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TherapeuticsJournal of Cardiovascular Pharmacology and

http://cpt.sagepub.com/content/early/2010/10/05/1074248410378506The online version of this article can be found at:

 DOI: 10.1177/1074248410378506

published online 11 October 2010J CARDIOVASC PHARMACOL THERAlaaeldin I. Saleh, Sahar M. Abdel Maksoud, Shohda A. El-Maraghy and Mohamed Z. Gad

AspirinProtective Effect of L-Arginine in Experimentally Induced Myocardial Ischemia: Comparison With

  

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Protective Effect of L-Arginine inExperimentally Induced MyocardialIschemia: Comparison With Aspirin

Alaaeldin I. Saleh, MSc1, Sahar M. Abdel Maksoud, PhD1,Shohda A. El-Maraghy, PhD2, and Mohamed Z. Gad, PhD1

AbstractObjective: Coronary artery diseases including myocardial ischemia (MI) remain one of the leading causes of death worldwide.This study was designed to compare the protective effect of L-arginine versus aspirin from the biochemical changes associatedwith MI injury. Experimental design: Four groups of male New Zealand white rabbits were investigated. Normal group (n ¼ 8)rabbits were fed standard chow pellets, untreated MI group (n ¼ 16), where hypercholesterolemia was induced by feeding theanimals with a diet containing 2% cholesterol for 28 days, L-arginine group (n¼ 12) rabbits were fed a 2% cholesterol-enriched dietin conjunction with L-arginine (2.25 g %) in drinking water for 28 days, and aspirin group (n ¼ 12) rabbits were fed 2% cholesterol-enriched diet in conjunction with aspirin administered orally (0.7 mg/kg per d) for 28 days. After 28 days, MI was induced in all groups,except the normal group, by a single subcutaneous (sc) injection of isoproterenol hydrochloride (0.2 mg/kg body weight [bw]). Animalswere sacrificed 6 hours later. Results: Our results showed that L-arginine was more effective than aspirin in reducing platelet aggrega-tion, reducing low-density lipoprotein (LDL) oxidizability, preventing aortic intimal thickening, andmaintaining histological architectureof the myocardium. Both drugs, however, had similar positive effects on plasma fibrinogen levels and on the prevention of myocardialrelease of cardiac troponin I and creatine kinase-MB. The effect on hypercholesterolemia was insignificant for both drugs. Aspirinwas more effective than L-arginine in prolonging prothrombin time. Conclusion: L-arginine supplementation represents a potentiallynovel nutritional strategy for preventing and treating coronary artery diseases especially in cases of aspirin resistance and/orhypersensitivity.

Keywords

myocardial ischemia, hypercholesterolemia, isoproterenol, aspirin, L-arginine

Introduction

Myocardial ischemia (MI) is caused by an insufficient supply

of oxygen-rich blood to the myocardium caused by increased

myocardial substrate demand and/or the narrowing or closure

of coronary arteries.1 Lack of oxygen and metabolic substrates

rapidly decreases the energy available to the cell and leads to

cell injury that might be reversible or irreversible in nature.

One of the most widely used experimental models to study

MI and the therapeutic effects of drugs is isoproterenol-

induced MI.2 Isoproterenol (isoprenaline) hydrochloride is a

synthetic sympathomimetic amine that is structurally similar

to epinephrine but selective for b receptors, activating b1 and

b2 receptors equally, and exerting positive inotropic and chron-

otropic effects on the heart.3 Indeed, pathophysiological

changes following isoproterenol administration are comparable

to those taking place in human MI/infarction.4

L-Arginine is a semi-essential amino acid found in most

mammals. Under conditions of stress and injury, L-arginine is

considered an essential amino acid. It is the precursor of

endothelial nitric oxide (NO) that plays a critical role in cardi-

ovascular protection5 and in maintaining an active vasodilator

tone in healthy blood vessels.6 Several studies in humans and

experimental animals indicated that oral L-arginine intake has

multiple beneficial cardiovascular effects when taken in doses

larger than normally present in diet (reviewed in ref 7). The pos-

itive role of L-arginine in cardiovascular health is due to both

NO-dependent and NO-independent effects.8 It has been shown

that both oral and parenteral administration of L-arginine can

1 Biochemistry Department, Faculty of Pharmacy, German University in Cairo,

New Cairo, Egypt2 Biochemistry Department, Faculty of Pharmacy, Cairo University, Giza, Egypt

