Platelets, February 2009; 20(1): 16–22

ORIGINAL ARTICLE

The effects of pitavastatin, eicosapentaenoic acid and combined therapyon platelet-derived microparticles and adiponectin in hyperlipidemic,diabetic patients

SHOSAKU NOMURA1, NORIHITO INAMI2, AKIRA SHOUZU3, SEITAROU OMOTO2,

YUTAKA KIMURA2, NOBUYUKI TAKAHASHI2, ATSUSHI TANAKA4,

FUMIAKI URASE5, YASUHIRO MAEDA5, HAJIME OHTANI2, & TOSHIJI IWASAKA2

1Division of Hematology, Kishiwada City Hospital, Kishiwada, Japan, 2Second Department of Internal Medicine, Kansai

Medical University, Moriguchi, Japan, 3Department of Internal Medicine, Saiseikai Izuo Hospital, Osaka, Japan,4Department of Cardiology, Wakayama Medical University, Wakayama, Japan, and 5Department of Hematology,

Kinki University, Sayama, Japan

(Received 26 April 2008; accepted 14 August 2008)

AbstractPlatelet-derived microparticles (PDMP) play an important role in the pathogenesis of diabetic vasculopathy, and statins oreicosapentaenoic acid (EPA) have been shown to have a beneficial effect on atherosclerosis in hyperlipidemic patients.However, the influence of EPA and statins on PDMP and adiponectin in atherosclerosis is poorly understood.We investigated the effect of pitavastatin and EPA on circulating levels of PDMP and adiponectin in hyperlipidemicpatients with type II diabetes. A total of 191 hyperlipidemic patients with type II diabetes were divided into three groups:group A received pitavastatin 2 mg once daily (n¼ 64), group B received EPA 1800 mg daily (n¼ 55) and group C receivedboth drugs (n¼ 72). PDMP and adiponectin were measured by ELISA at baseline and after 3 and 6 months of drugtreatment. Thirty normolipidemic patients were recruited as healthy controls. PDMP levels prior to treatment inhyperlipidemic patients with diabetes were higher than levels in healthy controls (10.4� 1.9 vs. 3.1� 0.4 U/ml, p50.0001),and adiponectin levels were lower than controls (3.20� 0.49 vs. 5.98� 0.42 mg/ml, p50.0001). PDMP decreasedsignificantly in group B (before vs. 6M, 10.6� 2.0 vs. 8.0� 1.7 U/ml, p50.01), but not in group A (before vs.6M, 9.4� 1.9 vs. 9.6� 1.7 U/ml, not significant). In contrast, group A exhibited a significant increase in adiponectinlevels after treatment (before vs. 6M, 3.29� 0.51 vs. 4.16� 0.60 mg/ml, p50.001). Furthermore, group C exhibitedsignificant improvement in both PDMP and adiponectin levels after treatment (PDMP, before vs. 6M, 11.2� 2.0 vs.4.5� 2.7 U/ml, p50.001; adiponectin, before vs. 6M, 3.24� 0.41 vs. 4.02� 0.70 mg/ml, p50.001). Reductions of PDMPin combined therapy were significantly greater than those observed with EPA alone ( p50.05 by ANOVA). In addition,soluble CD40 ligand exhibited almost the same change as PDMP in all therapy groups. These results suggest thatpitavastatin possesses an adiponectin-dependent antiatherosclerotic effect, and this drug is able to enhance the anti-plateleteffect of EPA. The combination therapy of pitavastatin and EPA may be beneficial for the prevention of vascularcomplication in hyperlipidemic patients with type II diabetes.

Keywords: pitavastatin, eicosapentaenoic acid, combined therapy, platelet-derived microparticles, adiponectin, type II

diabetes

Introduction

Diabetes mellitus and hyperlipidemia have

been clearly identified as risk factors for progression

of atherosclerosis and cardiovascular disease

[1]. Diabetic patients also show hypercoagulability

and platelet hyperaggregability [2, 3], with

increased levels of platelet activation-markers [4].

Platelet-derived microparticles (PDMPs) play a role

in the normal haemostatic responses to vascular

injury because they demonstrate prothrombinase

activity [5, 6]. PDMPs are also released from

platelets after physical stimulation under various

conditions [6, 7], but only a few studies on the

potential role of PDMPs in diabetic complications

have been published [6–11].

