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1 Division of Pediatric Cardiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032; Departments of 2 Physiology and 3 Pathology, New York Medical College, Valhalla, New York 10595; 4 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520; and 5 Division of Cardiology, Brigham and Women's Hospital, Boston, Massachusetts 02115
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Statin drugs can upregulate
endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial
cells independent of lipid-lowering effects. We investigated the effect
of short-term simvastatin administration on coronary vascular eNOS and
NO production in conscious dogs and canine tissues. Mongrel dogs were
instrumented under general anesthesia to measure coronary blood flow
(CBF). Simvastatin (20 mg · kg
1 · day1) was
administered orally for 2 wk; afterward, resting CBF was found to be
higher compared with control (P < 0.05) and
veratrine- (activator of reflex cholinergic NO-dependent coronary
vasodilation) and ACh-mediated coronary vasodilation were enhanced
(P < 0.05). Response to endothelium-independent
vasodilators, adenosine and nitroglycerin, was not potentiated. After
simvastatin administration, plasma nitrate and nitrite
(NOx) levels increased from 5.22 ± 1.2 to 7.79 ± 1.3 µM (P < 0.05); baseline and
agonist-stimulated NO production in isolated coronary microvessels were
augmented (P < 0.05); resting in vivo myocardial
oxygen consumption (M
O2) decreased from
6.8 ± 0.6 to 5.9 ± 0.4 ml/min (P < 0.05);
NO-dependent regulation of MO2 in
response to NO agonists was augmented in isolated myocardial segments
(P < 0.05); and eNOS protein increased 29% and eNOS
mRNA decreased 50% in aortas and coronary vascular endothelium.
Short-term administration of simvastatin in dogs increases coronary
endothelial NO production to enhance NO-dependent coronary vasodilation
and NO-mediated regulation of MO2.
statins; coronary vasodilation; myocardial oxygen consumption
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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3-HYDROXY-3-METHYLGLUTARYL (HMG) CoA reductase inhibitors are widely used as cholesterol-lowering agents based on their ability to block hepatic conversion of HMG-CoA to L-mevalonate in the cholesterol biosynthetic pathway (6). Clinical trials have demonstrated that these agents decrease the incidence of ischemic stroke and myocardial infarction in atherosclerotic and hypercholesterolemic patients (15, 18). Although the beneficial effects of HMG-CoA reductase inhibitors are primarily attributed to their lipid-lowering effects, data from these trials suggest that statins may have actions independent of their lipid effects. One of the earliest recognizable benefits after therapy with these drugs is normalization of endothelial-dependent relaxation in atherosclerotic coronary arteries (12). Laufs and colleagues (8, 9) previously showed that the HMG-CoA reductase inhibitors simvastatin and lovastatin reversed the downregulation of endothelial nitric oxide synthase (eNOS) by hypoxia and oxidized low-density lipoprotein under cholesterol-clamped conditions. It therefore appears that restoration of endothelial function in response to statin drugs may be related to increased eNOS gene expression.
NO plays an important role in both regulating coronary vascular tone
and modulating myocardial oxygen consumption
(MO2). Our previous studies have shown
that NO contributes to exercise-induced coronary vasodilation as well
as coronary vasodilation secondary to activation of the Bezold-Jarisch
reflex and is associated with increased eNOS gene expression (17,
25). In failing hearts, downregulation of NO production and eNOS
gene expression appears to contribute to the decompensation of heart
failure (10, 21). NO modulates mitochondrial respiration
and tissue oxygen consumption through reversible inhibition of
cytochrome oxidase, the terminal enzyme complex of the mitochondrial
electron transport chain (2). Recchia and co-workers
(13) showed that disappearance of endogenous NO from the
coronary microcirculation is associated with altered regulation of
MO2 and progression from compensated to
decompensated heart failure. Because statins can upregulate NO and NO
regulates myocardial blood flow and MO2,
the purpose of our study was to evaluate the effect of short-term
(i.e., 2 wk) simvastatin administration in normal dogs on the
following: 1) in vivo NO-dependent coronary vasodilation;
2) in vivo cardiac oxygen consumption and substrate
utilization; 3) basal and stimulated nitrite
(NO2) production in isolated canine coronary microvessels;
4) NO-dependent regulation of
MO2 in isolated cardiac tissue; and
5) eNOS protein and mRNA expression in freshly isolated
aortic and coronary vascular endothelium.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Surgical Preparation
Fourteen mongrel dogs (weighing 23-27 kg) were premedicated with acepromazine (0.3 mg/kg im), anesthetized with pentobarbital sodium (25 mg/kg iv), and then intubated and ventilated with room air. A thoracotomy was performed in the left fifth intercostal space using sterile surgical techniques. Tygon catheters (Cardiovascular Instruments) were placed in the descending thoracic aorta, coronary sinus, and left atrial appendage for the measurement of pressures, blood sampling, and injection of drugs. A solid-state pressure gauge (P-6.5, Konigsberg Instruments) was placed in the apex of the left ventricle for the measurement of left ventricular systolic pressure (LVSP) and calculation of the first derivative of the left ventricular pressure (LV dP/dt). A Doppler flow transducer (Craig Hartley) was placed on the left circumflex coronary artery for measurement of coronary blood flow (CBF). A pair of pacing electrodes was sutured on the left ventricle for short-term pacing. The chest was closed in layers. The wires and the catheters were run subcutaneously and exited from the back of the dog's neck.The dogs were allowed 10-14 days to recover fully and were trained to lie quietly on the laboratory table. The protocols were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the National Institutes of Health Guide for the Use and Care of Laboratory Animals.
