ORIGINAL CONTRIBUTION

Mitogen-activated protein kinases activation in T lymphocytesof patients with acute coronary syndromes

Ciro Indolfi • Cosimo Gasparri • Carla Vicinanza • Daniela De Serio •

Duino Boncompagni • Annalisa Mongiardo • Carmen Spaccarotella •

Valter Agosti • Daniele Torella • Antonio Curcio

Received: 28 July 2010 / Revised: 17 February 2011 / Accepted: 9 March 2011 / Published online: 20 March 2011

� Springer-Verlag 2011

Abstract Current available biomarkers cannot identify

myocardial ischemia without necrosis. To overcome this

issue and to increase diagnostic power, we evaluated the

activation of the three MAPK pathways, ERK1/2, JNK and

p38, in T lymphocytes of patients with acute coronary

syndromes (ACS). We included sixty consecutive patients

affected by either unstable angina (UA, N = 22), Non- ST-

segment elevation MI (NSTEMI, N = 19) or ST-segment

elevation MI (STEMI, N = 19). Two separate groups of

patients were matched as controls: healthy subjects (CTRL,

N = 20) and patients with stable coronary artery disease

(CAD, N = 21). MAPK activation in T lymphocytes,

measured by phospho-ERK1/2, phospho-JNK and phos-

pho-p38 levels, was assessed by flow cytometry analysis

which revealed significantly increased phosphorylated

levels of ERK1/2 in patients with UA, compared to con-

trols. In UA patients no significant changes were detected

for phospho-JNK compared to both control groups.

NSTEMI and STEMI groups showed a statistically

significant increase in both phospho-ERK1/2 and phospho-

JNK, compared to control groups. All ACS groups

demonstrated significantly increased phosphorylation of

p38 compared to CTRL, but not CAD. ROC curves showed

that a cut-off value of 22.5 intensity of fluorescence for

phospho-ERK1/2 was able to significantly discriminate UA

patients from patients with stable angina with 78% sensi-

tivity and 90% specificity. Therefore, a differential MAPK

activation in T lymphocytes denotes patients with ACS.

Indeed, patients with unstable angina are identified with

high specificity by activated ERK1/2 and normal JNK

levels. These data could represent a valuable new molec-

ular signature to be used as specific biomarkers for the

diagnosis of unstable angina within ACS.

Keywords Acute coronary syndromes � Biomarkers �T lymphocytes � Mitogen-activated protein kinases

Introduction

Acute coronary syndromes (ACS) are one of the major

causes of morbidity and mortality in developed countries

[26]. Early recognition of myocardial ischemia and accu-

rate diagnosis of ACS represent critical steps for selecting

and evaluating the response to therapeutic interventions [1,

36]. A combination of accurate history taking, physical

examination, measurement of circulating levels of cardiac

troponins (cTn) and the MB isoform of creatine kinase

(CK), EKG and echocardiography helps physicians in

identifying patients with ACS presenting to the emergency

department [37, 39]. However, the current approach is still

lacking of a diagnostic test able to identify without delay

patients with less-typical symptoms but yet at significant

risk for complications with ACS.

Systemic inflammation can trigger a cascade that

amplifies immune cell infiltration, platelet activation and

adhesion, plaque rupture and intermittent arterial occlu-

sion, hence representing a preeminent factor to the

C. Indolfi (&) � C. Gasparri � C. Vicinanza � D. De Serio �D. Boncompagni � A. Mongiardo � C. Spaccarotella �D. Torella � A. Curcio

Division of Cardiology, Laboratory of Molecular and Cellular

Cardiology, University Magna Graecia, Viale Europa,

Campus ‘‘S. Venuta’’ di Germaneto, 88100 Catanzaro, Italy

e-mail: indolfi@unicz.it

V. Agosti

Department of Experimental and Clinical Medicine,

Laboratory of Molecular Oncology, University Magna Graecia,

Viale Europa, Campus ‘‘S. Venuta’’ di Germaneto,

88100 Catanzaro, Italy

123

Basic Res Cardiol (2011) 106:667–679

DOI 10.1007/s00395-011-0172-1

instability of atherosclerotic plaques in acute patients [30].

Indeed, the investigation of novel circulating serum and

plasma biomarkers in patients with ACS has recently

unmasked the relationship between the inflammatory bio-

marker C-reactive protein (CRP) and the outcome in acute

patients [38]. Activated T lymphocytes are frequently

increased in peripheral blood of subjects affected by car-

diovascular diseases [9]. These evidences suggest that the

inflammatory component might be systemically detectable

in patients with ACS [2, 3, 8, 27]. However, even though

biomarkers of inflammation may provide unique informa-

tions to the clinician apart from that provided by bio-

markers of myocyte necrosis, the prognostic significance of

these new tools is still debated with regard to their potential

to enhance the care of patients [4, 25, 29, 34, 35].