Correspondence: Shosaku Nomura, MD, Division of Hematology, Kishiwada City Hospital, 1001 Gakuhara-cho, Kishiwada, Osaka 596-8501, Japan.

E-mail: shosaku-n@mbp.ocn.ne.jp

ISSN 0953–7104 print/ISSN 1369–1635 online � 2009 Informa Healthcare USA, Inc.

DOI: 10.1080/09537100802409921

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Plasma adiponectin concentrations are decreased

in obese individuals [12, 13] with type II diabetes

[14] and are closely related to whole-body insulin

sensitivity [15]. This protein is abundant in the

circulation [13] and suppresses the attachment of

monocytes to endothelial cells [16]. Adiponectin also

stimulates nitric oxide production in vascular

endothelial cells, which ameliorates endothelial

function [17, 18]. These observations suggest anti-

atherogenic properties for adiponectin, and that

hypoadiponectinemia may be associated with

a higher incidence of vascular disease in diabetic

patients.

Lipid lowering therapy with 3-hydroxy-3-methyl-

glutaryl-coenzyme A (HMG-CoA) reductase inhibi-

tors (statins) involves pleiotropic effects of statins.

Although the distinct mechanisms for these effects

of statins, which is responsible for the prevention of

atherosclerosis as well as their lipid lowering effects.

Pitavastatin is a HMG-CoA reductase inhibitor that

significantly reduces serum total cholesterol (TC)

and low-density lipoprotein cholesterol (LDL-C),

and triglycerides (TG) with modest elevation of high-

density lipoprotein cholesterol (HDL-C) [19, 20],

and has various pleiotropic effects [21–25]. Although

statins reduce the risk of cardiovascular events in

type II diabetic patients [26, 27], it is very important

for prevention of such events to continue with

a comprehensive program of risk modification, such

as lipid-lowering therapy, anti-thrombotic treatment

and improved lifestyle [28]. Therefore, combination

therapy, which targets lipid parameters associated

with cardiovascular risk, is thought to be required for

patients with type II diabetes mellitus. In the present

study, we selected eicosapentaenoic acid (EPA) as

the preference for combination therapy with pitavas-

tatin, since EPA has antithrombogenic and anti-

atherosclerotic properties [29]. Therefore, the main

purpose of this study was to compare the effects of

pitavastatin, EPA and combined therapy in patients

with diabetes and hyperlipidemia on PDMP and

adiponectin.

Methods

Patients

The study group included 30 normolipidemic con-

trols and 191 patients with diabetes and hyperlipi-

demia (Table I). Controls were recruited from

hospital staff and other sources. Between April

2005 and May 2007, patients were selected from

among patients admitted to our hospital for the

treatment of hyperlipidemia and diabetes mellitus.

The study protocol was approved by the Institutional

Review Board (IRB) of our institution and written

informed consent was obtained from each patient

prior to the start of the trial. Entry criteria required

the study participants could not have a history within

the 3 months prior to enrollment of inflammatory,

coronary artery, or cerebrovascular disease or have

had a clinically detectable renal (serum creatinine

2.0 mg/dl), hepatic (elevated serum transaminase),

infectious (fever or elevated white blood cells) or

malignant disease (on the basis of ultrasound or

computed tomography examination). Other anti-

lipidemic agents were prohibited, since these drugs

might have influenced data interpretation. These

medications were stopped at least 2 weeks prior to

initiation of pitavastatin or EPA therapy. There were

26 patients using aspirin and 13 patients using

a Ticlopidine due to a history of old cerebral

infarction or angina pectoris. There were 69 patients

using an angiotensin II receptor blocker (ARB) and

53 patients using a Ca-antagonists for hypertension

(Table I). Of 191 patients, 65 were treated with

sulfonylureas, 71 with �glucosidase inhibitors and

54 with insulin therapy. The dose of previous drugs

such as aspirin, ARB, and anti-diabetic agents

were not adjusted during the present study.

Hyperlipidemia was defined according to

Guidelines for Diagnosis and Treatment of

Hyperlipidemias in Adults published by the Japan

Atherosclerosis Society [30]. Type II diabetes was

defined according to the criteria of the American

Diabetes Association [31]. Table I shows the clinical

characteristics of the hyperlipidemic patients and the

control subjects.