Recording Techniques
Arterial pressure was measured by connecting the previously implanted catheter to a strain-gauge transducer (P23 ID, Statham), and mean arterial pressure (MAP) was derived using a 2-Hz low-pass filter. LV pressure was measured from the solid-state pressure gauge, and LV dP/dt was calculated using a microprocessor set as a differentiator and having a frequency response flat to 700 Hz (LM-324, National Semiconductor). Left circumflex CBF velocity was measured using a pulsed Doppler flowmeter (System 6, Triton Technology). Mean CBF was derived using a 2-Hz low-pass filter. Blood flow was calculated using the cross-sectional area of the blood vessel obtained from the external diameter determined by the flow probe, the instantaneous flow velocity, and the angle of the crystal. Mean coronary resistance was calculated as the quotient of MAP and CBF. Heart rate was monitored from the pressure-pulse interval using a cardiotachometer (Beckman Instruments). The double product, an index of mechanically related MO2, was calculated as heart rate × LVSP.
Studies in Chronically Instrumented Conscious Dogs
Effects of activation of cardiac receptors by veratrine. On the day of the control experiments, with the dog lying on the laboratory table quietly, baseline hemodynamics and CBF were recorded and blood samples were drawn from the aortic and coronary sinus catheters. The implanted pacing electrodes were attached to an external pacemaker (model EV-3434, Pace Medical), and the heart was paced at 150 beats/min. With the heart rate controlled, bolus intra-atrial injections of veratrine were given at 0.05, 0.1, 0.5, 1, 2.5, 5, and 10 µg/kg, and hemodynamic variables were recorded at each dose. Each measured variable was allowed to return to baseline for at least 10 min before the next injection was given. Veratrine activates the Bezold-Jarisch reflex, a cardioinhibitory reflex that causes reflexive cholinergic NO-dependent coronary vasodilation (25).
The animals were administered simvastatin tablets (20 mg · kg1 · day1) orally
once a day for 2 wk, and the effects of veratrine on hemodynamic
parameters were repeated. This dose of simvastatin was chosen because
maximum increase in eNOS expression was seen at this dose compared with
lower doses in mice (4). A dose 
160 mg/day has been
shown to be safe in humans (3). In a study in rats, 55
mg · kg1 · day1 of statins
failed to cause skeletal myopathy in adult rats, although myopathy was
seen in the young rats at doses of 15 mg · kg1 · day1 and higher
(14). We did not observe any evidence of skeletal myopathy
(weight loss, weakness) in our dogs with this dose. Our preliminary
studies showed no significant alteration of veratrine-mediated coronary
vasodilation following 7 days of simvastatin treatment (data not
shown); therefore subsequent hemodynamic studies were done after 14 days of simvastatin administration. Serum cholesterol levels and plasma
nitrate and nitrite (NOx) levels were measured at baseline
and after 14 days simvastatin treatment.
Effects of ACh, adenosine, and nitroglycerin. ACh (2.5 and 5 µg/kg), adenosine (0.5 µg/kg), and nitroglycerin (25 µg/kg) were administered intravenously via a catheter inserted into a peripheral vein and with heart rate controlled at 150 beats/min. ACh is an endothelium-dependent coronary vasodilator, and adenosine and nitroglycerin (an exogenous NO donor) are endothelium-independent coronary vasodilators. Hemodynamic variables were measured after each dose and hemodynamics were allowed to return to baseline between doses. These experiments were done in the control state and after 2 wk of simvastatin administration.