Inflammation and different forms of cellular stress are

capable to elicit biochemical responses [10, 46] and

subsequently trigger molecular cascades such as mitogen-

activated protein kinases (MAPK), which act by phos-

phorylating various intracellular substrates including

transcription factors, hence regulating signal transduction

and specific genetic responses to extracellular stimuli

[19–21]. The MAPK family includes three major sub-

groups, extracellular signal-regulated kinase-1 and -2

(ERK1/2) and two stress-activated protein kinase path-

ways, c-Jun NH2-terminal kinase (JNK) and p38 MAPK

[43]. Specifically, in cardiovascular system ERK1/2 has

been involved in several serine/threonine phosphorylation

cascades that control cellular proliferation, differentiation,

survival, and motility in response to diverse extracellular

stimuli including mitogens, growth factors, and cytokines

[13], whereas in the heart JNK activation has been dem-

onstrated by hyperosmotic shock, low concentrations of

protein synthesis inhibitors such as anisomycin, the car-

diotoxic agent daunomycin, hypoxia/reoxygenation and

reactive oxygen species but can also occur in response to

mechanical stretch or pacing [31].

Moreover, the activation of p38 has been demonstrated

in several pathological conditions affecting the heart such

as experimental ischemic preconditioning in rat [23], rabbit

[18] and pig [40, 41] models. More recently, inhibition of

p38 enhanced the number of circulating vasculogenic cells

and improved the functional capacity of different pro-

angiogenic cells, thus reducing atherosclerotic disease

progression [42].

To date, it is unknown whether MAPK proteins are

activated within circulating inflammatory cells in response

to acute myocardial ischemia in patients with acute coro-

nary syndromes. Furthermore, it has never been evaluated

whether a differential activation of one or more kinases

involved in this cascade could be elicited by the different

clinical subsets of ACS. If MAPK activation was propor-

tional to the extent of myocardial ischemia ranging from a

partial MAPK activation in unstable angina on one hand to

a complete MAPK activation in NSTEMI and STEMI

patients on the other, this differential molecular activation

could prove very useful as diagnostic testing to discern

between the different ACS conditions.

Therefore, the aim of this study was to assess MAPK

activity in T lymphocytes of patients with ACS in an

independent manner from antiplatelet, antithrombotic

and/or HMG-CoA reductase inhibitors treatment.

Methods

Patient population

Eighty-one consecutive patients who were admitted at the

Division of Cardiology of the University of Magna Graecia

were enrolled in the study. All patients gave written an

informed consent to the study that was carried out in

accordance with the principles of the Declaration of Hel-

sinki and approved by local ethical committee. All patients

were assigned to different groups depending on clinical,

laboratory and angiographic features.

Group I included 21 patients with stable angina pectoris

(coronary artery disease, CAD), clinical evidence of

Canadian Cardiovascular Society class II and III, and at

least 1 coronary artery stenosis detected at the angiographic

examination ([75% reduction of lumen diameter). Group

II included 22 patients with unstable angina (UA) admitted

to our Hospital. All patients with UA had experienced chest

pain at rest within the preceding 12 h without evidence of

myocardial infarction (MI) as shown by the absence of

elevation of CK-MB or cardiac troponin I (cTnI) blood

levels. In all UA patients, a culprit lesion was confirmed at

the coronary angiography. Group III included 19 patients

with Non ST-segment elevation MI (NSTEMI) who pre-

sented within 12 h of the onset of pain, elevated cTnI blood

levels and angiographic evidence of coronary lesions.

Finally, patients with ST-segment elevation MI (STEMI,

N = 19), documented with an increase of CKMB and cTnI,

admitted within 12 h from the onset of symptoms were

included in Group IV. All patients underwent coronary

angiography and coronary revascularization, if needed,

during hospitalization. Additional healthy volunteers

(CTRL, N = 20) with no clinical signs of CAD and

without coronary risk factors were recruited from the blood

donors bank.

Exclusion criteria were as follows: previous MI within

6 months, previous revascularization procedures, inflam-

matory conditions likely to be associated with an acute

phase response, autoimmune disease, neoplastic disease,

advanced liver disease, renal failure or severe heart failure

(NYHA class III-IV).

668 Basic Res Cardiol (2011) 106:667–679

123

In the groups I–IV, the blood samples for MAPK ana-

lysis were obtained immediately at the patients admission.

Cardiac TnI and CK measurements were performed

according to the current guidelines, as routinely performed

in our institution.

Isolation of peripheral blood mononuclear cells

and flow cytometry analysis

Peripheral blood samples were obtained from all patients

and collected in a Vacutest Kima tube with K3EDTA.

Isolation of mononuclear cells was performed by Ficoll

(biocoll separating solution; Biochrom AG) density gradi-

ent centrifugation. Mononuclear cells were resuspended in

cold incubation buffer (PBS 19, BSA 0.5%) (Becton–

Dickinson) and were incubated with 10 ll of CD3-PE or

CD19-PE antibody (markers of T and B lymphocytes,

respectively) at 4�C for 30 min in the dark. Then, after

antibody wash-out in the incubation buffer, cells were fixed

with paraformaldehyde 2% (Sigma-Aldrich) for 10 min at

37�C, and permeabilized with cold methanol 90% (Sigma-

Aldrich) for 30 min at 4�C. Hence, cells were resuspended

with incubation buffer and subsequently incubated with

phospho-ERK1/2, phospho-p38 and phospho-JNK anti-

bodies (Cell Signaling Technology) for 1 h at room tem-

perature followed by monoclonal antibodies anti-rabbit

FITC conjugated (Jackson ImmunoResearch Laboratories).

