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© 1997 American Heart Association, Inc.
Articles |
Hypercholesterolemia Impairs a Detoxification Mechanism Against Peroxynitrite and Renders the Vascular Tissue More Susceptible to Oxidative Injury
From the Division of Emergency Medicine (X.L.M., B.L.L., G.-L.L., T.A.C., F.G., Y.G.), Thomas Jefferson University, Philadelphia, Pa, and the Department of Cardiovascular Pharmacology (G.Z.F., R.R.R., F.C.B., T.-L.Y.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pa.
Correspondence to Xin L. Ma, MD, PhD, Division of Emergency Medicine, Jefferson Medical College, 1020 Sansom St, Philadelphia, PA 19107-5004. E-mail MA1{at}JEFLIN.TJU.EDU
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Abstract |
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Abstract Previous studies have shown that glutathione (GSH) plays a central role in the protection against peroxynitrite (ONOO-) toxicity. The present study evaluated the changes of the GSH cytoprotective system against ONOO- in hypercholesterolemia and determined the effects of carvedilol, a ß-blocker with free radical–scavenging activity, on these hypercholesterol-induced changes. New Zealand White rabbits were fed either a normal diet, a high-cholesterol diet, or a high-cholesterol diet supplemented with either carvedilol or propranolol. Eight weeks later, the rabbits were killed, and the thoracic aortas were isolated. Total GSH content of aortic tissue, vasorelaxation response of aortic rings to exogenous ONOO-, ·NO regeneration from ONOO- by aortic homogenate, and ONOO--induced aortic tissue injury were examined. Hypercholesterolemia decreased tissue GSH content (0.52±0.08 versus 0.86±0.04 µmol/g in control, P<.01), attenuated the vasorelaxation response to ONOO- (40±4.1% versus 76±3.2%, P<.01), reduced ·NO regeneration from ONOO- (387±40 versus 662±51 pmol, P<.01), and potentiated ONOO--induced vascular tissue injury (37±4.4% versus 14±2.6% of increase in lactate dehydrogenase release after 3-morpholinosydnonimine exposure, P<.01). Treatment of the hypercholesterolemic rabbits with carvedilol, but not propranolol, significantly preserved tissue GSH content (0.79±0.05 µmol/g, P<.01 versus nontreated hypercholesterolemic rabbits), restored the vasorelaxation to ONOO- (61±2%, P<.01), increased ·NO regeneration from ONOO- (583±39 pmol, P<.01), and attenuated ONOO--induced tissue injury (19±1.8%, P<.01). These results suggest that hypercholesterolemia impairs the GSH-mediated detoxification mechanism against ONOO- and renders the vascular tissue more susceptible to oxidative injury. Carvedilol, a novel vasodilating ß-blocker with antioxidant activity, significantly preserved this self-defense system and protected tissue from oxidant injury.
Key Words: free radical • atherosclerosis • glutathione • tissue injury
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peroxynitrite (ONOO-) is a powerful oxidant formed by the near-diffusion–limited reaction of nitric oxide (·NO) with superoxide (·O2-).1 Substantial evidence has emerged suggesting that ONOO- can be formed in significant concentrations in vivo and is capable of oxidizing lipid, protein, and DNA.2 3 4 5 In addition, experimental results suggest that ONOO- may contribute to cell death and tissue injury in a number of human diseases, including atherosclerosis, shock, and ischemia/reperfusion injury.6 7
Recent biochemical and physiological studies have revealed that there is a significant self-defense system against ONOO- toxicity under physiological conditions. Using isolated canine coronary vessels, Liu et al8 first reported that ONOO- induces a ·NO-like vasorelaxation. Subsequent biochemical studies demonstrated that ONOO- stimulates guanylyl cyclase in a GSH-dependent manner and induces cGMP accumulation in endothelial and smooth muscle cells.9 10 Further studies by Wu et al11 and Moro et al12 have demonstrated that ONOO- reacts with GSH to form an S-nitrosothiol compound and regenerates low concentrations of ·NO over a prolonged period. ·NO, in turn, stimulates soluble guanylate cyclase and results in both vasorelaxation and inhibition of platelet aggregation. In contrast to the cytotoxic effects of ONOO-, nanomolar concentrations of ·NO have been demonstrated in numerous studies to exert significant cytoprotective effects under many pathological conditions.5 13 Therefore, the reaction of ONOO- with GSH and the subsequent regeneration of ·NO has been proposed as a critical detoxification mechanism against ONOO-. However, the changes of GSH content and the status of the detoxification system against ONOO- under pathological conditions, such as hypercholesterolemia, have not been evaluated.