Cardiac metabolites. Blood samples from the aorta and coronary sinus were collected into plastic syringes treated with either heparin or EDTA and immediately stored in ice. Blood gases were measured in a blood-gas analyzer (model 170, Corning). PO2 was multiplied by 0.003 and added to oxygen content measured by a hemoglobin analyzer (CO-Oximeter, Instruments Laboratory) to obtain total oxygen content (vol:vol). Lactate was measured from whole blood with a YSI-1500 sport lactate analyzer (Yellow Springs Instruments). Glucose and free fatty acid (FFA) concentrations were determined in plasma after centrifugation of the blood samples at 1,000 g for 15 min at 0°C. Glucose was measured with a Beckman glucose 2 analyzer (Beckman Instruments). FFA analysis was performed in plasma from EDTA-treated samples using a colorimetric assay (NEFA C kit, WAKO Pure Chemical Industries) and a spectrophotometer (Kontron Instruments). The arterial-coronary sinus concentration difference of oxygen, lactate, glucose, and FFA contents was multiplied by mean CBF (MCBF) to calculate cardiac consumption.
Studies in Isolated Coronary Microvessels
Isolation of coronary microvessels from the left ventricle of the dog heart was performed using the method originally developed by Gerritsen and Printz, which we have previously described in detail (5, 22, 23). In brief, coronary microvessels were isolated free of large arteries, veins, and myocytes by a series of steps involving sequential dissection, homogenization, sieving, and glass-bead purification. Microvessels were incubated with drugs to be tested for 20 min. Sulfanilamide (450 µl of 1%) and N-(1-naphthyl)-ethylenediamine (50 µl of 0.2%) were then added to each tube for diazotization of sulfanilic acid by NO. After 5-10 min incubation at room temperature, the supernatant was removed from each tube. Formation of NO was measured as NO2, the major metabolite of NO in aqueous solution. Using a spectrophotometer (Uvicon 930, Kontron Instruments), we measured the increase in absorbance at 540 nm and compared it with known concentrations of NO2. Data were expressed as means ± SE in picomoles per milligram of wet weight per 20-min incubation.Angiotensin-converting enzyme (ACE) inhibitors and the calcium-channel
blocker amlodipine cause kinin-dependent NO release in coronary
microvessels (22, 23). The effect of increasing doses of
bradykinin (107 to 105 M), the ACE
inhibitor ramiprilat (1010 to 108 M), ACh
(107 to 105 M), and calcium ionophore
A-23187 (108 to 106 M) on NO2
production in coronary microvessels was studied with and without the NO
synthase inhibitor
NG-nitro-L-arginine methylester
(L-NAME, 104 M).
MO2
O2 was measured
polarographically in vitro using a Clark-type oxygen electrode
(YSI-5331, Yellow Springs Instruments, Yellow Springs, OH). Oxygen
consumption studies were performed at 37°C in a stirred bath
(YSI-5301) containing 3 ml Krebs solution buffered with 10 mM HEPES (pH
7.4). Tissue respiration was calculated as the rate of decrease in
oxygen concentration after the addition of muscle slices, assuming an
initial oxygen concentration of 224 nmol/ml, and was expressed as
nanomoles of oxygen consumed per minute per gram of tissue. After
measurement of baseline MO2, cumulative
dose-response curves were generated after the addition of various
pharmacological agents to separate tissue baths in increasing
concentrations. The observation time for each dose of agent was
~5-7 min, and new muscle segments were used for each drug
tested. Succinate, a substrate for the electron transport chain (1 mM)
followed by sodium cyanide, an inhibitor of cytochrome oxidase (1 mM),
was added at the end of each drug tested to confirm that the change in
MO2 was reversible and was from
mitochondrial sources.