A total of 30,000 events in a specific gate were analyzed on

a FACS Calibur flow cytometer with CellQuest acquisition

analysis software (Becton–Dickinson).

Immunoblotting analysis

In order to confirm the flow cytometry analysis, immuno-

blotting analysis was carried out including additional 28

patients. Briefly, proteins were extracted from T lympho-

cytes of CTRL subjects (N = 5) and both chronic and

acute coronary disease patients (CAD, N = 5; UA, N = 6;

NSTEMI, N = 6; STEMI, N = 6) obtained as above

described. Forty micrograms of total proteins were sepa-

rated by SDS-PAGE, transferred onto a nitrocellulose filter

through semi-dry trans-blot system (Biorad) and hybridized

with the different primary antibodies for activated MAPK:

phospho-ERK1/2, phospho-JNK, phospho-p38, and nor-

malized by antibodies recognizing the total ERK1/2, JNK

and p38 (Cell Signaling). The nitrocellulose filters were

blocked with 5% bovine serum albumin and the primary

antibodies were used at the dilution of 1:1,000 according to

the manufacturer’s recommendations (Cell Signaling

Technology). Specific HRP-conjugated secondary anti-

bodies were used according to the manufacturer’s recom-

mendations (Santa Cruz Biotechnology). Specific protein

bands were detected by chemiluminescence using the

Chemidoc XRS system (BioRad). Western blots for the

expression of total ERK1/2, JNK and p38 (antibodies from

Cell Signaling Technology) were performed to normalize

the phosphorylated levels of each protein kinase.

Statistical analysis

Significance between all groups was determined in multiple

comparisons by the analysis of variance (ANOVA). Bon-

ferroni’s post hoc method was used to locate the differences.

Receiver-operating characteristic (ROC) analysis was

also performed on the levels of phospho-ERK1/2, phospho-

JNK and phospho-p38 activation for UA, NSTEMI and

STEMI ACS. This analysis plots the true-positive fraction

(sensitivity) against the false-positive fraction (1-specifi-

city) by changing the cut-off value for the test. Areas under

the ROC curves indicate the relative accuracy of diagnostic

tests. Values of p \ 0.05 were considered statistically

significant.

Results

The baseline characteristics of all enrolled patients were well

matched among groups and are summarized in Table 1.

MAPK are activated in T but not B lymphocytes

in ACS patients

T lymphocytes are abnormally activated during the acute

phases of coronary atherosclerotic disease and can be

considered among putative novel biomarkers for early

diagnosis [16]. Then, we first ascertained whether MAPK

activation was confined to T lymphocytes or involved also

B lymphocytes in patients with ACS (UA, n = 5; NSTE-

MI, n = 5; STEMI, n = 5). Mononuclear cells freshly

isolated from blood samples were stained with antibodies

specific for the phosphorylated forms of ERK1/2, JNK and

p38 and either with CD3 antibody (recognizing T lym-

phocytes) or CD19 antibody (recognizing B lymphocytes)

and analyzed by fluorescent activated cell sorting (FACS).

Importantly, in all ACS patients ERK1/2, JNK and p38

were phosphorylated only in T lymphocytes while they

were mostly un-phosphorylated in B lymphocytes (Fig. 1).

Thus, we constrained the analysis of MAPK activation

in T lymphocytes isolated through CD3 immunomagnetic

bead cell sorting from mononuclear cells.

ERK1/2 is activated in T lymphocytes in all the subsets

of ACS patients

We first assessed ERK1/2 activation by FACS analysis and

reported the level of ERK1/2 phosphorylation (phospho-

Basic Res Cardiol (2011) 106:667–679 669

123

ERK1/2) as intensity of fluorescence for the specific

antibody. Importantly, ERK1/2 was significantly phos-

phorylated in UA, NSTEMI and STEMI patients (phospho-

ERK1/2 in UA = 40 ± 18, in NSTEMI = 26 ± 15 and in

STEMI = 34 ± 23) when compared with control subjects

(phospho-ERK1/2 in CTRL = 6 ± 3; p \ 0.05 vs. UA,

NSTEMI and STEMI). Of note, ERK1/2 was not signifi-

cantly activated in patients with chronic coronary artery

disease (phospho- ERK1/2 in CAD = 16 ± 8; p = NS vs.

UA, NSTEMI, STEMI and CTRL, Figs. 2, 3). Thus, these

data indicate that MAPK ERK1/2 is generally activated in

T lymphocytes of patients with ACS in response to myo-

cardial ischemia regardless of the presence of myocyte

necrosis.

JNK is activated in T lymphocytes only in ACS patients

with myocardial necrosis

A statistically significant increase of phospho-JNK, com-

pared to patients with stable angina pectoris, was observed

only in ACS patients with myocardial infarction (phospho-

JNK = 36 ± 19 in NSTEMI patients and 40 ± 28 in

STEMI patients; p \ 0.05 vs. UA, CAD and CTRL),

whereas JNK was not significantly activated in UA

(phospho-JNK = 12 ± 7) compared to CAD (10 ± 5;

p = NS vs. UA), or control patients (CTRL: 4 ± 2,

p = NS vs. UA and CAD, Figs. 2, 3).

