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Journal of the American College of Cardiology Vol. 45, No. 2, 2005© 2005 by the American College of Cardiology Foundation ISSN 0735-1097/05/$30.00P

ersistent Systemic Inflammation in Unstable Anginas Largely Unrelated to the Atherothrombotic Burdenlaudia Monaco, MD, PHD,* Elisabetta Rossi, MD,† Diego Milazzo, MD,† Franco Citterio, MD,‡rancesca Ginnetti, BSC,‡ Giuseppe D’Onofrio, MD,§ Domenico Cianflone, MD,� Filippo Crea, MD,†uigi M. Biasucci, MD, FACC,† Attilio Maseri, MD, FACC�ondon, United Kingdom; and Rome and Milan, Italy

OBJECTIVES The aim of this study was to assess the relationship between systemic inflammation,atherosclerosis, and thrombosis in two distinct clinical models of atherothrombosis.

BACKGROUND Persistent unstable angina (UA) is commonly associated with coronary thrombosis andpersistent systemic inflammation.

METHODS We assessed circulating markers of activation of the thrombotic and fibrinolytic cascades andsystemic soluble and cellular markers of inflammation on admission in 40 patients withpersisting UA (Braunwald class IIIB; group 1) and 30 patients with Leriche-Fontaine stageIIB-III peripheral artery disease awaiting revascularization (group 2).

RESULTS The extent of atherosclerosis (p � 0.01) and activation of the coagulation system were greaterin group 2, which had higher thrombin-antithrombin III complexes and D-dimer levels (2.7and 24.4 �g/l, respectively), than in group 1 (2.0 �g/l and 12.9 �g/l, p � 0.02 and p �0.0001, respectively). In contrast, C-reactive protein and interleukin-6 levels were higher ingroup 1 (7.6 pg/ml and 7.8 pg/ml, respectively) than in group 2 (4.5 pg/ml and 3.0 pg/ml,p � 0.01 and p � 0.03, respectively). Moreover, neutrophil activation was only found ingroup 1 (neutrophil myeloperoxidase content �4.0 arbitrary units vs. �3.4 arbitrary units ingroup 2, p � 0.0001). These differences persisted during the initial three days ofhospitalization.

CONCLUSIONS Such a large, consistent discrepancy between atherothrombotic burden and systemic inflam-mation suggests that atherothrombosis, by itself, is an unlikely cause of persisting, recurringUA. An understanding of the primary inflammatory mechanisms of persistent and recurrentcoronary instability could open the way to novel therapeutic strategies. (J Am Coll Cardiol

ublished by Elsevier Inc. doi:10.1016/j.jacc.2004.09.064

2005;45:238–43) © 2005 by the American College of Cardiology Foundation

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he spectrum of clinical presentation of acute coronaryyndromes is varied. At one extreme end of the spectrum,ome patients present with acute myocardial infarction notreceded by anginal symptoms and, after this event, mayemain totally asymptomatic for years or decades. At thether end, some patients present with infarction preceded

See page 244

nd followed by unstable angina (UA). Indeed, UA, whichowadays accounts for about 50% of coronary care admis-ions, is typically characterized by persistence or recurrencef symptoms and by an incumbent threat of myocardialnfarction and sudden death over periods of weeks and

onths. The actual triggers of the occasional transitionrom stable to unstable atherosclerosis may not be the samen these two extreme groups. By definition, angina isonsidered “unstable” when onset or sudden worsening ofymptoms dates back up to two months before admission

From the *Cytokine Biology of Vessels, Kennedy Institute of Rheumatology andurgery, Anesthetics and Intensive Care, Faculty of Medicine, Imperial College,ondon, United Kingdom; Institutes of †Cardiology, ‡Vascular Surgery, andHaematology, Universita Cattolica del Sacro Cuore, Rome, Italy; and �Cardiotho-acic and Vascular Department, Vita-Salute San Raffaele University, Milan, Italy.upported by a MURST grant (ICS 030 4RF9891 IVASC) and the Fondazionenternazionale di Ricerca per il Cuore Onlus, Rome, Italy.

iManuscript received December 23, 2003; revised manuscript received July 19,

004, accepted September 3, 2004.