Corresponding Author:

Mohamed Z Gad, Biochemistry Department, Faculty of Pharmacy, The German

University in Cairo, El-Tagmoa El-Khamis, New Cairo, Egypt

Email: mohamed.gad@guc.edu.eg

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by Alaaeldin Saleh on October 12, 2010cpt.sagepub.comDownloaded from

increase NO synthesis in various tissues.9 In hypercholesterole-

mia, the synthesis and/or release of NO is severely impaired,

leading to attenuated endothelium-dependent vasodilation,

enhanced platelet aggregation, intimal thickening, and subse-

quent development of atherosclerosis.10 The impairment of

endothelial NO liberation in hypercholesterolemia is related to

low-density lipoprotein (LDL) cholesterol uptake into the

endothelium,11 and both native and oxidized LDLs are reported

to interfere with the biological activity of NO in vitro.12

Evidence suggests that L-arginine may play a therapeutic role

in tissue preservation during reperfusion after a period of

hypoxia, as animal studies showed protective effects of

L-arginine administration against ischemia/reperfusion inju-

ries in several tissues, including the myocardium.13

Among the traditional medicines used for cardiovascular

disease (CVD) is aspirin, the prototype of traditional nonster-

oidal anti-inflammatory drugs (NSAIDs), which had been

officially approved by the US Food and Drug Administration

(FDA) in 1939. The history of aspirin use in protection from

CVD started in the 1950s, after the appearance of evidence

that aspirin prolongs prothrombin time (PT).14 During the

1960s, aspirin was found to inhibit thromboxane A2 synthesis

that enhances platelet aggregation.15 Today, use of low-

dosage aspirin is a common practice in the prophylaxis and

treatment of CVD.16 This study is among the few studies that

address the potential role of L-arginine in the prevention of

MI injury. The molecular mechanisms behind this prevention

were investigated and compared to the widely used cardiovas-

cular protective agent, aspirin.

Materials and Methods

Animals

A total of 48 male New Zealand white rabbits weighing 1.55 to

1.95 kg were used in the study. They were kept in standard rab-

bit cages in the animal house of the Faculty of Pharmacy, Cairo

University. The animals were adapted for 10 days prior to

experimental use. All rabbits were maintained at almost con-

stant environmental conditions throughout the study with free

access to food and water. The study protocol was approved

by the local ethics committee.

Drugs and Chemicals

L-Arginine powder (NOW FOODS, Bloomingdale, Illinois),

Cholesterol fine powder (Sigma-Aldrich, Germany), Isoprena-

line hydrochloride ampules (0.2 mg/mL, Monico SPA, Italy),

Aspirin tablets (acetylsalicylic acid 75 mg, CID, Egypt), and

Tween 80 (Adwic, Egypt) were used from the indicated manu-

facturers. All other reagents and chemicals were of high analy-

tical grade.

Experimental Protocol

After the initial adaptation, rabbits were divided into four

groups: group I, normal group of 8 rabbits fed standard chow

pellets; group II, myocardial ischemic (untreated MI) group

of 16 rabbits fed 2% cholesterol-enriched diet for 28 days17;

group III, L-arginine group of 12 rabbits fed 2% cholesterol-

enriched diet in conjunction with L-arginine in drinking water

(2.25 g %) for 28 days18; and group IV, aspirin group of 12 rab-

bits fed 2% cholesterol-enriched diet in conjunction with orally

administered aspirin (0.7 mg/kg per d) for 28 days.19 After

completion of the experimental period, animals in the untreated

MI, L-arginine, and aspirin groups were then injected with a

single bolus subcutaneous (sc) injection of isoproterenol hydro-

chloride (0.2 mg/kg body weight [bw])20 and sacrificed 6 hours

later. Control group rabbits were injected with vehicle and were

sacrificed 6 hours later. All animals were subjected to a

12-hour fast, with free access to water and were anesthetized

with intravenous injection of 20 mg/kg bw thiopental sodium21

before killing.

Sample Collection

Fasting blood samples from each rabbit ear vein were drawn

before killing, for the preparation of serum or plasma. Later,

hearts were removed and used for histopathological examina-

tion and determination of aorta intimal thickness.