Therefore, in T lymphocytes from ACS patients, in

contrast to ERK1/2, JNK activation could distinguish

patients with myocardial infarction in whom JNK is sig-

nificantly phosphorylated from patients with unstable

angina in whom JNK is un-activated.

p38 is activated in T lymphocytes in both acute

and chronic myocardial ischemia

Finally, FACS analysis showed that p38 was significantly

phosphorylated in T lymphocytes isolated from patients

with both chronic and acute myocardial ischemia (Figs. 2,

3). Indeed, a statistically significant increase of the phos-

phorylated form of p38 was detected in patients with

ST-segment elevation myocardial infarction (34 ± 27),

non ST-segment elevation myocardial infarction

(21 ± 20), unstable angina (15 ± 13) and patients with

chronic coronary artery disease (16 ± 13) compared to

healthy control subjects (p \ 0.05, Figs. 2, 3). Importantly,

Table 1 Clinical characteristics and biological parameters of the study populations

CTRL CAD UA NSTEMI STEMI

Patients, n 20 21 22 19 19

Age (years) 59 ± 13 61 ± 7 61 ± 8 60 ± 13 60 ± 11

Male sex, n (%) 16 (80%) 16 (76%) 17 (77%) 15 (79%) 15 (79%)

Risk Factors, n (%)

Hypertension 2 (10%) 5 (25%)* 6 (26%)* 5 (26%)* 5 (26%)*

Diabetes mellitus 0 (0%) 1 (3%)* 1 (3%)* 1 (5%)* 1 (5%)*

Smoking 0 (0%) 5 (23%)* 6 (25%)* 4 (23%)* 5 (28%)*

Hypercholesterolemia 0 (0%) 4 (18%)* 4 (18%)* 4 (19%)* 4 (20%)*

Familial history of CAD 2 (10%) 6 (27%)* 7 (30%)* 5 (28%)* 6 (29%)*

Single-vessel disease 0 (0%) 10 (48%)* 11 (50%)* 10 (53%)* 9 (47%)*

Multi-vessels disease 0 (0%) 11 (52%)* 11 (50%)* 9 (47%)* 10 (53%)*

BMI 26.5 ± 4.2 26.7 ± 2.0 27.1 ± 5.0 26.7 ± 2.9 27.8 ± 3.5

EF (%) 55 ± 10 50 ± 12 52 ± 7 50 ± 7 42 ± 16*,#,?

hs CRP (mg/l) 0.9 ± 0.9 1.9 ± 0.6* 8.4 ± 3.4*,# 25.0 ± 8.3*,#,? 21.1 ± 8.6*,#,?

Lymphocyte count (9103/ll) 2.1 ± 0.9 2.1 ± 0.6 2.3 ± 0.6 2.0 ± 0.5 2.0 ± 0.5

cTnI (ng/ml) \0.01 ± 0 \0.01 ± 0 \0.01 ± 0 0.88 ± 0.87*,#,? 1.6 ± 4*,#,?

CK (U/l) 89.0 ± 57.0 99.0 ± 87.0 154.2 ± 26.4* 266.3 ± 23.8*,#,? 2085.0 ± 999.0*,#,?,§

Pro-BNP (pg/ml) 198.3 ± 13.6 213.7 ± 140.7 216.0 ± 120.0 1800.2 ± 230.0*,#,? 5650.0 ± 521.0*,#,?,§

All values are expressed as mean ± SD or percentage

Statistical comparisons were performed by applying ANOVA test. For hs CRP evaluation, a Student’s t test was applied with Bonferroni

correction

CTRL healthy volunteers from donors bank, CAD stable coronary artery disease, UA unstable angina, NSTEMI non-ST-elevation myocardial

infarction, STEMI ST-elevation myocardial infarction, EF ejection fraction, hs CRP high sensitivity C-reactive protein, cTnI cardiac isoform of

the I subunit of the troponin complex, CK creatine kinase, Pro-BNP precursor of the brain natriuretic peptide

* p \ 0.05 versus CTRL, #p \ 0.05 versus CAD, ?p \ 0.05 versus UA, §p \ 0.05 versus NSTEMI

670 Basic Res Cardiol (2011) 106:667–679

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p38 activation was higher in STEMI patients compared to

all the other groups.

Immunoblotting analysis confirms differential

activation of MAPK in ACS patients

We further validated the data by western blotting analysis

of activated MAPK in T lymphocytes protein extracts

obtained from additional patients in all the groups included

in the study. In agreement with the FACS data, western

blots showed that phospho-ERK1/2 was significantly

increased in all ACS patients when compared to CAD and

CTRL groups (Fig. 4). Furthermore, JNK was found to be

significantly phosphorylated in both ST- and non-ST-ele-

vation MI when compared to UA, CAD and CTRL patients

(Fig. 4). Finally, p38 was significantly phosphorylated in

all ischemic patients when compared to control subjects

and higher levels of phospho-p38 were detected in T

lymphocytes of STEMI patients.

These data strongly suggest that the acute and intense

myocardial stress produced by coronary occlusion is scouted

by T lymphocytes activating ERK1/2. The latter molecular

switch turns into the concomitant JNK activation when

ischemia time lasts enough to induce myocardial necrosis.

Moreover, according to above results, both acute and chronic

myocardial ischemia activates p38 in T lymphocytes

whereby the highest levels of phospho-p38 are associated

with more intense myocardial damage in STEMI patients.

