractions were obtained with A23187, but not
acetylcholine. This heterogeneity in response is consistent
with the findings in the same arteries from rats with
streptozotocin-induced type I
diabetes[8]. The difference in response to A23187 and acetylcholine could be explained by
different increases in intracellular calcium in the
endothelium evoked by these 2
agents[23]. Therefore, the calcium ionophore A23187 was used as a tool to investigate the
endothelium-dependent contractions in further experiments.
The endothelium-dependent response was augmented in
older rats, confirming that aging enhances the production
or the action of the endothelium-derived contracting
factor[10]. The endothelium-dependent contractions observed in
the present study must be due to the production of COX and
the subsequent activation of thromboxane-prostanoid
receptors on the vascular smooth muscle, since indomethacin
and terutroban, a blocker of these
receptors[36], completely prevented the
response[1,8,37]. However in aging, unlike in
the aorta of the spontaneously hypertensive
rats[3] and the femoral arteries of diabetic
rats[8], both COX-1 and COX-2 contribute to endothelium-dependent contractions, since at
the concentrations used, either preferential inhibition of
COX-1 (valeryl salicylate[3,38]) or COX-2
(NS-398[3,39]) reduced the response. The constitutive isoform COX-1 plays a
prominent role in the augmented endothelium-dependent
contraction in hypertension[3,27] and type I
diabetes[8]. COX-2, the inducible isoform of COX, is present in macrophages and
vascular smooth muscle in response to inflammatory stress,
cytokines, and oxidative stress[42]. It is upregulated in the
senescent endothelium[17,30] and by
atherosclerosis[41_43]. The increased presence of both COX-1 and COX-2 observed in
the present study with Western blotting supports an active
contribution of both COX-1 and COX-2 in the occurrence of
endothelium-dependent contractions with aging. The data
of the present study concur with the results obtained in the
aorta of 2-year-old Wister-Kyoto rats where both the
constitutive and inducible isoforms of COX were involved in the
blunted endothelium-dependent relaxations observed with
aging[17,29].
The oxidative state was studied in the present study with
fluorescence measurement under confocal microscopy and
the measurement of catalase activity and glutathione levels.
Both glutathione and catalase transform hydrogen peroxide
to water[25]. Since the activity of catalase, but not the
glutathione net level was significantly reduced in the arteries
from the 1-year-old rats, the present data indicate that the
reduced activity of catalase is a likely source of oxidative
stress in the endothelium with aging as it is in the same
artery from rats with streptozotocin-induced
diabetes[14]. In the presence of
L-NAME, the calcium ionophore A23187 increased the fluorescence of ROS. An increased production
of ROS in response to A23187 during the inhibition of nitric
oxide has been reported in the aorta of the spontaneously
hypertensive rats and in the femoral arteries of diabetic
rats[14,23]. Since indomethacin, but not terutroban, reduced the free
radical release, these data are in line with the
endothelium-dependent contraction observed in organ chambers and
demonstrate that the ROS derived from COX inside the
endothelium play an important role in the genesis of the
endothelium-dependent
contractions[3,14,22,23,44].
In conclusion, endothelium-dependent contractions are
augmented in the femoral arteries of 1-year-old rats.
Oxygen-derived free radicals take part in the occurrence of this
response augmented by aging. Both the constitutive and
inducible isoforms of COX contribute to this endothelial
dysfunction.
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