1), and the average risk of major cardiac events at four to sixonths exceeds 10% under the current optimal medical (2)

r interventional strategies (3). Eventually, only after sixonths, the increased risk transiently conferred by instabil-

ty reverts back to that of stable or “quiescent” phases ofschemic heart disease. Coronary thrombi composed ofayers of different ages are common at autopsy in unstableatients (4), consistent with a “staccato” pattern of clinicalxacerbations of instability and with a nearly 50% reductionf infarction rates achieved by antiplatelet and antithrom-otic treatments (1). However, thrombosis, by itself, mayot be the only plausible explanation of the long persistencend common recurrence of coronary instability. A possibleole of an acute coronary inflammatory process is suggestedy: 1) elevated levels of circulating inflammatory markersbserved in patients with refractory UA and with acutenfarction preceded by UA (5–9); 2) persistence of elevated-reactive protein (CRP) serum levels after acute instability

ssociated with a seven-fold greater risk of recurring UA orf infarction (10,11); and 3) the adverse prognostic value ofncreasing levels of interleukin (IL)-1 receptor antagonistnd IL-6 during the first two days after admission inatients with persisting UA (12).Thus, the intriguing association between coronary

hrombosis and inflammation observed in patients with UAaises the question of whether systemically detectable

nflammation is largely a reflection of ongoing atherothrom-

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239JACC Vol. 45, No. 2, 2005 Monaco et al.January 18, 2005:238–43 Atherothrombosis, Inflammation, and UA

osis or whether it may reflect independent, primary causalechanisms of inflammation directly responsible for per-

isting, recurrent coronary instability.To investigate the relative role of atherothrombosis and

hat of primary inflammatory triggers in UA, we comparedhe levels of circulating markers of systemically detectablehrombotic and inflammatory activation in two distinctlinical syndromes, both characterized by atherothrombosis.

We assessed systemic soluble and cellular markers ofnflammation together with markers of activation of thehrombotic and fibrinolytic cascades and seropositivity toutative infectious agents in 40 patients with Braunwaldlass IIIB UA and negative troponin T, as well as in 30atients with Leriche-Fontaine stage IIB-III peripheralrtery disease (PAD) awaiting revascularization procedures,s potential distinct clinical models of atherothromboticiseases.

ETHODS

tudy population. GROUP 1: UNSTABLE ANGINA. Wenrolled 40 consecutive patients (34 men, age 60 � 12ears) admitted to our coronary care unit with Braunwaldlass IIIB UA, without evidence of acute myocardial necro-is detected by troponin T release, and submitted to coro-ary angiography. We excluded patients with positive tro-onin T (�0.1 mg/l), known or suspected thromboticisorders, malignancy, infection, inflammatory disease, orecent surgery or trauma (�4 weeks). All patients werereated with aspirin, intravenous nitrates, calcium antago-ists, and/or beta-blockers. Heparin was not used as arst-step treatment. In addition, we also excluded patientsith Doppler evidence of PAD or a systolic ankle/brachialressure ratio �0.95.

ROUP 2: PERIPHERAL ARTERY DISEASE. We enrolled 30onsecutive patients (21 men, age 63 � 16 years) witheriche-Fontaine stage IIB to III PAD with claudicationppearing after �100 m of walking, admitted for revascu-arization by a Rotablator percutaneous procedure or sur-ery. General exclusion criteria adopted for UA patientsere also applied to PAD. In addition, we also excludedatients with evidence of trophic limb lesions and thoseith a previous myocardial infarction and echocardiographic

vidence of regional wall motion alteration at rest or after aipyridamole stress test.nformed consent. The Ethics Committee of the Catholicniversity of Rome approved the protocol, and all patients

Abbreviations and AcronymsCRP � C-reactive proteinIL � interleukinPAD � peripheral artery diseaseUA � unstable angina

ave their written, informed consent. n

lood sampling and laboratory methods. The number oforonary and peripheral artery flow-limiting stenoses with70% diameter reduction and total occlusion and of steno-

es between 50% and 70% was assessed visually by twondependent observers, and discordant findings were re-olved by a third observer.

Peripheral blood samples were taken on hospital admis-ion and after 6, 12, 24, 48, and 72 h in both groups ofatients.We measured high-sensitivity CRP and IL-6 as markers

f an acute-phase response, neutrophil myeloperoxidaseontent as an indicator of neutrophil activation, thrombin-ntithrombin III complexes and prothrombin fragment�2 as markers of thrombin generation, and D-dimers asarkers of formed intravascular fibrin undergoing lysis.

eropositivity for Chlamydia pneumoniae, cytomegalovirus,nd Helicobacter pylori was also assessed. Troponin T, apecific marker of myocardial necrosis, was measured to ruleut the possible role of myocardial cell damage in inducinghe inflammatory response.