Biochemical Analysis

Commercially available kits were used for the determination of

serum total cholesterol ([TC]; Diamond Diagnosis Co,

Germany),22 serum creatine kinase-MB activity ([CK-MB];

Greiner Diagnostics GmbH, Germany),23 cardiac Troponin

I ([cTnI]; DRG International Inc, Mountainside, New Jersey),24

PT, and fibrinogen measurements (Diagnostica stago,

France).25 Spectrophotometric measurements were performed

using a Jasco UV/VIS spectrophotometer (Japan) and a micro-

titer well reader Perkin Elmer (Perkin Elmer, Waltham, Massa-

chusetts). In the LDL oxidizability experiment, metal ion

copper was used for measuring the susceptibility of LDL and

very-low-density lipoprotein (VLDL) to become oxidized.26

Plasma was used for the determination of platelet aggregation

using a dual channel platelet aggregometer (Chrono-log,

Havertown, Pennsylvania).27

Histological Assessment

Autopsy samples were taken from the heart’s myocardium,

coronaries, and aorta of rabbits and fixed in 10% formol

saline. Samples were then subjected to histopathological

examination and intimal thickness measurement.28 For each

animal, 10 specimens from the aorta were subjected to

measurement of intimal thickness (mm) by a Leica image ana-

lyzer. The mean of the 10 sections represented the intimal

thickness of the animal. The average intimal thickness for all

animals of the group was then calculated to represent the mean

of intimal thickness for each group. These measurements were

done in the Histology department, Faculty of Medicine, Cairo

University.

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Statistical Analysis

Results are expressed as mean + standard error of the mean

(SEM). For statistical analysis of the data, multiple compari-

sons were carried out using 1-way analysis of variance

(ANOVA) followed by Tukey-Kramer test for post hoc analy-

sis. Statistical significance was accepted for a level of P < .05.

Results

Biochemical ChangesHypercholesterolemia. Administration of 2% cholesterol-

enriched diet resulted in significant rise in serum levels of

TC in untreated MI, aspirin, and L-arginine groups as compared

to the normal group

No significant difference was observed between the

means of untreated MI, aspirin, and L-arginine groups

(Figure 1).

Markers of MI. Cardiac markers CK-MB and cTnI in the

untreated MI group were significantly increased over the

normal group (Figure 2A, B). Administration of aspirin or

L-arginine produced a significant decrease in cTnI level by

48% and 50%, respectively (P ¼ .016 and P ¼ .0153) and in

CK-MB activity by 43% and 44%, respectively (P ¼ .048 and

P ¼ .04), as compared with untreated MI group. No significant

differences were observed between the results of aspirin and

L-arginine; both drugs reduced CK-MB and cTnI to control

group levels (Figure 2A, B)

Blood clotting parameters. Platelet aggregation and fibrinogen

levels were significantly elevated by 57.4% and 52.5% in the

untreated MI group as compared with the normal group (P ¼.006 and P ¼ .00127, respectively; Figure 3A, B, C). However,

PT was significantly lowered by 51% (P ¼ 7 � 10�5).

Administration of aspirin produced a decrease in platelet

aggregation by 49% (P ¼ .0014), a decrease in fibrinogen by

30% (P ¼ .002), and an increase in PT by 122% (P ¼ 2 � 105)

as compared with untreated MI group. All changes were statisti-

cally significant (P < .05). L-arginine administration also resulted

in significant changes of these parameters. Platelet aggregation

decreased by 61% (P ¼ 3 � 10�5), fibrinogen decreased by

24% (P ¼ .0713), and PT increased by 59% (P ¼ .00142).

Comparing the results of aspirin with those of L-arginine revealed

that L-arginine was more effective in lowering platelet aggrega-

tion (P ¼ .0196), whereas aspirin in the dosage used was more

powerful in extending PT (P¼ .0079). No significant differences

were observed for the decrease in fibrinogen. Platelet aggregation

in the L-arginine group was even below the normal group, despite

cholesterol-rich diet (Figure 3A), whereas PT in the L-arginine

group improved compared to the untreated MI group but

remained below normal levels (Figure 3C). Differences from nor-

mal group was observed for L-arginine in platelet aggregation

results (P¼ .0346) and in PT (P¼ .0254). Other results for aspirin

and L-arginine in blood clotting parameters were insignificantly

different from those of normal results.