Sensitivity and specificity

Given a cut-off value of 22.5 intensity of fluorescence for

phospho-ERK1/2 by FACS analysis in T lymphocytes, this

activated kinase could significantly discriminate UA patients

Fig. 1 Increased amounts of

ERK1/2-, JNK-, and

p38-activated forms in T but not

B lymphocytes during ACS.

The representative fluorescent

activated cell sorting (FACS)

analysis shows that ERK1/2,

JNK and p38 are specifically

activated (phosphorylated) in

CD3? T lymphocytes while

they are practically un-

phosphorylated in CD19? B

lymphocytes from patients with

acute coronary syndrome (in

particular, the data presented are

from a patient with NSTEMI)

Basic Res Cardiol (2011) 106:667–679 671

123

from patients with chronic stable coronary artery disease

(CAD) and showed 78% sensitivity and 90% specificity for

the diagnosis of unstable angina (Table 2). Moreover, con-

sidering a cut-off of 13.5 intensity of fluorescence for

phospho-ERK1/2, this protein could significantly discrimi-

nate NSTEMI patients from control subjects with 56% sen-

sitivity and 79% specificity, while setting a cut-off value of

12 intensity of fluorescence for phospho-ERK1/2, STEMI

patients could be easily discriminated from patients with

chronic stable coronary artery disease, with a 56% sensitivity

and 84% specificity (Fig. 5). Interestingly, a cut-off value of

18.5 and 21 intensity of fluorescence for phospho-JNK could

discriminate patients with NSTEMI and STEMI, respec-

tively, from patients with chronic stable coronary artery

disease (NSTEMI sensitivity 94% and 90% specificity,

STEMI sensitivity 95% and 100% specificity).

ERK1/2 activation in T lymphocytes is a transient event

in patients with unstable angina

Since ERK1/2 was found significantly activated in T

lymphocytes from unstable-angina patients in response to

acute myocardial ischemia, it was important to investigate

whether this molecular activation was related to the acute

phase of cell stress or it was related to the disease inde-

pendently from the time of assessment. Therefore, in

patients successfully treated with percutaneous revascu-

larization for unstable angina (N = 22), we evaluated

ERK1/2 activation in T lymphocytes at a follow-up of

180 days after the acute event. Importantly, ERK1/2 acti-

vation expressed by the levels of its phosphorylated form

was significantly reduced at 6-months follow-up when

compared with the acute event (from 40 ± 18 to 16 ± 8,

Fig. 2 Differential activation of

MAP Kinases in T lymphocytes

from patients with acute

coronary syndromes. The

representative FACS analysis

shows the differential pattern of

phospho- ERK1/2, phospho-

JNK and phospho-p38

expression in T lymphocytes of

patients with acute coronary

syndromes, (UA unstable

angina, NSTEMI non

ST-segment elevation

myocardial infarction, STEMIST-segment elevation

myocardial infarction), patients

with stable coronary artery

disease (CAD), and healthy

controls (CTRL)

672 Basic Res Cardiol (2011) 106:667–679

123

p \ 0.05, Fig. 6a, b), returning at values similar to coro-

nary artery disease patients. Moreover, considering an

intermediate follow-up, such as 60 days, it may be argued

that the reduction in ERK1/2 activation levels is a pro-

gressive phenomenon which starts after a successful

revascularization proceeding until complete normalization

after 6 months. Thus, the latter indicates that ERK1/2 is

specifically activated in T lymphocytes during acute

myocardial ischemia in the setting of UA. Indeed, the acute

administration of antiplatelet therapy, such as clopidogrel,

did not affect phospho-ERK1/2 levels in UA patients

0

1

2

3

4

5

42 kDa

42 kDa

46 kDa

46 kDa

40 kDa

40 kDa

Phospho-ERK 1/2

Total-ERK 1/2

Phospho-JNK

Total-JNK

Phospho-p38

Total-p38C

AD

STE

MI

NS

TEM

I

UA

CTR

L

T Lymphocytes

Fo

ld in

du

ctio

n (

ove

r co

ntr

ol)

CA

D

STE

MI

NS

TEM

I

UA

CA

D

STE

MI

NS

TEM

I

UA

CA

D

STE

MI

NS

TEM

I

UA

Phospho-ERK 1/2

Phospho-JNK

Phospho-p38

*

*

*

* * ***

(A)

(B)

Fig. 4 Immunoblotting analysis confirms differential activation of

MAPK in T lymphocytes from ACS patients. a Representative

western blot analysis showing ERK1/2 phosphorylation and the

MAPK levels in patients with acute coronary syndromes (UA, N = 6;

NSTEMI, N = 6; STEMI, N = 6), stable angina pectoris (CAD,

N = 5) and control subjects (CTRL, N = 5). b Densitometric anal-

ysis of activated forms of ERK1/2, JNK, and p38 (*p \ 0.05 vs.

stable angina pectoris)

Fig. 3 ERK1/2, JNK and p38 activation in T lymphocytes from

patients with acute coronary syndromes. Phospho-ERK1/2 fluores-

cence intensity was significantly increased in patients with unstable

angina, non-ST-elevation and ST-elevation myocardial infarction but

not in patients with stable angina pectoris and in control subjects

(*p \ 0.05 vs. CTRL). A statistically significant increase in fluores-

cence intensity of phospho-JNK was observed only in patients with

acute coronary syndromes associated with an increase of biomarkers

of necrosis (p \ 0.05 vs. CTRL), whereas P-JNK levels in UA were

not significantly elevated in comparison to CAD and control subjects.