The following agonists of NO were added: bradykinin (107
to 104 M), ramiprilat (107 to
104 M), amlodipine (107 to
105 M), and
S-nitroso-N-acetyl-penicillamine (SNAP,
107 to 104 M). To assess the role of
endogenous NO production, the above studies were repeated after
preincubation of the muscle segments with L-NAME
(104 M), an inhibitor of NO synthase.
eNOS mRNA Measurement: Northern Blotting and PCR
Total RNA was isolated as previously described (17, 19) from the endothelium of thoracic aortas from control dogs and dogs receiving simvastatin. Equal amounts of total RNA (~15 µg) were separated by formaldehyde-agarose gel electrophoresis and transferred to nylon membrane blots. eNOS cDNA radiolabeling, hybridization, and washing of Northern blots were performed as previously described. The specific mRNA bands for eNOS (4.4 kb) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1.5 kb) were detected on phosphor screens and quantified by Image Quant Software (Storm Molecular Dynamics). These results were corroborated using competitive PCR developed by us for canine eNOS in isolated coronary vascular endothelium.eNOS Protein Measurement: Western Blotting
Proteins were prepared and separated on SDS-PAGE as described previously (19) from control and simvastatin-treated dogs. Proteins were transferred to polyvinyl difluoro membranes (Millipore) in 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS, pH 10) buffer containing 10% methanol and 0.5 mmol dithiothreitol. The 135-kDa eNOS band was detected by scanning blots in the fluorescent mode on a Storm Molecular Dynamics Scanner and Image Quant Software as described for Northern blots. The primary antibody was rabbit polyclonal anti-bovine eNOS peptide antibody (Affinity Bioreagents); rabbits were immunized with a COOH-terminal peptide (amino acids 599-613) coupled to keyhole limpet hemocyanin. Immunoreactivity was detected by a chemifluorescence method (Vistra kit, Amersham) in which secondary antibody is an alkaline phosphatase conjugate.Source of Drugs and Chemicals
Drugs (bradykinin, ACh, A-23187, and atropine) and chemicals (L-NAME and NOx) were purchased from Sigma Chemical (St. Louis, MO). Amlodipine was supplied by Pfizer Pharmaceutical (Groton, CT). Ramiprilat was supplied by Hoechst-Roussel (Somerville, NJ). Simvastatin was obtained from Merck, Sharp, & Dohme.Statistical Analysis
Hemodynamic results, percentage change in MO2 from baseline, and coronary
microvessel NO2 production are expressed as means ± SE. Data are analyzed using two-way ANOVA, with a Student-Newman-Keuls post hoc analysis to identify which means are different (Sigma Stat,
version 1.0, Jandel Scientific, San Rafael, CA). Results of Northern
and Western blots in control and statin-treated dogs are expressed as
means ± SE. The data were analyzed by unpaired Student's
t-test. Serum cholesterol and plasma NOx levels
were analyzed by a paired Student's t-test. A value of
P < 0.05 was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Data from 6 control dogs and 14 simvastatin dogs are presented. Total cholesterol levels were 3.6 ± 1.6 mM (138 ± 6 mg/dl) at control and decreased to 2.1 ± 1.8 mM (82 ± 7 mg/dl) after simvastatin administration. Plasma NOx levels increased significantly after 2 wk simvastatin (5.22 ± 1.2 in control vs. 7.79 ± 1.3 µM after simvastatin; P = 0.02). There was no significant correlation between plasma cholesterol and NOx levels (r = 0.53).
Coronary and Systemic Hemodynamics
The heart was paced at 150 beats/min to avoid the effects of veratrine-induced bradycardia on CBF. Mean resting hemodynamics in the control state were as follows: CBF, 26 ± 1 ml/min; mean coronary resistance, 4 ± 0.2 mmHg · ml1 · min1; MAP,
104 ± 2 mmHg; LVSP, 125 ± 2 mmHg; and LV dP/dt,
2,855 ± 78 mmHg/s.
Effects of Veratrine
Baseline.
Intra-atrial injections of veratrine (n = 8) at 0.05, 0.1, 0.5, 1, 2.5, 5, and 10 µg/kg caused dose-dependent increases in CBF to 56 ml/min at the highest dose and decrease in coronary resistance to 1.6 mmHg · ml1 · min1
(P < 0.05 from baseline). The actual changes in blood
flow and resistance are shown in Fig. 1.
This was associated with significant decreases in MAP to 82 ± 7 mmHg, LVSP to 108 ± 6 mmHg, and LV dP/dt to 1,816 ± 144 mmHg/s at the highest dose (P < 0.05).