Low-density lipoprotein oxidizability. Low-density lipoprotein

oxidizability in the MI group showed an increase compared to

the normal group, which was, however, not statistically signifi-

cant (P¼ .45; Figure 4). Aspirin did not elicit significant changes

in LDL oxidizability (P ¼ .0633 and P ¼ .201, respectively, as

compared to normal and untreated MI groups). In contrast,

administration of L-arginine produced a significant decrease in

LDL oxidizability reaching 57% of normal (P ¼ .017), 32% of

untreated MI (P¼ .008), and 43% of aspirin (P ¼ 6� 10�6).

Histopathological ChangesAortic intimal thickness. Aortic intimal thickness in the normal

group was 5.96 + 0.38 mm (Figure 5). A significant increase in

the intimal thickness in the untreated MI group was observed

with a mean of 71.59 + 11.3 mm (P ¼ 6.6 � 10�6). Adminis-

tration of aspirin or L-arginine reduced aortic intimal thickness

significantly to 52.88 + 7.5 and 9.86 + 1 mm as compared

with untreated MI group (P ¼ .0056 and P ¼ 9.6 � 10�6,

respectively). In the L-arginine group the decrease of the

intimal thickness was more pronounced than in the aspirin

group (P ¼ 5.3 � 10�6). Indeed, aortic intimal thickness in the

L-arginine group was close to normal levels.

Histopathological examination of heart, coronaries, and aorta.Examination of sections from the normal group revealed no

histopathological findings in the heart, coronaries, and aorta

(Figures 6-8).

In contrast, sections from the untreated MI group showed

ischemia in myocardial muscle cells characterized by granular

degeneration and hyalinization (d in Figure 6) as well as focal

inflammatory cell infiltration and fatty changes (v in Figure 6)

between the myocardial bundles. Examination of coronaries

showed severe congestion (Figure 7) and swelling of the

Figure 1. Effect of cholesterol-enriched diet either alone (untreatedMI group) or in combination with aspirin or L-arginine on serum levelsof total cholesterol (mg/dL). Data are expressed as mean + SEM.Number of animals were 8, 16, 12, and 12, respectively, for normal,untreated MI, aspirin, and L-arginine groups. a indicates significant dif-ference from normal at P < .05; MI, myocardial ischemia; SEM, stan-dard error of mean.

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aortic endothelial cells in the tunica intima associated with vacuo-

lization in the media (m in Figure 8) and edema in the adventitia (d

in Figure 8). In the aspirin group, hearts showed focal inflamma-

tory cell infiltration, with focal degeneration and hyalinization (d

in Figure 6) in the myocardial bundles, which indicates the occur-

rence of MI. Severe congestion was observed in the coronaries (c

in Figure 7). The aorta showed swelling, vacuolization, and stra-

tification in the endothelial cells of the intima and the subintimal

layer (Figure 8). In the L-arginine group, no abnormal histopatho-

logical findings were observed (Figure 6). Severe congestion was

observed in the myocardial coronaries (Figure 7). The aorta of

L-arginine group animals showed swelling, vacuolization, and

stratification in the endothelial cells of the intima as well as the

underlying layer (Figure 8).

Discussion

Experimental Induction of MI by Cholesteroland Isoproterenol

The current study aimed to investigate the protective effect of

L-arginine in comparison to aspirin from the biochemical

changes caused by hypercholesterolemia-associated MI.

Several studies have shown that acute hypercholesterolemia

increases the severity of MI.29-31 Experimental ischemia and

hypercholesterolemia were induced in our study by sc injection

of isoproterenol to rabbits that had been fed a 2% cholesterol-

enriched diet for 28 days. Isoproterenol produces relative ische-

mia or hypoxia due to myocardial hyperactivity and coronary

hypotension, associated with cytosolic Ca2þ overload.32 This

method seems to have an advantage over the method using cor-

onary ligation and reperfusion, as it is nonsurgical and results in

very small morbidity or mortality. The cholesterol-rich diet

caused a significant increase in serum TC in the untreated

MI, aspirin, and L-arginine groups as compared to the normal

group. Similar results have been reported previously by many

other groups.33-35

Isoproterenol-induced MI was verified by significant

increases in the serum levels of the ischemic markers CK-

MB and cTnI in the untreated MI group. Hypercholesterolemia

and MI are associated with Ca2þ overload and generation of

reactive oxygen species (ROS), which lead to cardiac muscle

injury, thus raising the levels of cTnI in the blood. The

observed increase in serum cTnI after isoproterenol injection

in our study is in agreement with previous reports.36,37 In sup-

port of our cTnI results, serum CK-MB also significantly

increased in the untreated MI group over the normal group.