A statistical increase in fluorescence intensity of phospho-p38 was

detected in all patients with coronary atherosclerosis (acute and stable

patients) compared to control subjects (*p \ 0.05 vs. CTRL)

b

Basic Res Cardiol (2011) 106:667–679 673

123

compared to additional evaluations performed 24 h after

clopidogrel intake (42 ± 9 vs. 39 ± 11, p = NS).

HMG-CoA reductase inhibition decreases MAPK

activation in activated T lymphocytes in vitro

The role of HMG-CoA reductase inhibitors is mandatory

in stabilizing atherosclerotic plaques, and this issue has

been addressed from several studies. Moreover, published

data from our lab already demonstrated that statin

administration can reduce vascular smooth muscle cell

proliferation in vitro and in vivo [22], suggesting that the

pleiotropic effects of these medications is involved also in

migration of the cells into the plaque. In order to inves-

tigate the effective role of pharmacological treatment on

T lymphocytes activation long after the acute event, T

lymphocytes were harvested from healthy subjects and

MAPK signaling pathway was assessed by immunoblot-

ting. Briefly, in order to mimic an inflammatory state,

cultured T cells were treated with 1,000 UI/ml interleu-

kin-2 (IL-2) at 37�C, 5% CO2 for 15 h; subsequently,

10 lmol rosuvastatin (Rosu) was administered in a single-

shot and cells were collected after 15 min or at the end of

the stimulation. IL-2 administration markedly increased

ERK1/2 activation levels (Fig. 6c), while Rosu treatment

inhibited the phosphorylation levels of ERK1/2 in cul-

tured T lymphocytes in a time-dependent manner, indi-

cating that ERK1/2 activation in T lymphocytes is a

continuous mechanism leading to worsening of the

inflammatory state, and mostly, that inflammation affects

circulating T lymphocytes both acutely and chronically,

therefore suggesting that standard optimal medical ther-

apy, including statins, should be started as soon as pos-

sible in acute patients.

Discussion

The major finding of the present study is that MAPK are

differently activated in T lymphocytes of patients with

ACS depending on the clinical presentation. In particular,

in all patients with ACS, regardless of the presence of

myocyte necrosis, we documented a significant increase of

phospho-ERK1/2, whereas in ACS, phospho-JNK was

activated only in patients with acute myocardial infarction

but not in unstable angina patients. Finally, phospho-p38

was found activated in all patients with atherosclerosis,

including ACS and CAD. Of note, these findings were not

correlated with the extension of coronary artery disease, in

terms of angiographic findings considering neither single-

nor multi-vessels disease (see Table 1), since no significant

difference was found among the groups.

Biomarkers are integral to the care of patients with

cardiovascular disease, in particular those presenting with

ACS with chest discomfort, for whom biomarkers are

central to establishing a diagnosis and guiding evidence-

based therapy [5]. The I subunit of cardiac troponin com-

plex (cTnI) established a paradigm for the modern clinical

use of a biomarker for identifying high-risk patients for

whom specific therapeutic interventions could be imple-

mented to modify the associated risk [33]. Indeed, in the

clinical setting, once it is established that no cTnI has been

released, the patient with suspected ACS may be consid-

ered to have experienced unstable angina. On the other

hand, the diagnosis of myocardial infarction (MI) is

established if a biomarker, i.e. cTnI, is released in the

bloodstream [25]. Moreover, a previous work from another

laboratory reported that beta2-integrin activation of T cells

is increased in patients with UA and severe CAD inde-

pendent of cardiac specific troponin levels [24], suggesting

that evidences that precede myocardial necrosis are needed

in these patients.

Therefore, from a clinical point of view, the diagnosis of

NSTEMI or STEMI is relatively straightforward by mea-

suring the markers of myocyte necrosis, with the caveat of

the delay of their raise in the bloodstream up to several

hours after the onset of ischemic chest pain [2, 3, 8, 9, 37,

38]. However, unstable angina still represents a difficult

diagnosis in a substantial percentage of patients. In the

United States each year, 5.3 million patients present to

emergency departments with chest discomfort and related

symptoms. Ultimately, 1.4 million individuals are hospi-

talized for unstable angina and NSTEMI. Due to the lack of

a biomarker for detecting myocardial ischemia in absence

of necrosis, the correct diagnosis and consequent treatment

of UA patients represent a significant challenge for phy-

sicians [15, 16].