Table I. Baseline characteristics of the study population.

Healthy controls Patients p-Value

Variables 30 191

Gender (male/female) 17/13 101/90

Age, y 56� 3 65� 3 50.01

BMI (kg/m2) 25.4� 2.2 27.3� 3.9 N.S.

TC (mg/dL) 196� 34 243� 15 50.001

TG (mg/dL) 138� 21 242� 46 50.0001

HDL-C (mg/dL) 53� 11 46�12 50.05

LDL-C (mg/dL) 115� 22 152� 33 50.001

HbA1c (%) 4.9� 0.5 7.0�1.1 50.001

PDMP (U/ml) 3.1� 0.4 10.4� 1.9 50.0001

Adiponectin (mg/ml) 5.98� 0.42 3.20�0.49 50.0001

Risk factors, n (%)

Current smoking 4 (13.3) 21 (11.0)

Complications, n (%)

Angina pectoris 1 (3.3) 8 (4.2)

Heart failure 0 (0) 8 (4.2)

Cerebral infarction 1 (3.3) 11 (5.7)

Hypertension 4 (13.3) 84 (44.0)

Medications, n (%)

Aspirin 1 (3.3) 26 (13.6)

Ticlopidine 0 (0) 13 (6.8)

ARBs 3 (10.0) 69 (36.1)

Ca-antagonists 1 (3.3) 53 (27.7)

Data are shown as mean�SD. N.S.: not significant; BMI: bodymass index; TC: total cholesterol; LDL-C: low-density lipoproteincholesterol; PDMP: platelet-derived microparticle.

Effects of pitavastatin/EPA on PDMP 17

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Study design

Pitavastatin 2 mg once daily, EPA 1800 mg, or

pitavastatin 2 mg and EPA 1800 mg was adminis-

tered daily during 6 months. The patients for

treatments were randomly selected. There were no

other changes in any patient’s pharmacologic regi-

men during treatment. Clinical and biochemical data

obtained before and after pitavastatin and EPA

administration were compared.

Measurements of platelet-derived microparticles

An enzyme-linked immunosorbent assay (ELISA) kit

for platelet-derived microparticles (PDMP) [32–34]

was obtained from JIMRO Co., Ltd (Tokyo, Japan).

In brief, blood samples were collected from periph-

eral veins using vacutainers containing EDTA-ACD

(NIPRO Co. Ltd, Japan) with 21-gauge needle to

minimize platelet activation. The samples were

gently mixed by turning the tubes up-side down

once or twice and stored at room temperature for

2–3 h, then centrifuged at 8000g for 5 min at room

temperature. Immediately after centrifugation, we

collected 200 ml of the upper-layer supernatant from

2 ml samples to avoid the contamination of the

platelets and stored the samples at �40�C until

analysis. The ELISA results obtained under our

conditions were reproducible. The PDMP values

were measured twice and the mean values were

recorded. This kit used two monoclonal antibodies

against glycoprotein CD42b and CD42a (glycopro-

tein Ib and IX). One (1) U/ml of PDMP is defined

as 24 000 platelets/ml of solubilized platelets in this

ELISA.

Measurements of adiponectin, soluble CD40 ligand

and soluble E-selectin

Blood samples from patients and controls were

collected into tubes with sodium citrate or tubes

without anticoagulant. Blood samples were allowed

to clot at room temperature for a minimum of 1 h.

Serum or citrated plasma was isolated by centrifuga-

tion for 20 min at 1000g (4�C) and stored at �30�C

until analysis. Adiponectin ELISA kit acquired from

Otsuka Pharmaceuticals Co. Ltd (Tokyo, Japan).

Soluble CD40 ligand (sCD40L) was measured

with an ELISA kit from Chemikon International

Inc. (Temecula, CA, USA), and sE-selectin was

measured with a monoclonal antibody-based ELISA

kit from BioSource International Inc. (Camarillo,

CA, USA). The recombinant products and

standard solutions provided with the commercial

kits were used as positive controls in each assay. All

kits were used according to the manufacturer’s

instructions.

Statistics

Data are expressed as the mean�SD and were

analysed by two-factor ANOVA for repeated mea-

sures as appropriate. Between-group comparisons

were made with the Bonferroni test and within-group

differences were determined with the Student’s t-test

for paired values with a level of significance being

p50.05. Correlations between adiponectin and other

parameters (BMI, TC, TG, HDL-C, LDL-C,

HbA1c, PDMP) were analysed first with a simple

logistic regression analysis (Pearson’s coefficient of

correlation), and then by multivariate analysis using

a stepwise method.