Like cTnI, CK-MB is a well-known marker for MI; it is even

more specific to cardiac muscle than to skeletal muscle.38

Low-density lipoprotein oxidizability was increased, but not

to the level of statistical significance, after the induction of

hypercholesterolemia and MI. Hypercholesterolemia is associ-

ated with increased production of ROS and increased oxidation

of LDL cholesterol.39 Production of ROS decreases the bioa-

vailability of NO, thus leading to inadequate endothelium-

dependent responses.40 It is also known that isoproterenol itself

generates free radicals and stimulates lipid peroxidation.

Indeed, this may be a causative factor for the irreversible dam-

age to the myocardial membrane.41 All the blood-clotting para-

meters (platelet aggregation, fibrinogen, and PT) exhibited

significant changes in the untreated MI group as compared to

the normal group. The increase in platelet aggregation might

be induced by thromboxane A2, which is synthesized by plate-

lets and other cells. Hypercholesterolemia was found to

enhance the sensitivity of platelets to agonists such as ADP,

collagen, or epinephrine.42 The increase in plasma fibrinogen

in the untreated MI group is in support of the reported role of

fibrinogen as a major risk factor in cardiovascular disease.43

Prothrombin time, however was significantly reduced in the

untreated MI group, as expected, because hypercholesterole-

mia is one of the factors known to decrease PT.44 Furthermore,

it has been reported that PT was significantly decreased

in MI.45

Histopathological examination of the hearts of untreated MI

group animals revealed an abnormal histological structure of

the myocardial bundle. The aorta intimal thickness of untreated

MI group was dramatically increased as compared to the nor-

mal group. These results are in agreement with a study showing

Figure 2. Effect of cholesterol-enriched diet either alone (untreatedMI group) or in combination with aspirin or L-arginine on serum levelsof troponin ([cTnI] ng/mL) (A) and creatine kinase-MB (CK-MB; U/L)(B). Data are expressed as mean + SEM. Number of animals were 8,16, 12, and 12, respectively, for normal, untreated MI, aspirin, andL-arginine groups. 1 U/L CK-MB ¼ change of abosorbance/min �6666. a indicates significant difference from normal at P < .05; b, signif-icant difference from untreated MI group at P < .05; MI, myocardialischemia; SEM, standard error of mean.

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that patients with hyperlipidemia had significantly lower levels

of endothelium-dependent vasodilatation and higher intima

media thickness of the common carotid artery when compared

to controls.46

Protective Effect of Aspirin

It was evident in the current study that hypercholesterolemia

itself was not corrected by oral administration of aspirin. This

result is in accordance with that of Yi et al,47 which showed no

effect of aspirin in decreasing the cholesterol level in athero-

sclerotic rabbits. They concluded from their study that the

mechanism of atherosclerosis suppression by aspirin in

cholesterol-fed rabbits is related to the inhibition of cyclooxy-

genase 2 (COX-2) expression together with the reduced inflam-

mation followed by but not related to the hypolipidemic effects.