Samples for thrombin-antithrombin III complexes, pro-hrombin fragment 1�2, D-dimers, and IL-6 were immedi-tely transferred into pre-cooled tubes containing citrate, the-phylline, adenosine, and dipyridamole (CTAD tubes; Dade-ehring, Marburg, Germany) and centrifuged at 2,000 g andt 4°C for 20 min. Plasma aliquots were stored at �80°C.ommercially available ELISA assays (Enzygnost thrombin-

ntithrombin III complex, micro-prothrombin fragment 1�2,nd D-dimer ELISA kit, Dade-Behring; Quantikine humanL-6, R&D Systems, Minneapolis, Minnesota) were used.

Serum samples were obtained for CRP and troponin Tnalysis and for assessment of seropositivity for C. pneumoniae,ytomegalovirus, and H. pylori. C-reactive protein wasssayed in an ultra-sensitive CRP assay by nephelometryBN-II, Dade-Behring) with a sensitivity of 0.2 mg/l.roponin T was measured by a commercial enzyme immu-oassay (Boehringer Mannheim, Mannheim, Germany).eropositivity for C. pneumoniae was assessed by the micro-

mmunofluorescence method (Chlamydia MIF kit, MRLiagnostics, Cypress, California); seropositivity for C. pneu-oniae was defined as the presence of specific antibodies atserum dilution of 1:16. Seropositivity for cytomegalovirus

nd H. pylori was assessed by a commercial ELISA forpecific immunoglobulin G (Dade-Behring); seropositivityor cytomegalovirus and H. pylori was defined as theresence of a serum antibody titer �6 U/l and 10 U/l,espectively.

Samples for myeloperoxidase intracellular content weremmediately transferred into tubes containing 1:9 EDTAolution and quickly analyzed using a Bayer H*3 hematol-gy analyzer (Bayer-Diagnostic Co., Tarrytown, Nework), as previously described (8,13). The computer soft-are calculates an index of intracellular myeloperoxidase

ctivity (intracellular myeloperoxidase index), which quan-ifies the mean myeloperoxidase activity of the whole

eutrophil population in arbitrary units. In normal subjects,

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240 Monaco et al. JACC Vol. 45, No. 2, 2005Atherothrombosis, Inflammation, and UA January 18, 2005:238–43

his index is about 0. Positive values appear when theeutrophils are rich in myeloperoxidase, and negative values

ndicate depletion of myeloperoxidase, due to neutrophilctivation and degranulation.tatistical analysis. As the data were not normally distrib-ted, non-parametric tests were used. Results are expresseds the median value, 25% to 75% interquartiles, and range.he Mann-Whitney U test was used to evaluate differencesetween groups, and discontinuous variables were tested byhe chi-square test. Correlation between variables was as-essed by the Spearman correlation test. Continuous vari-bles containing clinical data are expressed as the meanalue � SD and were evaluated by the Student t test. A palue of �0.05 was considered statistically significant. Allests were two-tailed.

ESULTS

linical variables and angiographic findings. The tworoups were similar in terms of age, gender, smoking status,ypertension, diabetes, and family history of ischemic heartisease (Table 1). The number of flow-limiting arterialtenoses was significantly higher in PAD than in UAstenosis �70% and occlusion: 5.5 � 1.6 vs. 2.5 � 0.9,espectively; stenoses between 50% and 70%: 4.7 � 2.3 vs..1 � 0.5, respectively; p � 0.01) (Table 1, Fig. 1). In PAD,he presence of flow-limiting coronary stenoses was ex-luded by the echocardiographic dipyridamole stress test,nd in UA, by the presence of peripheral artery stenosis bysystolic ankle/brachial pressure ratio �0.95.ctivation of thrombotic and fibrinolytic cascade. Acti-

ation of the thrombotic and fibrinolytic cascades was signifi-antly greater in patients with PAD than in those with UA.n admission, thrombin-antithrombin III complexes and-dimers levels were significantly higher in patients with PAD

han in those with UA, being 2.7 �g/l (range 0.9 to 47.0 �g/l)ersus 2.0 �g/l (range 1.2 to 7.4 �g/l) (p � 0.02) (Fig. 2A) and4.4 �g/l (range 9.1 to 182.5 �g/l) versus 12.9 �g/l (range 1.9o 60.0 �g/l) (p � 0.0001) (Fig. 2B), respectively. Moreover,he levels of thrombin-antithrombin III complexes and

able 1. Clinical and Angiographic Findings

UnstableAngina

(n � 40)

PeripheralArtery

Disease(n � 30)

ge (yrs) 60 � 12 63 � 16ale gender 34 (85%) 21 (70%)

amily history of CAD 28 (41%) 6 (20%)moking 16 (40%) 15 (50%)ypertension 20 (50%) 15 (50%)iabetes 13 (32%) 16 (55%)ypercholesterolemia 22 (55%) 11 (36%)rterial stenoses �70% and occlusions 2.5 � 0.9 5.5 � 1.6*rterial stenoses 50% to 70% 1.1 � 0.5 4.7 � 2.3*

p � 0.01 vs. unstable angina. Data are presented as the mean value � SD orumber (%) of patients.