It is well recognized that thrombosis underlies most

acute complications of atherosclerosis, notably UA and

Table 2 Diagnostic performances of the assays

Phospho-ERK1/2 Phospho-JNK

UA

Cut-off 22.5 10.5

Sensitivity 78 67

Specificity 90 60

NSTEMI

Cut-off 13.5 18.5

Sensitivity 56 94

Specificity 79 90

STEMI

Cut-off 12 21

Sensitivity 56 95

Specificity 84 100

The cut-off, sensitivity and specificity of phospho-ERK1/2 and

phospho-JNK in patients with unstable angina (UA), non-ST-eleva-

tion myocardial infarction (NSTEMI) and STEMI

674 Basic Res Cardiol (2011) 106:667–679

123

acute MI. A consensus has emerged that inflammation

plays a key role in the pathophysiology of these acute

thrombotic events [15]. Indeed, patients with cardiovas-

cular risk factors, such as tobacco smoke, have a reduced

number of circulating endothelial progenitor cells and a

high rate of arterial thrombotic complications [11]. To this

regard, it has been proven that the effect of glutathione

S-transferase gene polymorphism was most marked among

smokers with CAD [44]. Accordingly, smoking-induced

chronic inflammation is sufficient to increase activated

levels of all MAPK, but our study demonstrated for the first

time that only when an ACS with unstable angina occurs,

we detect significantly higher levels of phospho-ERK1/2

compared to CAD group.

Active investigation has brought forward an increas-

ingly large number of novel candidate markers of inflam-

mation in the setting of myocardial ischemia; however,

these markers have yet to be incorporated into routine

clinical use [5]. Non-invasive indicators of separate

pathobiologically diverse contributors to the progression of

cardiovascular disease, such as inflammation and throm-

bosis, could add complementary informations to the

available biomarkers of myocardial ischemia [32]. In fact,

novel biomarkers of inflammation, such as C-reactive

Fig. 5 Sensitivity and specificity data obtained with the ROC curves.

Receiver-operating characteristic (ROC) curves of phospho-ERK1/2

and phospho-JNK for diagnosis of ACS (a–c) and ACS without ST

elevation (d) among consecutive patients undergoing coronary

angiography. True-positive fraction (sensitivity as y-axis) was plotted

versus false-positive fraction (1-specificity as x-axis) by changing cut-

off values for the test

Basic Res Cardiol (2011) 106:667–679 675

123

protein (CRP) and myeloperoxidase, and of pathways for

thrombosis, such as soluble CD40 ligand and von Wille-

brand factor, have been shown to add independent prog-

nostic information in a variety of clinical settings,

including those involving stable and unstable ischemic

heart disease [5, 33]. Also, serum lectin-like oxidized LDL

receptor-1 (LOX-1) levels were found to be significantly

high in ACS [16]. Additionally, we have recently shown

through the use of proteomic serum analysis that vitamin D

binding protein is significantly increased in the serum of

STEMI patients [14].

In patients with ACS, the role of systemic inflammation

and lymphocyte activation has been previously reported

[34]. In fact, several laboratories demonstrated an increase

in circulating activated T cells and IgM in patients with

unstable angina. Patients with unstable angina have sig-

nificant higher admission percentage of circulating

T-helper (CD41) cells than controls. Activated T lympho-

cytes are also frequently found in peripheral blood of

patients with ACS [9, 26, 28, 30]. A previous report has

shown the usefulness of tailored interventions in ACS

patients, such as intravascular ultrasound with virtual his-

tology technique for preventing periprocedural coronary

microembolization in distal territories after PCI, thus

reinforcing the importance of appropriate diagnosis and

treatment [6, 7]. To this regard, the specificity in treating

inflammatory plaque rupture exerted by statins and anti-

platelet agents is translated in reduction of thrombus bur-

den release [17]. Indeed, our experimental study

demonstrates for the first time that ERK1/2 phosphorylated

levels measured at fluorescent activated cell sorting could

represent an important marker in stratifying risk for acute

patients and no correlation was found with previous phar-

macological therapy; in fact, FACS spectra have been

compared analyzing twice the same patients before anti-

platelet agents administration at the time of first admission

for chest pain and 24 h after clopidogrel intake for treat-

ment of ACS according to current guidelines. No signifi-

cant difference was found in sera 24 h after clopidogrel

administration, since phospho-ERK1/2 levels in UA with-

out clopidogrel versus same patients evaluated 24 h after

clopidogrel administration were 42 ± 9 vs. 39 ± 11

(p = NS). On the other hand, the majority of patients

admitted for an ACS were already under treatment with

different types and doses of statins, hence making the

impact of statins on MAPK difficult to assess. However,

HMG-CoA reductase inhibitors may affect MAPK in acute

coronary syndromes, since our in vitro data from cultured T

lymphocytes demonstrate that statin administration abo-

lished interleukin 2-induced ERK1/2 activation.

It is well known that the mitogen-activated protein

(MAP) kinase family plays a critical role in intracellular

signal transduction and regulation. These proteins form

complex signalling networks that can be induced by a large

array of external stimuli and can achieve highly specific

cellular effects through multitudes of regulatory mecha-

nisms [31]. Inflammation as well as different cellular

stresses activate MAP kinases proteins and specifically the

three different pathways, JNK, ERK1/2 and p38 [10, 22].

The prototypic ERK1/2 pathway is found to be responsive

mainly to stimulation of growth signalling as well as in

response to inflammatory cytokines. JNK and p38 are

collectively called stress-activated MAP kinases because of

their selective response to physical, chemical and different

stressors (such as ultraviolet rays, osmotic shocks, infection

and cytokines).