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Simvastatin for 2 wk. There was a significant increase in resting CBF (34 ± 1 vs. 26 ± 1 ml/min) after 14 days of simvastatin as well as an augmented coronary vasodilation in response to veratrine compared with the control state (P < 0.05 from control, Fig. 1). Resting coronary resistance and MAP were lower after simvastatin administration. Also, veratrine caused a significantly larger reduction in MAP and coronary resistance after simvastatin administration (P < 0.05 from control). Veratrine-induced decreases in LVSP (108 ± 8 mmHg) and LV dP/dt (1,867 ± 210 mmHg/s) were not significantly different after simvastatin compared with the control state (P > 0.05).
Effects of ACh, Adenosine, and Nitroglycerin
Bolus injection of ACh (2.5 and 5 µg/kg iv) (n = 6) resulted in significant increase in CBF and decrease in coronary resistance as shown in Table 1 (P < 0.05 from baseline). This was associated with significant decreases in MAP and LVSP and no significant change in LV dP/dt. After 2 wk of simvastatin, ACh caused a significantly larger increase in CBF and decrease in coronary resistance at a 2.5 µg/kg dose (P < 0.05 from control, Table 1). Both adenosine and nitroglycerin increased CBF and decreased coronary resistance (P < 0.05 from baseline). This response was not significantly different after simvastatin administration (Table 1).
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Cardiac Metabolism
Arterial PO2 and concentrations of oxygen, lactate, glucose, and FFA did not change significantly after 14 days of simvastatin. In vivo MO2
was measured in 7 dogs. Cardiac oxygen consumption decreased
significantly after 14 days simvastatin, P < 0.05 (Fig. 2). It is important to note that
this change in MO2 occurred in the
absence of a change in rate-pressure product as an index of cardiac
work, which was 12.4 ± 0.6 mmHg/s both before and after simvastatin. Although lactate uptake showed a tendency to decrease after 14 days simvastatin from 33 ± 7 to 23 ± 10 µM/min
(n = 5), the results did not reach statistical
significance (P = 0.18). There was no change in basal
FFA uptake (4.4 ± 1 to 4.0 ± 1 µeq/min) after simvastatin
administration. There was no significant uptake of glucose before or
after simvastatin treatment.
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Nitrite Production in Isolated Coronary Microvessels of Control and Simvastatin Dogs
There was a significant elevation in basal NOx production in microvessels from simvastatin-treated dogs (108 ± 13 pmol/mg) (n = 5) compared with controls (73 ± 6 pmol/mg), P < 0.05 (n = 6). All the agonists caused significantly greater increases in NO2 production in coronary microvessels from dogs receiving simvastatin compared with controls (Fig. 3). The highest dose of ACh increased NO2 production from 71 ± 4 to 146 ± 12 pmol/mg in control dogs and from 109 ± 12 to 170 ± 21 pmol/mg in simvastatin-treated dogs (P < 0.05 from control). A-23187-stimulated NO2 production was significantly higher after simvastatin administration compared with controls (209 ± 25 vs. 161 ± 17 pmol/mg). The same was true of bradykinin (213 ± 25 vs. 197 ± 19 pmol/mg) and ramiprilat (219 ± 28 vs. 140 ± 18 pmol/mg), P < 0.05. L-NAME, an inhibitor of NO synthase, inhibited agonist-stimulated NO production in both control and simvastatin groups.
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In Vitro MO2 in Control and
Simvastatin-Treated Dogs
O2 in isolated LV muscle
segments of control dogs (n = 6) was 182 ± 21 nmol · g1 · min1. This was
not significantly different in simvastatin-treated dogs (162 ± 17 nmol · g1 · min1)
(n = 9). All the agonists caused a dose-dependent
reduction in MO2 (Fig.
4). After simvastatin administration,
these agonists caused significantly larger reductions in
MO2 compared with controls: bradykinin
(
27 ± 1% vs. 21 ± 5%, respectively), ramiprilat (22 ± 2% vs. 10 ± 3%, respectively), and amlodipine
(25 ± 3% vs. 11 ± 1%, respectively). The effect of
SNAP on MO2 (39 ± 5% vs.
43 ± 6%) was not significantly different between the two
groups. Pretreatment of the tissue with L-NAME did not
significantly alter baseline MO2;
however, L-NAME attenuated the effect of bradykinin,
ramiprilat, and amlodipine (but not SNAP) on
MO2 in both control and simvastatin dogs
(P < 0.05, Fig. 5).