CAD � coronary artery disease.

-dimers remained consistently higher in patients with PADo1

han in those with UA throughout the first 72 h of hospital-zation (p � 0.01; data not shown). Prothrombin fragment�2 median levels were not significantly different between UA1.0 nmol/l, range 0.2 to 8.3 nmol/l) and PAD (1.0 nmol/l,ange 0.3 to 7.5 nmol/l) (p � NS).nflammatory markers. In contrast, the inflammatoryarkers CRP and IL-6 were significantly higher in patientsith UA than in those with PAD, being 7.6 mg/l (range 1.4

o 114 mg/l) versus 4.5 mg/l (range 0.7 to 22.2 mg/l) (p �.01) for CRP (Fig. 2C) and 7.8 pg/ml (range 3 to 90g/ml) versus 3.0 pg/ml (range 3 to 15 pg/ml) (p � 0.03) forL-6 (Fig. 2D), respectively. Furthermore, neutrophil acti-ation was only observed in UA (intracellular myeloperox-dase index �4.0 arbitrary units [�14.5 to �4.7] vs. �3.4rbitrary units[�5 to �8.5] in PAD, p � 0.0001) (Fig. 2E).hese differences were maintained throughout the first 72 hf hospitalization (p � 0.01). The neutrophil count did notiffer between UA and PAD patients. A correlationetween CRP and IL-6 levels was statistically significant inA and PAD patients (r � 0.45, p � 0.01 and r � 0.34,� 0.05, respectively). The correlation between the intra-

igure 1. Typical discrepancy in atherosclerotic burden between patientith unstable angina (UA) and peripheral artery disease. Right (A) and left

B) coronary angiograms in a patient with UA with a single coronary arterytenosis in the proximal segment of the left anterior descending coronaryrtery; the C-reactive protein (CRP) level was 57 mg/l and the D-dimeralue was 3.7 �g/l. Femoral (C) and tibial (D) angiograms of a patient witheripheral vascular disease with multiple atherosclerotic plaques and

cclusions; the CRP level was 3.7 mg/l and the D-dimer value was07.4 �g/l.

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241JACC Vol. 45, No. 2, 2005 Monaco et al.January 18, 2005:238–43 Atherothrombosis, Inflammation, and UA

ellular myeloperoxidase index and CRP was statisticallyignificant in patients with UA (r � �0.62, p � 0.03) butot in patients with PAD (r � �0.13, p � NS). Noorrelation was found between CRP and thrombin-ntithrombin III complexes, D-dimers, or prothrombinragment 1�2 levels in UA or PAD patients.

The prevalence of seropositivity for infectious agents wasimilar in UA and PAD: 62% versus 40% for C. pneumoniaep � NS), 89% versus 87% for cytomegalovirus (p � NS),nd 83% versus 78% for H. pylori (p � NS). There was noorrelation between CRP levels and antibody titers against. pneumoniae, cytomegalovirus, and H. pylori in UA orAD groups.

ISCUSSION

ur study shows that atherothrombosis is unlikely by itselfo account for the elevation of systemic markers of inflam-ation, which characterizes patients with persisting UA.

ndeed, levels of thrombin-antithrombin III complexes (aarker of thrombin generation) and D-dimers (a marker of

ormed intravascular fibrin) were nearly twice as high inatients with severe PAD (Leriche-Fontaine IIB-III)waiting revascularization than in patients with severe UABraunwald class IIIB), consistent with a significantlyreater number of flow-limiting stenosis, total occlusions, ortenoses �70% and of stenoses 50% to 70%. By contrast,

igure 2. A discrepancy between activation of the coagulation cascade aneripheral artery disease (PAD) (shaded boxes). Thrombin-antithrombinatients with PAD than in patients with UA. Conversely, interleukin (ILatients with UA than in patients with PAD. Moreover, activation of neutMPXI) (E) was only present in patients with UA. Data are shown as the

evels of CRP and IL-6, two major markers of inflamma- i

ion, were significantly higher in patients with UA than inatients with PAD. Moreover, neutrophil activation, asssessed by its intracellular content of myeloperoxidase, wasnly observed in UA but not in PAD patients and wasinearly correlated with CRP levels. Thus, although severeAD requiring revascularization procedures is associatedith a much greater systemic activation of the coagulation

ascade, as compared with UA, it is characterized by a muchmaller elevation of CRP and IL-6 levels and by noetectable neutrophil activation in the peripheral blood.uch differences remained significant in all successive bloodamples taken up to 72 h after admission.