CADUA

Phospho-ERK1/2

15 m

in

15 h

rs

IL-2

CON

IL-2+Rosu

Phospho-ERK1/2

Total-ERK1/2

Ph

osp

ho

-ER

K1/

2 fl

uo

resc

ence

inte

nsi

ty in

T ly

mp

ho

cyte

s

0

20

40

60

80

UA

UA-FU

2m 6m CAD CAD6mFU

*

UA-FU

6m2mCAD

6mFU

(A)

(B)

(C)

Fig. 6 In vivo and in vitro transient MAP kinases activation in T

lymphocytes. a Representative immunoblot comparing phospho-

ERK1/2 in unstable angina (UA) and in stable coronary artery

disease (CAD), together with respective follow-up evaluations. b The

bar graphs display phospho-ERK1/2 fluorescence intensity in T

lymphocytes of twenty-two patients at the time of presentation for UA

and after 2 and 6 months (40 ± 18; 35 ± 11; 16 ± 8, respectively,

p \ 0.05). No significant changes in levels of phospho-ERK1/2 were

observed in CAD population. c T lymphocytes were harvested

from healthy subjects and cultured in the absence (CON) or with

1,000 UI/ml interleukin-2 (IL-2); ERK 1/2 activation was assessed

after 15 min and 15 h of rosuvastatin (Rosu, 10 lmol) administration

676 Basic Res Cardiol (2011) 106:667–679

123

The evaluation of MAPK in patients with ACS before

and after PCI was not accompanied by a significant change

in phospho-ERK1/2 activity. This finding has been previ-

ously underlined in other studies, which demonstrated that

the liver-X-receptors activation inhibits chemokine-

induced migration of CD4-positive lymphocytes without

abolishing atherogenesis [45].

Recently, increased levels of activated MAPK have

been found in white blood cells isolated from hypertensive

patients with uncontrolled blood pressure values, therefore

corroborating the hypothesis that in several human cardiac

diseases, such as uncontrolled hypertension, MAPK can be

used as early biomarkers [12]. Thus, we set to investigate

whether MAPK proteins are activated within circulating T

lymphocytes in response to acute myocardial ischemia in

patients with ACS.

Phosphorylated ERK1/2 in T lymphocytes was remark-

ably higher in UA, NSTEMI and STEMI compared with

stable CAD and control subjects. On the other hand, phos-

pho-ERK1/2 was not significantly activated in patients with

stable CAD compared to normal healthy controls. More

interestingly, JNK was significantly phosphorylated in T

lymphocytes from patients with ACS associated with an

increase of biomarkers of necrosis, i.e. NSTEMI and

STEMI, whereas phospho-JNK was not significantly acti-

vated in UA when compared to CAD, NSTEMI or STEMI

patients. A mechanistical explanation for differential MAPK

activation might be due to different triggers in the settings of

either unstable angina or myocardial infarction. Some per-

tinent studies already demonstrated that hypoxia, ischemia

and ischemia/reperfusion induce mitogen activated protein

kinase and transcriptional changes in cardiac myocytes.

Cellular stresses, including infarction, activate p38 and

JNK, whereas the activation of ERK by ischemia–reperfu-

sion is still controversial. In the present study, the phos-

phorylation of all MAPK was increased by myocardial

infarction, whereas ERK1/2 was increased in unstable

angina with normal JNK. In this setting, we found that the

temporal window of ERK1/2 and p38MAPK activation was

different from JNK. ERK1/2 and p38MAPK activities

increased more rapidly than that of JNK. Perhaps, JNK is

activated by proinflammatory cytokines and environmental

stress, such as a tumor necrosis factor and ultraviolet irra-

diation, and is not mediated via a Ras-dependent pathway.

In contrast, ERK1/2 is activated by growth factors via a

Ras-dependent signal-transduction pathway. Interestingly,

although the signal pathways of JNK is very similar to

p38MAPK in the majority of in vitro studies, the timing of

JNK activation has been reported to be distinct from

p38MAPK activation in a rat model of coronary artery

ligation. Myocardial infarction causes inflammation in the

infarcted region and this process may induce JNK activa-

tion, hence the assumption can be made that each MAPK

family may play a different role during myocardial

ischemia.

In conclusion, in the present study, the evaluation of

MAPK activation in T lymphocytes from ACS patients was

able to discriminate acute myocardial ischemia in absence

of myocyte necrosis. Indeed, patients with unstable angina

presented with T lymphocytes showing ERK1/2 but not

JNK phosphorylation. On the other hand, STEMI and

NSTEMI patients had T lymphocytes with increased levels

of both phosphorylated forms of ERK1/2 and JNK.

Study limitations

The present study was performed to assess the molecular

activation of T lymphocytes in a relative small selected

population of patients with ACS. Further studies, however,

should be performed in a larger population to establish the

potential role of MAP kinases activation as biomarker for

the diagnosis and perhaps risk stratification of acute coro-

nary syndromes. Additional studies should also be per-

formed to assess the exact time-course of MAPK activation

in T lymphocytes during ACS and its potential role to

direct timely the appropriate therapy.

Acknowledgments This study was supported, in part, by Grants for

Scientific Research from the Ministry of the Education, University and

Research of Italy (PRIN2003065977_002, RBAU018WWP_001), by

Ricerca sanitaria finalizzata 2007—Programma Strategico APICE

‘‘Activity of Platelets after Inhibition and Cardiovascular Events’’, and

by GENECOR, a non-profit organization.

Conflict of interest The authors declare that they have no conflict

of interest.

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