Substantial evidence indicates that ROS play a pivotal role in the pathogenesis of atherosclerosis14 15 and that GSH exerts significant protective effects against ROS-induced tissue injury in this pathological condition.16 17 Recently, it has been suggested that ONOO- may also play a significant role in the formation of atherosclerosis and the subsequent tissue injury.18 19 However, whether GSH may protect ONOO--induced tissue injury in hypercholesterolemia has not been determined.
Carvedilol is a new vasodilating ß-adrenergic receptor antagonist that has been used clinically for the treatment of mild to moderate hypertension and congestive heart failure.20 Recent experiments have demonstrated that carvedilol is a powerful antioxidant agent and exerts significant protective effects on free radical–induced cell injury in vitro and attenuates postischemic myocardial injury in vivo.21 22 However, whether treatment with carvedilol in hypercholesterolemia restores the balance between the GSH-centered detoxification system and ONOO- toxicity and thereby attenuates ONOO--induced tissue injury has not been studied.
Therefore, the purposes of the present study were to (1) determine the status of the GSH-centered self-defense system in hypercholesterolemia, (2) evaluate the effects of an altered GSH-centered detoxification mechanism on ONOO--induced tissue injury, and (3) test the effects of antioxidant treatment with carvedilol on this altered GSH-centered detoxification system in hypercholesterolemia.
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Materials and Methods |
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Materials
Chemicals used in the present studies were purchased from Sigma Chemical Co, except those stated specifically.
Animals and Dietary Protocol
Adult male New Zealand White rabbits weighing 2.1 to 2.7 kg were
provided with food and water ad libitum. Blood was drawn from the
central ear artery of each rabbit for determination of baseline plasma
lipids (cholesterol and triglyceride) by
SmithKline Beecham Clinical Laboratories. On day 0 of the study, the
rabbits were assigned to one of four dietary groups (14 to 16 rabbits
in each group): (1) normal rabbit diet, (2) rabbit diet containing 1%
cholesterol, (3) rabbit diet containing 1%
cholesterol supplemented with carvedilol (1200 ppm), and
(4) rabbit diet containing 1% cholesterol supplemented
with propranolol (1200 ppm). All rabbit diets were prepared
by and purchased from Zeigler Bros, Inc.
Chemical Synthesis of ONOO-
ONOO- was prepared by the methods described
previously.8 Briefly, NaNO2 (0.6 mol/L) was
rapidly mixed with H2O2 (0.7 mol/L) in HCl (0.6
mol/L) in a polytetrafluoroethylene
cylindrical quench-flow reactor. The peroxynitrous acid was immediately
combined with a stream of NaOH (1 mol/L) entering near the bottom of
the flow reactor. All solutions were precooled on ice, and the flow
rates were set at 25 mL/min. The yellowish ONOO- was
collected in a beaker on ice. Excess H2O2 was
removed by passing the ONOO- solution over solid granular
manganese dioxide packed into a small column. ONOO- was
stored at -20°C in 15-mL capped centrifuge tubes and used
within 1 week. Before each experiment, an aliquot was taken from the
concentrated liquid layer on top of the ice crystals, and the
concentrations were determined using the extinction coefficient (1670
per mol/L per centimeter at 302 nm). The concentrations of
ONOO- in this fraction were typically 0.3 to 0.5 mol/L.
Incubation of ONOO- at room temperature in 0.5 mol/L
sodium phosphate buffer (pH 7.4) for 5 minutes results in complete
decomposition of ONOO-. This decomposed ONOO-
solution was used as a blank when measuring ONOO-
concentrations. It was also used as a control solution in all the
experiments.
Aortic Tissue GSH Measurement
Rabbits were carefully scheduled to start their special diets so
that two rabbits were mature on the same day 8 weeks later. On the day
of an experiment, two rabbits from different groups were studied.