Results

When baseline values before each treated period

were compared among the three treatment arms, no

significant differences were noted in any of the

parameters measured. Pitavastatin alone or com-

bined therapy significantly reduced TC and LDL-C

after 6 months administrations compared with base-

line. These reductions were significantly greater than

that observed with EPA alone ( p50.01 by ANOVA).

EPA alone or combined therapy significantly lowered

TG levels compared with those observed with

pitavastatin alone ( p50.05 by ANOVA).

Pitavastatin alone or combined therapy significantly

elevated HDL-C after 6 months administration

compared with baseline, but not EPA alone. On

the other hand, there were no significant differences

in HbA1c between pitavastatin monotherapy and

combined therapy (Table II).

Pitavastatin therapy exhibited no remarkable

change of PDMP after 3 and 6 months administra-

tion compared with baseline (Figure 1).

Combination therapy and EPA monotherapy sig-

nificantly decreased the plasma PDMP levels relative

to baseline (Figure 1, EPA: before vs. 3M vs.

6M, 10.6� 2.0 vs. 9.0� 1.7 vs. 8.0� 1.7 U/ml,

3M & 6M; p50.01, Combined: before vs. 3M vs.

6M, 11.2� 2.9 vs. 7.0� 2.9 vs. 4.5� 2.7 U/ml, 3M;

p50.01, 6M; p50.001). These reductions were

significantly greater than those observed with pita-

vastatin alone (EPA: p50.05 by ANOVA,

Combined: p50.001 by ANOVA, Figure 1).

In addition, reductions in combined therapy were

significantly greater than those observed with EPA

alone ( p50.05 by ANOVA, Figure 1).

Pitavastatin alone and combined therapy exhibited

a significant increase in adiponectin levels after 3 and

6 months administration compared with baseline

(Figure 2, pitavastatin: before vs. 3M vs. 6M,

3.29� 0.51 vs. 3.79� 0.51 vs. 4.16� 0.60 mg/ml,

3M; p50.01, 6M; p50.001; Combined: before vs.

3M vs. 6M, 3.24� 0.41 vs. 3.67� 0.65 vs.

4.02� 0.70 mg/ml, 3M; p50.05, 6M; p50.01). On

the other hand, EPA monotherapy exhibited an

18 S. Nomura et al.

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elevation in adiponectin levels after only 6 months

administration compared with baseline (Figure 2,

EPA: before vs. 3M vs. 6M, 3.03� 0.57 vs.

3.09� 0.70 vs. 3.30� 0.68 mg/ml, 6M; p50.05).

These increases were significantly greater than

those observed with EPA alone (pitavastatin:

p50.05 by ANOVA, Combined: p50.01 by

ANOVA, Figure 2). However, there were no

significant differences observed between pitavastatin

alone and combined therapy (Figure 2).

Table III shows the correlation of adiponectin with

other parameters. Since the distribution of adipo-

nectin was skewed, logarithmically transformed

values were used for statistical analysis.

Adiponectin levels showed a significant correlation

with PDMP in combined therapy. In order to resolve

the significance of adiponectin and PDMP in

combined therapy, we investigated the changes of

sCD40L and sE-selectin (Table IV). Pitavastatin

exhibited a significant decrease of sE-selectin, not

but sCD40L. In contrast, EPA exhibited a significant

decrease in sCD40L, but not in sE-selectin. On the

other hand, combined therapy exhibited significant

decrease of both sCD40L and sE-selectin.

In particular, the decrease of sCD40L is more

significant than that of EPA.

Table II. Effects of pitavastatin, combined therapy and EPA on lipids and HbA1c in hyperlipidemic, diabetic patients.