In our experiments, aspirin showed only an insignificant

effect on LDL oxidizability. This was opposite to the findings

by Yada et al,48 who reported that patients with acute myocar-

dial infarction (AMI), taking aspirin for 4 weeks experienced

significant decrease in oxidized LDL. Aspirin was found to

decrease the progression of atherosclerosis by protecting LDL

from oxidative modification49 and also improves endothelial

dysfunction in atherosclerotic vessels.50

However, aspirin administration has demonstrated signifi-

cant improvement for all blood-clotting parameters. It normal-

ized platelet aggregation and PT and decreased plasma

fibrinogen levels significantly. Aspirin action is supposed to

involve acetylation of platelet COX and subsequent inhibition

of the synthesis of prostaglandins and thromboxane A2.51

Aspirin promotes inhibition of platelet aggregation by neutro-

phils, an effect that appears to be mediated by a nitric oxide

(NO/cyclic guanosine monophosphate [cGMP])-dependent

process.52 It was also found that aspirin acetylates fibrinogen

and enhances fibrinolysis.53 Aspirin affects clotting factors

including thrombin, fibrinogen, factor XIII, and tissue plasmi-

nogen activator, thereby potentially having a direct effect on

clot formation and lysis.54 Furthermore, it has been suggested

that aspirin modifies thrombin generation by acetylating

macromolecules of platelet membrane and/or prothrombin.55

In a clinical study by Jacek et al,56 75 mg daily aspirin had

no effect on decreasing PT in patients with plasma cholesterol

levels >5.2 mmol/L (200 mg/dL). This indicates that individu-

als with hypercholesterolemia might receive less benefit than

others from preventive aspirin treatment.

Histopathological examination of the aspirin group showed

ischemic myocardial bundles with the appearance of severe

congestion in the blood vessels. However, a significant

decrease in aorta intimal thickness was observed when com-

pared with the untreated MI group. Guo et al57 found that

aspirin treatment of cholesterol-fed rabbits resulted in a signif-

icant decrease in maximum plaque thickness, the degree of

artery stenosis and the proportion of the intimal circumference

occupied by artheroma. Furthermore, a study found a 20%reduction in ischemic heart disease events in men at increased

risk after taking low-dose aspirin.58

Protective Effect of L-Arginine

L-arginine currently receives increased attention in the thera-

peutic field. Numerous studies have shown a beneficial role

of L-arginine administration when taken in amounts greater

than those taken in diet. In the current study, administration

of L-arginine resulted in insignificant changes in hypercholes-

terolemia, similar to aspirin. It was also reported that L-arginine

Figure 3. Effect of cholesterol-enriched diet either alone (untreatedMI group) or in combination with aspirin or L-arginine on plasma levelsof platelet aggregation (%) (A), plasma levels of fibrinogen (mg/dL) (B),and prothrombin time (seconds) (C). Data are expressed as mean +SEM. Number of animals were 8, 16, 12, and 12, respectively, for nor-mal, untreated MI, aspirin, and L-arginine groups. a indicates significantdifference from normal at P < .05; b, significant difference fromuntreated MI group at P < .05; c, significant difference from aspiringroup at P < .05; MI, myocardial ischemia; SEM, standard error ofmean.

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supplementation in a model of hypercholesterolemia had no

effect on serum lipids and lipoproteins.59,60 Nevertheless,

L-arginine administration was associated with a remarkable

reduction in aortic intimal lesions. Similar results were previ-

ously demonstrated in another study from our laboratory.34

In this study, L-arginine showed remarkable positive results

with regard to the prevention of MI. Both serum cTnI and

CK-MB were significantly lower in the L-arginine group than

in the untreated MI group, both returning to levels of the nor-

mal group. This is in agreement with a study by Carrier

et al,61 where serum cardiac troponin T level was reduced in

patients receiving L-arginine–enriched blood cardiologic

solution, suggesting an overall improvement in myocardial

protection. Furthermore, Kumar et al62 also found a significant

decrease in serum CK level in L-arginine-treated rabbits

compared to hypercholesterolemic untreated animals, both

subjected to experimental MI.

The antioxidant property of L-arginine is evident in this

study from the remarkable reduction of LDL oxidizability as

compared to all other groups in the study. This antioxidant

property of L-arginine was previously described in a number

of reports.7,63 In these studies, L-arginine was found to elicit

anti-inflammatory effects by scavenging ROS and to prevent

the oxidation of LDL cholesterol, thereby retarding the pro-

gression of atherosclerosis. Reduced superoxide production

by endothelial cells has been shown to be specific for L-argi-

nine but not for D-arginine,64 suggesting that it may be related

to NO synthase activity.