Rabbits were anesthetized with sodium pentobarbital (30 mg/kg
body wt IV). A 3-mL blood sample was drawn for analysis of
plasma lipids. After a midsternal thoracotomy, the thoracic aortas were
carefully removed and placed in ice-cold 5% SSA. The segment was
rinsed three to four times with SSA solution to ensure no blood
contamination, and the fat and perivascular connective tissue were then
carefully excised. Aortic tissue (
50 mg) was homogenized
in 5% SSA solution (1:100 [wt/vol]). The homogenate was
centrifuged at 6000g for 10 minutes at 4°C, and
protein-free supernatant was stored at -70°C. The total GSH content
(µmol/g wet tissue) was measured within 1 week using the enzyme
recycling method initially described by Tietze23 and
recently modified by Vandeputte et al24 for use with a
multiwell-plate reader (Molecular Devices Corp).
Isolated Aortic Ring Studies
Isolated thoracic aortas were obtained from each rabbit as
described above and placed into ice-cold K-H buffer consisting of
(mmol/L) NaCl 118, KCl 4.75, CaCl2·2H2O
2.54, KH2PO4 1.19,
MgSO4·7H2O 1.19, NaHCO3 25, and
glucose 10.0. Isolated vessels were cleaned of adhering fat and
connective tissue and cut into rings 4 to 5 mm in length. The
rings were then mounted onto stainless steel hooks, suspended in 7.5-mL
tissue baths, and connected to FORT-10 force transducers (World
Precision Instruments) to record changes in force on WindoGraf
recorders (Gould Inc). The baths were filled with 7.5 mL of K-H
buffer and aerated at 37°C with a gas mixture of 95%
O2/5% CO2. Aortic rings were initially
stretched to give an optimal preload of 2 g of force and
equilibrated for 60 minutes. During this period, the K-H buffer in the
tissue bath was replaced every 20 minutes.
After equilibration, 100 nmol/L U-46619 (Biomol Research Laboratories), a thromboxane A2 mimetic, was added to generate a maximal vasoconstriction. After the response stabilized, the rings were washed several times, and force was allowed to return to baseline values. Those rings responding with a generated force <1.5 or >2.5 g were discarded to ensure a comparable contraction in the subsequent studies. Rings were then contracted again by the addition of 75 nmol/L U-46619, and cumulative relaxation curves to ONOO- or decomposed ONOO- (10-7 to 10-4 mol/L) were obtained. After the response had stabilized, the rings were washed several times and allowed to equilibrate to baseline once again. The procedure was repeated with acidified NaNO2, a chemical ·NO donor (10-7 to 10-4 mol/L). Acidified NaNO2 was prepared by dissolving the compound in 0.1N HCl and titrating it to pH 2.0. Titrating distilled water to pH 2.0 and adding aliquots to the bath did not produce any vasorelaxation.
·NO Analysis
·NO measurements were determined on a
·NO chemiluminescence analyzer (model 270B,
Sievers Instruments) by using the modified method of Wu et
al.11 Briefly, the aortic segment was obtained from each
rabbit and cleaned of fat and connective tissue as described above.
Approximately 500 mg of arterial tissue was placed in a
10-mL plastic tube and homogenized in Dulbecco's PBS (1:4
[wt/vol]) for 1 minute. The tube was then sealed and degassed with
100% nitrogen for 15 minutes. With the use of Hamilton gas tight
syringes, aliquots of nitrogen-equilibrated ONOO- (final
concentration, 0.5 mmol/L) or decomposed ONOO-
(0.5 mmol/L) were injected into the homogenate under
vortex. After 30 minutes of incubation in 37°C water bath, a 0.5-mL
aliquot of the head space gas from each sealed tube was removed and
injected into the ·NO analyzer. The amount of
·NO regenerated from ONOO- was quantified on
the basis of ·NO standards and recovery of authentic
·NO from the testing tube containing 2 mL of Dulbecco's
PBS.