Pitavastatin (P) (n¼ 64) PitavastatinþEPA (C) (n¼ 72)

Variables Baseline (0) Treatment (6M) Baseline (0) Treatment (6M)

Lipids (mg/dl)

TC 254� 24 193�29*** 251�45 183� 39***

TG 198� 57 171� 38* 248�61 177� 42***

HDL-C 48� 14 54�12** 46�17 53� 15***

LDL-C 169� 21 108�23*** 156�31 95� 22***

HbA1c (%) 7.5� 1.2 7.5� 1.0NS 6.5�1.1 6.3�0.9NS

EPA (E) (n¼ 55) ANOVA

Baseline (0) Treatment (6M) P/C P/E C/E

Lipids (mg/dl)

TC 229� 37 202� 35** NS 50.01 50.01

TG 258� 72 209�61*** 50.05 5 0.05 NS

HDL-C 43� 15 45� 10NS NS NS NS

LDL-C 128� 32 114� 33** NS 50.05 50.05

HbA1c (%) 7.0� 1.3 7.0� 0.9NS NS NS NS

Data are expressed as means�SD. There were no significant differences among each baseline values. *p50.05, ** p50.01, *** p50.001for comparison with each base line value. P/C¼pitavastatin vs. combined therapy; P/E¼pitavastatin vs. EPA; C/E¼ combined therapy vs.EPA. NS: not significant.

Figure 1. Changes in PDMP levels before and after administra-

tion of pitavastatin, EPA and combined therapy to hyperlipidemic

patients with type II diabetes. PDMP: platelet-derived

microparticle; Bars are shown as mean�SD. 0: before; M:

month (after); p-Values for comparison with each baseline value

(before vs. after). ANOVA: analysis of variance (pitavastatin or

EPA vs. EPA or combined therapy).

Figure 2. Changes in adiponectin levels before and after admin-

istration of pitavastatin, EPA and combined therapy to hyperlipi-

demic patients with type II diabetes. Bars are shown as

mean�SD. 0: before; M: month (after); p-Values for comparison

with each baseline value (before vs. after). ANOVA: analysis of

variance (pitavastatin or EPA vs. EPA or combined therapy).

Effects of pitavastatin/EPA on PDMP 19

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Discussion

Plasma adiponectin concentration is decreased in

obese individuals [12] and is closely related to whole-

body insulin sensitivity [14]. A significant decrease in

plasma adiponectin concentrations is also found in

patients with type II diabetes [14]. Adiponectin has

been noted to suppress the attachment of monocytes

to endothelial cells [16] and plays a role in protection

against vascular damage. We recently reported that

the administration of pitavastatin significantly

increased concentrations of adiponectin in hyperlipi-

demic patients [25, 35]. Although pitavastatin did

not appear to change of PDMP and sP-selectin levels

in these patients, this drug significantly improved

sE-selectin and sL-selectin in hyperlipidemic patients

with type II diabetes [25]. These results suggest the

possibility of pitavastatin possessing an anti-athero-

sclerotic effect via modification of adiponectin levels.

In the previous reports [25, 35], we speculated that

the mechanism by which pitavastatin treatment leads

to an increase in circulating adiponectin levels was

the improvement of reactive oxygen species (ROS),

or the activation for sterol regulatory element binding

protein (SREBP)-1c by pitavastatin. However, the

third possible mechanism has appeared. That is the

participation of the peroxisome proliferator-activated

receptor gamma (PPARg). PPARg, a member of the

nuclear hormone receptor family of ligand-depen-

dent transcription factors, has been well character-

ized as a regulator of adipogenesis and is abundant

in fat cells [36]. In particular, synthetic ligands of

PPARg have been shown to induce expression of the

adiponectin gene and increase adiponectin levels

both in vivo and in vitro [37–39]. Furthermore, it has

recently been reported that statins including pitavas-

tatin can activate PPARg via extracellular signal-

regulated kinase 1/2 and p38 mitogen-activated

protein kinase activation [40].