Elevation of CRP and IL-6 and neutrophil activationave been consistently reported in patients with UA but not

n patients with chronic, stable effort angina and a greatereverity of coronary atherosclerosis (6–8) or in patients withctive variant angina and a greater ischemic burden (8,14).t a variance from CRP and IL-6 serum levels, which inatients with Braunwald class IIIB UA may remain elevatedor several months after discharge (12,15), neutrophil acti-ation appears limited to the acute symptomatic phase ofnstability of the disease, as it is not found at discharge (8),nd appears to take place selectively in the coronary vasculared of unstable patients (13). These observations are con-istent with the prognostic value of myeloperoxidase levelsn patients with acute coronary syndromes (16). Conversely,

ulating inflammatory markers in unstable angina (UA) (open boxes) andmplexes (TAT) (A) and D-dimers (DD) (B) were significantly higher inD) and C-reactive protein (CRP) levels (C) were significantly higher inls, indicated by the significant reduction of their myeloperoxidase contentian values, 25% to 75% interquartiles, and range.

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242 Monaco et al. JACC Vol. 45, No. 2, 2005Atherothrombosis, Inflammation, and UA January 18, 2005:238–43

eported after induced claudication (17), but not at rest17,18).

The greater systemic levels of inflammatory markers inA, compared with PAD, cannot be explained by differ-

nces in the prevalence of traditional risk factors (Table 1)r in seropositivity for infectious agents possibly involved intherosclerotic syndromes, as the prevalence of seropositiv-ty for C. pneumoniae, cytomegalovirus, and H. pylori wasot significantly different between patients with UA andhose with PAD, in agreement with previous studies19,20).

Our data show significant differences not only in theagnitude but also in the pattern of systemic markers of

nflammation and activation of the coagulation system inwo prototypical clinical models of atherothrombosis, UA,nd peripheral vascular disease. In particular, UA is char-cterized by a marked systemic up-regulation of inflamma-ory activation, which considerably exceeds the amountttributable to the atherothrombotic burden in PAD. Theersistence and recurrence of coronary instability is moreuggestive of persistent, recurrent thrombogenic stimulihan of a process of repair of a single initial injury resultingrom a fissured or eroded atherosclerotic plaque.tudy limitations. We assessed the atherosclerotic burdennly on the basis of the number of stenoses �70% and 50%o 70%, which was more than double in patients witheripheral vascular disease (Table 1). However, in postmor-em studies, the number of coronary flow-limiting stenosestrongly correlated with the percentage of coronary intimalurface covered by raised fibrous plaque (21). This correla-ion is likely to hold true also for PAD. A greater athero-clerotic burden and a larger endothelial involvement areonsistent with the markedly higher levels of D-dimers andhrombin-antithrombin III complexes in patients withAD also observed in previous studies (22–24). Yet, sucharkedly larger atherosclerotic burden in peripheral vascular

isease was not associated with neutrophil degranulation,hich was present in UA and with lower high sensitivityRP and IL-6 serum levels, as compared with UA.therothrombosis, inflammation, and plaque instability.ollectively, our findings suggest the hypothesis that the

ecurring phases of coronary instability may not necessarilyevelop when the atherosclerotic burden has reached aritical threshold, but may as a result of transient superim-osed “primary” coronary inflammatory stimuli. Such trig-ers of instability eventually wane with a return of theatient to a stable phase and baseline ischemic risk, relatedo atherosclerotic and risk factor burden. This hypothesisould be consistent with findings of multiple complex

25,26), fissured (27,28), and inflamed (29) coronary arterylaques found in patients with persistent, recurrent insta-ility, suggestive of simultaneous multifocal plaque instabil-ty and with widespread coronary inflammation, also involv-ng angiographically normal arteries (13). Such widespreadoronary inflammation could be related to perturbation of

he immune response (30,31), antigenic mimicry, and au-

oimmune mechanisms (32,33). Understanding these in-ammatory mechanisms could open the way to the devel-pment of novel therapeutic strategies designed to blocknflammatory triggers of persistent, recurrent coronaryhrombus formation, without interfering heavily withemostatic mechanisms—so far the only available strategyo prevent local coronary thrombosis—at the price of anncreased risk of bleeding.

eprint requests and correspondence: Prof. Attilio Maseri,ita-Salute San Raffaele University, Via Olgettina 58, 20132ilan, Italy. E-mail: maseri.attilio@hsr.it.

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