Oxidative Tissue Injury Caused by ONOO-
The oxidative injury caused by ONOO- was determined
in isolated aortic segments using the method described by Keaney et
al.14 Rabbits were treated as described above, and
thoracic aortas were isolated from each group and placed into ice-cold
K-H solution. After being cleaned of fat and connective tissue, each
aorta was cut into segments 15 to 18 mm in length. The segments
were weighed and placed into the 37°C water-jacketed tissue baths
containing K-H solution (1:20 [wt/vol]). After 20 minutes of
equilibration, the K-H solution was replaced with fresh solution, and
1 mmol/L SIN-1 (a molecule that releases
·O2- and ·NO
simultaneously and generates ONOO- in
crystalloid solution) or vehicle (PBS solution, pH 5.0) was added to
the tissue baths. After an additional 2-hour incubation, the K-H
solution LDH content was assayed using a kinetic plate reader
(Molecular Devices). LDH values were determined from a standard curve
obtained from sequential dilutions of an LDH enzyme solution (Sigma
enzyme control 2-E). Tissue injury was assessed by comparing the LDH
release in SIN-1–treated segments with that of vehicle-treated
segments from the same rabbits.
Statistical Analysis
All values in the text and figures are presented as
mean±SEM of n independent experiments. All data were subjected to
one-way ANOVA followed by Scheffé's correction for post hoc
t test comparison. Values of P
.05 were
considered to be statistically significant.
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Results |
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Aortic GSH Content
To determine the effects of hypercholesterolemia on aortic antioxidant activity, GSH content of aortic tissue was measured after 8 weeks of normal or cholesterol diet feeding. In the 11 rabbits fed a normal diet, the content of total GSH in the aortic tissue ranged from 0.73 to 1.08 µmol/g tissue, with a mean value of 0.86±0.04 µmol/g tissue. The 8-week cholesterol-enriched diet feeding significantly decreased tissue total GSH content (0.52±0.08 µmol/g, P<.01 versus control). Supplementation with carvedilol, a nonselective ß-blocker with significant antioxidant activity, in the cholesterol-enriched diet markedly preserved tissue GSH content (0.79±0.05 µmol/g, P<.01 versus cholesterol alone). In contrast, supplementation with propranolol, a compound possessing similar ß-blocking activity but lacking significant antioxidant activity at the concentration used, failed to exert any protective effect on depletion of tissue GSH content (0.59±0.04 µmol/g, P=NS versus cholesterol alone) (Fig 1
).
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Aortic Vascular Effects of ONOO-
To determine whether ONOO- can induce
endothelium-independent vasorelaxation in rabbit aortic
rings, as has been reported in canine coronary and bovine
pulmonary arteries,8 11 the dose-response
relationship of ONOO- in rabbit aortic rings was tested in
both endothelium-intact rings and
endothelium-denuded rings. Addition of
ONOO- to the rabbit aortic rings with intact
endothelium resulted in significant vasorelaxation in a
dose-dependent manner, with nearly complete vasorelaxation
(75.9±3.2%) at an ONOO- concentration of 100
µmol/L. Mechanical disruption of intimal endothelium
did not alter the ONOO--induced relaxation (77.8±3.8% at
100 µmol/L ONOO-, P=NS versus intact
endothelium). In contrast, addition of decomposed
ONOO- resulted in a slow and markedly decreased relaxation
(29.7±2.7% at 100 µmol/L decomposed ONOO-,
P<.01 versus ONOO-). These results indicate
that ONOO- is an endothelium-independent
vasodilator in rabbit aortic arteries, as was observed previously in
canine coronary and bovine pulmonary arteries.
It has been recently shown that in cat cerebral arterioles, ONOO--induced relaxation results from the activation of KATP channels rather than from the activation of soluble guanylate cyclase.25 To determine whether the vasorelaxation to ONOO- in rabbit aortic arteries is mediated by a similar mechanism (ie, activation of KATP channels), vascular rings were preincubated for 20 minutes with 5 µmol/L glibenclamide, a KATP channel blocker that has been shown in our preliminary studies to completely block vasorelaxation induced by pinacidil, a KATP channel opener. Preincubation of rabbit aortic rings with glibenclamide did not significantly alter the vasorelaxation to ONOO-. Specifically, when 100 µmol/L of ONOO- was added to the rings that had been incubated with glibenclamide, a 69.1±5.2% relaxation was observed (P=NS versus rings without glibenclamide treatment). These results indicate that the vasorelaxation of ONOO- in rabbit aortic rings appears unrelated to KATP channels.