One of the most prominent effects of L-arginine in this study

was its effect on platelet aggregation. L-arginine reduced plate-

let aggregation dramatically to a value even below that of the

normal group. In comparison to aspirin, the reducing effect

of L-arginine was more pronounced, demonstrating a platelet

aggregation of 64% of that observed for aspirin. This result is

in accordance with many investigations, which show that

chronic dietary supplementation with L-arginine decreased pla-

telet aggregation in hypercholesterolemic rabbits,33,34 and in

individuals with hypercholesterolemia.65 In addition, L-argi-

nine was previously shown to inhibit platelet aggregation in

vitro, indicating that its effects are exerted directly on luminal

platelets and not on the platelets attached to the vascular

endothelium.66 The antiplatelet activity of L-arginine may be

secondary to the increase in NO production.

L-arginine also stimulates fibrinogenolysis by inhibiting the

formation of thromboxane B2 and the platelet�fibrin complex

while enhancing plasmin generation and fibrin degradation.67

Indeed, Cylwik et al68 showed that fibrinogen concentration

decreased after both acute and long-term administration of

L-arginine. Both results support our observations. L-arginine

also produced a significant increase in PT as compared to

untreated animals; however, aspirin was more effective in

increasing PT than L-arginine.

Superior to aspirin treatment, histopathological examination

of the L-arginine group showed no abnormal histopathological

findings in the heart. Oral administration of L-arginine to

animals69 and humans70 has been demonstrated to slow the

progression of atherosclerosis and its component processes.

The initial stages of atherosclerosis and plaque formation are

both inhibited by treatment with L-arginine.71

In conclusion, the salient findings of this study can be

summarized in the following points:

(1) L-arginine exerts protective effects against myocardial

ischemic injury through different mechanisms. Evidence

includes reduction in cardiac, coronary, and aortic

histopathological changes, prevention of LDL oxidation,

reduction in myocardial injury, and improvement in

blood-clotting parameters.

Figure 4. Effect of cholesterol-enriched diet either alone (untreatedMI group) or in combination with aspirin or L-arginine on LDL oxidiz-ability (nmole MDA/mL). Data are expressed as mean + SEM. Num-ber of animals were 8, 16, 12, and 12, respectively, for normal,untreated MI, aspirin, and L-arginine groups. a indicates significant dif-ference from normal at P < .05; b, significant difference from untreatedMI group at P < .05; c, significant difference from aspirin group at P <.05; MI, myocardial ischemia; SEM, standard error of mean; LDL,low-density lipoprotein; MDA, malodialdehyde.

Figure 5. Effect of cholesterol-enriched diet either alone (untreatedMI group) or in combination with aspirin or L-arginine on aortic intimalthickness (mm). Data are expressed as mean + SEM. Number of ani-mals were 8, 16, 12, and 12, respectively, for normal, untreated MI,aspirin, and L-arginine groups. a indicates significant difference fromnormal at P < .05; b, significant difference from untreated MI groupat P < .05; C, significant difference from aspirin group at P < .05; MI,myocardial ischemia; SEM, standard error of mean.

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Figure 6. A photomicrograph of a section in heart of a rabbit in the normal, untreated MI, aspirin and L-arginine group. Hx and E,�64. Hx and Eindicates hematoxylin and eosin.

Figure 7. A photomicrograph of a section in coronary blood vessel of a rabbit in the normal, untreated MI, aspirin, and L-arginine group. Hx andE, �40. Hx and E indicates hematoxylin and eosin.

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(2) L-arginine and aspirin have no effect on blood cholesterol.

L-arginine demonstrated better effect in the reduction

of LDL oxidizability, platelet aggregation, and aortic

intimal thickness. Prolongation of PT is the only para-

meter in this study where aspirin was found to be

superior to L-arginine.

(3) Given that L-arginine is a nutritional supplement with a

relatively high safety margin and no tolerance on long-

term use, we recommend considering L-arginine supple-

mentation as a potentially novel nutritional strategy for

preventing and modulating CVD, especially in cases of

aspirin resistance or hypersensitivity.

Declaration of Conflicting Interests

The authors declared no conflicts of interest with respect to the author-

ship and/or publication of this article.

Financial Disclosure/Funding

The authors received no financial support for the research and/or

authorship of this article.

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Figure 8. A photomicrograph of a section in ascending aorta of normal, untreated MI, aspirin, and L-arginine-treated rabbit. Hx and E,�400. Hxand E indicates hematoxylin and eosin.

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