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eNOS mRNA in Control and Simvastatin-Treated Dogs
Simvastatin administration was associated with a significant (~60%) reduction (P < 0.05) in the ratio of aortic eNOS to GAPDH (147 ± 9 to 95 ± 2 Phosphorimage volume units for control and simvastatin-treated dogs, respectively; n = 4 in each group) as demonstrated by Northern blotting (Fig. 5). These results were corroborated using a competitive PCR for canine eNOS in coronary microvascular endothelium. Simvastatin decreased steady-state eNOS mRNA levels by ~50% (from 43 ± 6.6 to 19 ± 0.4 amol/500 ng RNA in control and simvastatin-treated dogs, respectively; n = 7 for control and n = 5 for simvastatin) (Fig. 6).
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eNOS Protein in Control and Simvastatin-Treated Dogs
There was a significant elevation in eNOS protein in aortas from simvastatin-treated dogs compared with controls as illustrated by Western blotting (n = 4 in each group; Fig. 7). Scanning densitometry showed a 29% increase in eNOS protein in the simvastatin-treated dogs compared with control dogs despite the ~50% reduction in eNOS mRNA.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The most important finding of the present study is that short-term
administration of simvastatin increases NO production in canine
coronary vascular endothelium. This conclusion is supported by the
observation that simvastatin treatment enhances NO-dependent coronary
vasodilation, increases NO production in coronary microvessels, and
enhances NO-mediated regulation of MO2
in association with an increase in aortic endothelial eNOS protein.
Treatment with HMG-CoA reductase inhibitors can cause rapid improvement in endothelium-dependent vasomotor function in the human forearm circulation with 2-12 wk of statin therapy (12), suggesting a beneficial effect on endothelial function that extends beyond the ability to lower cholesterol. Statins improve stress-induced contractile-defect size and perfusion abnormalities in patients with >50% coronary stenoses as early as 3 mo after onset of therapy (7). Our study reports a rapid improvement in endothelial function and NO-dependent coronary blood flow in animals with no underlying coronary artery disease after administration of a statin drug.
Administration of simvastatin enhanced NO-dependent coronary vasodilation in response to veratrine. This was evident after 14 days, but not after 7 days (data not shown), indicating a time-dependent response. Our previous studies have shown that veratrine-activated reflex cholinergic coronary vasodilation is entirely mediated by NO (24, 25). To assess endothelial function, we performed studies using endothelium-dependent (ACh) and endothelium-independent (adenosine and nitroglycerin) coronary vasodilators. ACh (2.5 µg/kg) caused significantly greater coronary vasodilation in dogs after 2 wk simvastatin administration. The response to the 5 µg/kg dose was not enhanced after simvastatin treatment. This may reflect either a ceiling effect or the involvement of factors other than NO such as endothelium-derived hyperpolarizing factor at the higher dose of ACh. The coronary vasodilation in response to the endothelium-independent vasodilators adenosine and nitroglycerin, an exogenous NO donor, was not enhanced after simvastatin administration. Simvastatin also increased resting CBF in association with elevated basal aortic and coronary NOx production. In a recent study, Endres and colleagues (4) showed that HMG-CoA reductase inhibitors upregulate cerebral eNOS and increase cerebral blood flow to decrease infarct size after experimental cerebral ischemia in mice. Lefer and co-workers (11) showed that statin drugs inhibit leukocyte-endothelial cell interactions and increase endothelial NO release to preserve cardiac contractile function and coronary perfusion after myocardial ischemia and reperfusion in isolated rat hearts. Therefore the ability of simvastatin to increase resting and agonist-mediated CBF in our study may account in part for the cardioprotective effect of statins and may underlie some of the beneficial effects in reducing cardiac mortality and morbidity and decreasing episodes of myocardial ischemia (15, 16, 18).
Although there was a significant reduction in serum total cholesterol levels in dogs receiving simvastatin, it is important to recognize that all the dogs were normocholesterolemic and coronary vasodilation did not significantly correlate with degree of lipid lowering, which indicated that lipid lowering alone did not account for this improvement in endothelial NO release. Most of the changes in coronary hemodynamics appear to be related to increased aortic and coronary NO production as reflected by elevated basal plasma NOx levels and coronary microvessel NOx levels. Also, simvastatin potentiated NO production in response to agonists like ramipril, which is an ACE inhibitor, and other endothelium-dependent agents such as ACh and A-23187.