Effects of Hypercholesterolemia on
ONOO--Induced Vasorelaxation
Fig 2 shows a typical tracing of the vasorelaxant
effect of ONOO- in four sets of rings isolated from
rabbits fed a normal diet, a cholesterol-enriched diet, and
a cholesterol-enriched diet supplemented with either
carvedilol or propranolol. As illustrated in Fig 2 and
summarized in Fig 3, vasorelaxation of the rings from
cholesterol-fed rabbits was markedly decreased, and the
concentration-response curve was shifted significantly to the right. At
the highest concentration of ONOO- tested (ie, 100
µmol/L), these aortic rings only relaxed 39.6±4.1%
(P<.01 versus control). Supplementation with carvedilol,
but not propranolol, significantly improved the
vasorelaxation of the aortic rings to ONOO-. Thus, when
100 µmol/L of ONOO- was added to the rings isolated
from the hypercholesterolemic rabbits treated with
carvedilol, a significant vasorelaxation occurred (60.7±2.3%,
P<.01 versus cholesterol alone).
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To determine whether the decreased vasorelaxation to ONOO-
in hypercholesterolemic rings was due to a decreased
smooth muscle response to exogenous ·NO, the
vasorelaxation to acidified NaNO2, a chemical
·NO donor, was examined. As illustrated in the right
panel of Fig 3
, all rings relaxed completely when 100 µmol/L of
acidified NaNO2 was added, indicating that the
vasorelaxation response to exogenous ·NO is not impaired
in hypercholesterolemic rabbit aortic rings.
To determine the role of GSH depletion in the observed diminished vasorelaxation to ONOO- in the rings from hypercholesterolemic rabbits, a separate study was conducted in which the aortic segments isolated from nontreated cholesterol-fed rabbits were separated into two parts. One half of these segments were placed in K-H solution containing 10 mmol/L GSH monoethyl ester, and the other half was placed in regular K-H solution. After 30 minutes of incubation, the K-H solution containing GSH monoethyl ester was completely replaced with regular K-H solution, and the vasorelaxation to ONOO- was then studied. Direct measurement of tissue GSH content indicated that preincubation of aortic rings with GSH monoethyl ester significantly restored the GSH concentration (from 0.498±0.037 to 0.725±0.027 µmol/g in six pairs of aortic vascular segments). Correspondingly, the vasorelaxation to ONOO- was markedly improved after GSH incubation (57.9±4.1%, P<.01 versus hypercholesterolemic rings without GSH incubation).
·NO Release from ONOO-
To determine whether the above-mentioned diminished vasorelaxant
response to ONOO- is ·NO dependent, the
·NO regeneration from ONOO- by aortic tissue
was directly measured by using a recently established method of Wu et
al.11 The addition of 0.5 mmol/L ONOO-
to 500 mg aortic tissue homogenate obtained from normal
rabbits resulted in a significant ·NO release. At 30
minutes after ONOO- injection, 662±51 pmol of
·NO was detected in eight independent studies. These
results are consistent with those reported previously by Wu et
al using bovine pulmonary arteries. However, when the same
amount of ONOO- was added to tissue homogenate
obtained from the rabbits subjected to
hypercholesterolemia, detectable
·NO was markedly decreased (387±40 pmol,
P<.01 versus control rabbits). Moreover, treatment with
carvedilol significantly preserved the ability of aortic tissue to
regenerate ·NO from ONOO- (583±39 pmol,
P<.01 versus nontreated cholesterol-fed
rabbits). In contrast, treatment with propranolol failed to
exert significant effects on ·NO release from
ONOO- by aortic tissue (399±36 pmol, P<.01
versus control rabbits, P=NS versus nontreated
cholesterol-fed rabbits) (Fig 4).