Activated platelets may cause capillary microem-

bolization secondary to the formation of microag-

gregates [41]. PDMP, which are derived from

activated platelets, also play an important role in

the process of coagulation. Therefore, an increase of

PDMP may causes hypercoagulability [42]. We

previously reported that PDMP were significantly

increased in diabetic patients with high LDL levels

compared with similar patients who had low LDL

levels [8]. Because PDMP enhanced the expression

of adhesion molecules on monocytes and endothelial

cells [43], it seems possible that PDMP may

participate in the development or progression of

atherosclerosis in diabetics. Intensive anti-platelet

drugs such as cilostazol or ticlopidine can inhibit the

elevation of PDMP [9–11]. However, the use of

these drugs for primary prevention of atherothrom-

bosis is problematic. For this reason, we turned our

attentions to EPA. EPA is a polyunsaturated fatty

acid found at high levels in fish oil. Its antithrombo-

genic and antiatherosclerotic actions were suggested

by an epidemiological study of Greenland Eskimos

[44]. The mechanisms of these actions have been

postulated to include the inhibition of platelet

aggregation and the improvement of blood rheologic

properties [45]. In the present study, combined

therapy with pitavastatin and EPA significantly

decreased the plasma PDMP levels after treatment

compared to monotherapy with EPA. Although we

Table III. Correlation of adiponectin with age, gender, BMI, TC,

TG, HDL-C, CRTN, HbA1c and PDMP after 3 and 6 months

of combined therapy.

Combined therapy

Regression coefficient p-Value

After 3 months

Age �0.02 0.8027 (NS)

Gender 0.07 0.0479 (5 0.05)

BMI 0.14 0.5377 (NS)

TC 0.21 0.2258 (NS)

TG 0.09 0.5009 (NS)

HDL-C 0.11 0.4263 (NS)

LDL-C 0.13 0.3988 (NS)

HbA1c 0.07 0.8269 (NS)

PDMP 0.33 0.0259 (50.05)

After 6 months

Age �0.05 0.8197 (NS)

Gender 0.16 0.6109 (NS)

BMI 0.19 0.6547 (NS)

TC 0.13 0.6704 (NS)

TG 0.22 0.2628 (NS)

HDL-C 0.18 0.1195 (NS)

LDL-C �0.14 0.2645 (NS)

HbA1c 0.11 0.6396 (NS)

PDMP 0.45 0.0001 (50.001)

These were analysed by the stepwise method. Correlationcoefficients were derived a simple logistic regression analysis(Pearson’s coefficient of correlation). BMI¼ body mass index;TC¼ total cholesterol; TG¼ triglycerides; HDL-C¼ high densitylipoprotein-cholesterol; HbA1c¼ hemoglobin A1c; PDMP¼ pla-telet-derived microparticle; NS: not significant.

Table IV. Changes of soluble factors before and after treatments

in hyperlipidemic patients with diabetes.

Treatment

Baseline (0) 3M 6M

Pitavastatin

sCD40L (ng/ml) 16.5� 4.2 16.1� 4.1 15.8� 3.9

sE-selectin (ng/ml) 73� 22 72� 19 48� 12**

EPA

sCD40L (ng/ml) 16.2� 4.6 14.1� 4.8 12.2� 4.5*

sE-selectin (ng/ml) 70� 25 66� 23 62� 24

Pitavastatin þ EPA

sCD40L (ng/ml) 17.4� 5.1 13.5� 4.4 10.2� 3.9***

sE-selectin (ng/ml) 75� 21 62� 23 44� 17**

Data are expressed as means�SD. *p50.05, **p50.01,***p50.001 for comparison with each baseline valuesCD40L¼ soluble CD40 kigand; sE-selectin¼ soluble E-selectin.

20 S. Nomura et al.

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could not show any direct hallmarks of platelet

function, combined therapy improved another plate-

let activation marker, sCD40L, in hyperlipidemic

patients with diabetes. EPA has reported effects on

coronary events in hyperlipidemic patients [46] and

biological functions of this drug include reduction of

platelet activation [29, 47]. Our results suggest that

pitavastatin could enhance the effect of EPA and this

action is dependent on adiponectin.

In conclusion, pitavastatin, EPA or combined

therapy directly or indirectly increased circulating

adiponectin in hyperlipidemic patients with type II

diabetes. In addition, pitavastatin could enhance the

antiplatelet effect of EPA. Hyperlipidemic patients

with type II diabetes possess the risk of atherothrom-

bosis in which platelets participate. Combined

therapy with pitavastatin and EPA may be beneficial

as a primary prevention therapy for atherothrombosis

in hyperlipidemic patients with type II diabetes.

Acknowledgements

This study was partly supported by a grant from

the Japan Foundation of Neuropsychiatry and

Hematology Research, a Research Grant for

Advanced Medical Care from the Ministry of

Health and Welfare of Japan, and a Grant

(13670760 to S.N.) from the Ministry of

Education, Science and Culture of Japan.

Conflict of interest: There is no conflict of interest

in this study.

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