Our results further show that increased coronary microvessel NO release
after simvastatin treatment is associated with enhanced NO-dependent
regulation of MO2. Basal cardiac oxygen
consumption measured in vivo was significantly lower following 2 wk of
simvastatin administration. Rate-pressure product, which relates
linearly to cardiac work (1), did not change after
simvastatin treatment; therefore oxygen consumption was lower for
comparable cardiac work after simvastatin administration. Bernstein and
colleagues (1) reported that inhibition of NO synthesis in
conscious dogs causes the heart to consume more oxygen for the same
amount of cardiac work during exercise. To determine whether change in
MO2 was secondary to change in
myocardial substrate utilization, we measured fasting lactate, FFA, and
glucose uptake. A healthy heart utilizes mostly FFA and, to a lesser
extent, lactate during fasting (20). Recchia and
colleagues (13) recently reported that inhibiting NO
production shifts cardiac substrate utilization from FFA to glucose and
lactate. In our study, there was no demonstrable change in basal FFA or
glucose uptake after simvastatin treatment. It is likely that because
the heart already extracts predominantly FFAs at rest, there may not be
a further increase in FFA uptake at rest despite increased NO.
Simvastatin not only decreased cardiac oxygen consumption in vivo, but
also enhanced the ability of ramipril and amlodipine to lower
MO2 in vitro. The decrease in
MO2 in response to the exogenous NO
donor SNAP, on the other hand, was not enhanced after simvastatin
treatment. The lack of difference to SNAP suggests that the underlying
mitochondrial function was not altered by simvastatin and that the
decrease in MO2 with other drugs was related to enhanced NO availability.
We attempted to correlate the upregulation of NO production in coronary
vascular endothelium with eNOS gene expression and eNOS protein in
aortic vascular endothelial cells. One of the surprising findings of
our study was that although eNOS protein increased 
29% in aortas
from simvastatin-treated dogs, eNOS mRNA decreased almost 50% in both
aorta and coronary microvessels. These data are supported by using both
Northern blotting and a canine-specific competitive PCR we developed.
Laufs and co-workers (9, 10) reported that inhibition of
vascular endothelial HMG-CoA reductase upregulates the expression of
eNOS through an increase in eNOS mRNA stability secondary to changes in
isoprenoid synthesis. However, their study measured eNOS expression
after only 48-96 h of in vitro statin exposure, as opposed to our
study, which measured eNOS after 14 days of in vivo simvastatin
administration. The decrease in gene expression in our study occurred
in the face of elevated NO production. Because statins cause
posttranscriptional regulation of eNOS to stabilize the message
(10), we speculate that stabilization of the message and
chronic elevation of NO may have a feedback effect on eNOS mRNA
resulting in a decrease in eNOS mRNA over time. Support for this
concept also comes from our previous studies in conscious dogs in which
1 wk administration of CAS-936, an NO-releasing agent, produced a
selective reduction in eNOS mRNA (24).
These findings may have important clinical implications especially in conditions of lowered NO availability as seen in failing hearts. Heart failure is associated with impaired ability of vascular endothelium to produce NO secondary to reduced eNOS expression, and this may contribute to progression of heart failure (13, 19, 21). Statins, by virtue of their ability to upregulate eNOS, may act synergistically with ACE inhibitors or amlodipine to augment NO production in heart failure.
In conclusion, simvastatin administration increases coronary
endothelial NO production, enhances NO-mediated regulation of CBF, and
decreases MO2 in association with an
increase in eNOS protein. This finding may have important clinical
implications beyond the well-recognized lipid-lowering effect of these
drugs. Because myocardial ischemic events often occur in individuals with normal cholesterol levels, upregulation of NO production by
HMG-CoA reductase inhibitors may prevent or limit the severity of
coronary events even in normocholesterolemic individuals. By increasing
NO production in the systemic vasculature, these drugs may also have
beneficial effects in other cardiovascular conditions where endothelial
dysfunction can contribute to pathologic processes such as
atherosclerosis, diabetes, pulmonary hypertension, and congestive heart failure.
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ACKNOWLEDGEMENTS |
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The authors thank Jing-Zhi He for doing the Northern and Western blots.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-50142, HL-53053, HL-52233, HL-61290, and PO-1HL-43023.
Address for reprint requests and other correspondence: T. H. Hintze, Dept. of Physiology, Basic Science Bldg., Rm. 636, New York Medical College, Valhalla, NY 10595 (E-mail: thomas_hintze{at}nymc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 September 1999; accepted in final form 5 June 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Bernstein, RD,
Ochoa FY,
Xu X,
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