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To further verify the role of GSH depletion in the observed diminished ·NO release from ONOO- in the aortic tissue from hypercholesterolemic rabbits, the aortic segments isolated from nontreated cholesterol-fed rabbits were preincubated with 10 mmol/L GSH monoethyl ester as described above. Incubation of the hypercholesterolemic aortic segments with GSH monoethyl ester for 30 minutes significantly increased the ·NO regeneration from ONOO- (571±49 versus 368±31 pmol without GSH incubation, P<.01).
Effect of Hypercholesterolemia on
ONOO--Induced Tissue Injury
The above-mentioned results suggest that
hypercholesterolemia significantly decreased
tissue GSH content and thus impaired one of the most important
detoxification mechanisms against ONOO-. To determine
whether this alteration in tissue antioxidant activity has a
significant effect on ONOO--induced tissue injury, aortic
tissues obtained from four experimental groups were exposed to 1
mmol/L SIN-1, a compound that releases ·NO and
·O2- simultaneously
with subsequent ONOO- formation.26 Incubation
of aortic tissue isolated from control rabbits with 1 mmol/L SIN-1
for 2 hours resulted in a moderate increase in LDH release (14±2.6%
over vehicle-treated aortic segments). However, SIN-1 caused a marked
increase in LDH release in aortic vascular segments from
hypercholesterolemic rabbits (36.8±4.4% over
vehicle-treated hypercholesterolemic aortic vascular
segments, P<.01 compared with SIN-1–treated control aortic
vascular segments). Moreover, SIN-1–induced tissue injury was
dramatically attenuated in the hypercholesterolemic
vascular segments from rabbits treated with carvedilol (18.9±1.8%
over vehicle-treated aortic vascular segments; P<.01
compared with nontreated hypercholesterolemic segments,
P<.05 compared with propranolol-treated
hypercholesterolemic segments). Interestingly,
propranolol treatment also significantly attenuated
SIN-1–induced tissue injury (28.9±1.6% over vehicle-treated aortic
vascular segments; P<.05 compared with nontreated
hypercholesterolemic segments), although to a lesser
extent than in segments from carvedilol-treated rabbits (Fig 5). This effect may be related to its direct protection
on the arterial wall, as reported
previously.27
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To determine whether acute restoration of tissue GSH content by incubation of hypercholesterolemic aortic tissue with GSH monoethyl ester may also attenuate SIN-1–induced tissue injury, isolated vascular segments were incubated with 10 mmol/L GSH monoethyl ester as described above. Incubation of the vascular segments with GSH monoethyl ester, a pharmacological intervention that acutely restores tissue GSH content, markedly attenuated SIN-1–induced tissue injury (LDH release decreased to 23.9±2% over vehicle-treated segments; P<.01 versus segments without GSH incubation).
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Discussion |
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It is well recognized that the endothelium-dependent vasorelaxation is significantly decreased in hypercholesterolemia and atherosclerosis.28 Numerous mechanisms have been proposed to explain this pathological alteration, including deficiencies of arginine supply, alteration of signaling mechanisms, alterations of ·NO synthase expression or one of the cofactors involved in ·NO synthase activation, and increased destruction of ·NO.29 30 Among these proposed mechanisms, superoxide inactivation of ·NO has been thought to be the most important mechanism of endothelial dysfunction in hypercholesterolemia.31 32 33
The destruction of ·NO by ·O2- may not only have relevance to vasomotor impairments in the vessel wall but may also participate in the atherosclerotic process. It is well recognized that oxidative modification of lipoproteins and their uptake via the scavenger receptors in endothelial cells and macrophages is an early and critical event in the pathogenesis of atherosclerosis,34 but the nature of the oxidants leading to lipoprotein oxidation is far from certain. ONOO-, the reaction product between ·NO and ·O2- at diffusion-limited speed, is a powerful oxidant. Using cultured human umbilical vein endothelial cells and bovine aortic endothelial cells, Pritchard et al35 and Deliconstantinos et al36 have demonstrated that incubation of the endothelial cells with native LDL or cholesterol significantly increases ·O2- and ·NO production and causes an 8-fold increase in ONOO- production. In a recent study, Buttery et al37 have confirmed an earlier report by Beckman et al,19 clearly demonstrating that inducible ·NO synthase is present within human atherosclerotic lesions and promotes the formation and activity of ONOO-. Several recent studies have demonstrated that addition of either chemically synthesized ONOO- or SIN-1, a compound that generates ·NO and ·O2- simultaneously, results in significant formation of oxidized LDL that is recognized by macrophage scavenger receptors.18 38 39 40 41 Taken together, it is conceivable to postulate that ONOO- is a likely candidate for the in vivo oxidation of LDL and may thus play a critical role in development of the atherosclerotic lesion.
On the other hand, increasing evidence suggests that under physiological conditions, there is a highly efficient detoxification system against ONOO- toxicity in which GSH plays a central role.8 9 10 11 12 However, the status of this system under severe pathological conditions, such as hypercholesterolemia and atherosclerosis, remains unclear. Moreover, the impact of an alteration of the GSH-centered anti-ONOO- system on tissue oxidative injury has not been fully studied. In the present study, the alteration of total GSH content in aortic tissue, the vasorelaxation response of aortic rings to exogenous ONOO-, ·NO regeneration from ONOO- by aortic homogenate, ONOO--induced aortic tissue injury, and finally, the effects of antioxidant treatment on these pathological changes were investigated in a rabbit hypercholesterolemic model.
Several interesting results were obtained from the present
study. First, the total GSH content in the
hypercholesterolemic rabbits was found to be
significantly decreased. Although several early studies have shown that
the activities of antioxidant enzymes, such as superoxide dismutase,
GSH peroxidase, and catalase are increased or unchanged in early
hypercholesterolemia,42 43 44 the
metabolism of GSH is not well defined. Exposure of isolated
macrophages to oxidized LDL has been shown to significantly
increase intracellular GSH content.45 In contrast,
exposure of cultured bovine endothelial cells to
oxidized LDL has been shown to result in a concentration- and
time-dependent depletion of intracellular GSH.46
Specifically, incubating endothelial cells with 530
µg/mL of cholesterol for 8 hours decreased intracellular
total GSH to 10% of the control value. In a very recent study,
Gokkusu et al47 demonstrated that the total GSH content in
rabbit aortic tissue decreased to 60% of the control value after
2.5 months of high-cholesterol diet feeding.
Consistent with this finding, our present study
demonstrated that the total GSH content in aortic tissue decreased from
a value of 0.86±0.04 µmol/g in control rabbits to
0.52±0.08 µmol/g in rabbits after 8 weeks of
high-cholesterol diet feeding. The exact mechanism of this
alteration could not be precisely determined from the present
study. One explanation for this decrease in total GSH content is that
GSH biosynthesis might be impaired in
hypercholesterolemic rabbits. In this connection, it
has been recently reported that myocardial ischemia followed by
reperfusion, a pathological condition that shares many common features
with hypercholesterolemia in the redox state,
significantly decreases the enzyme activity of 
-glutamylcysteine
synthetase, a regulating enzyme in the GSH synthesis, and causes
depletion of myocardial total GSH content.48 A more likely
explanation for the decrease in GSH is the constant onslaught of
ONOO- formed by reactions of
·O2- and ·NO, both
of which are increased in
hypercholesterolemia.
Second, the vasorelaxation response to exogenous ONOO- was found to be significantly decreased, and ·NO regeneration from ONOO- by aortic tissue from hypercholesterolemic rabbits was markedly reduced. Increasing the tissue GSH content by incubating the aortic tissue isolated from hypercholesterolemic rabbits with GSH monoethyl ester, a cell-permeable form of GSH that has been shown to increase intracellular GSH concentration,17 49 not only markedly restored vasorelaxation to exogenous ONOO- but also improved ·NO regeneration by aortic tissue from ONOO-. To our knowledge, these results indicate for the first time that the depletion of GSH in hypercholesterolemic tissue markedly impairs one of the most important detoxification mechanisms against ONOO-. GSH is the most prevalent low-molecular-weight peptide present in animal cells, and its antioxidant effects against ROS (including ·O2-, hydrogen peroxide, and hydroxyl radical)-induced tissue injury is well documented. Several recent studies have demonstrated that GSH also plays a critical role in the detoxification process against reactive nitrogen species (eg, ·NO, NO2, and ONOO-) through the formation of the S-nitrosothiol compound. Under physiological conditions, GSH is present in most living cells at a relatively high concentration (ie, in the millimolar range),17 and the generation of ·NO and ·O2- is relatively low. In this setting, ONOO- may not cause significant cell injury. However, under pathological conditions, such as hypercholesterolemia and atherosclerosis, ·O2- generation is markedly increased through multiple pathways, and ·NO generation is increased through the increased expression of endothelial nitric oxide synthase and the de novo expression of inducible nitric oxide synthase. In this setting, the formation of ONOO- is very likely to be significantly increased. In contrast, as demonstrated by the present study, the detoxification system against ONOO- toxicity is impaired in hypercholesterolemia. This alteration would render tissue unprotected against the cytotoxic effects of ONOO- and would lead to severe tissue oxidative injury.
Third, the vascular tissue from hypercholesterolemic
rabbits was found to be more susceptible to ONOO--induced
injury when compared with normal tissue. In the present study, we
used SIN-1 as a chemical ONOO- donor. Although SIN-1 has
been used as a ·NO donor in some early studies and has
been shown to be protective in myocardial ischemia and
reperfusion in vivo,50 recent biochemical studies using
direct ·NO measurement, including one from our
laboratory, have revealed that SIN-1 does not release measurable
·NO in physiological solutions in
vitro unless superoxide dismutase is present. Rather, it releases
ONOO-.51 52 On the basis of these
observations, SIN-1 has been used extensively as an ONOO-
donor in recent studies.53 54 As illustrated in Fig 5, exposure of aortic segments isolated from
hypercholesterolemic rabbits to 1 mmol/L SIN-1, a
concentration that has been shown to generate ONOO- at a
rate of 10 µmol/L/min,26 markedly increased LDH
release, and preincubation of these vessels with GSH monoethyl ester
significantly attenuated the tissue injury caused by SIN-1. These
results provide direct evidence that depletion of tissue GSH
significantly increases the susceptibility of tissues to oxidative
injury.
Fourth, treatment of hypercholesterolemic rabbits with carvedilol, a novel ß-blocker with strong antioxidant activity, significantly preserved the vascular tissue GSH content and thus maintained the detoxification process against ONOO-, as characterized by increased vasorelaxation to ONOO-, improved ·NO regeneration from ONOO-, and attenuated SIN-1–induced tissue injury. Previous pharmacological studies have demonstrated that carvedilol exerts a ß-adrenergic receptor blocking effect comparable to a traditional nonselective ß-blocker, propranolol.55 However, it possesses unique antioxidant effects and exerts significant protective effects on free radical–induced injury in cultured cells that are comparable to those produced by vitamin E or probucol.56 It has been previously reported that both vitamin E and probucol significantly preserve aortic antioxidant activity in hypercholesterolemia.42 43 In addition, our previous study has shown that administration of carvedilol in hypercholesterolemic rabbits significantly preserves endothelium-dependent relaxation to acetylcholine in isolated aortic arteries and markedly reduces postischemic myocardial injury.22 Our present experiment has demonstrated that carvedilol, but not propranolol, maintains the balance between oxidants and antioxidants in hypercholesterolemia and improves the endothelium-independent vasorelaxation to ONOO-. It is well recognized that hypertension and hypercholesterolemia are two major risk factors for coronary artery disease and that these two pathological conditions are closely related and often coexist in the same patients.57 Treatment with carvedilol thus may not only reduce blood pressure but also attenuate oxidative tissue injury occurring in hypercholesterolemia.
In summary, our present study strongly suggests that although there is a highly efficient detoxification system against ONOO- under physiological conditions, the balance between ONOO- toxicity and a GSH-centered defense mechanism is significantly disturbed in pathological conditions such as hypercholesterolemia, in which significant amounts of ONOO- can be formed in vascular tissue. The reduced detoxification ability against ONOO- renders vascular tissue more susceptible to oxidative injury. Given that it is likely that ONOO- may exert significant deleterious effects in tissue injury observed in hypercholesterolemia and atherosclerosis, the administration of antioxidants, such as carvedilol, may attenuate oxidative tissue injury caused by ONOO- as well as by ROS.
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Selected Abbreviations and Acronyms |
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Received February 11, 1997; accepted April 14, 1997.
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