Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a calcium-independent enzyme produced mainly by macrophages. Lp-PLA2 may directly promote atherogenesis by generating potent proinflammatory and proatherogenic products, such as lysophosphatidylcholine and oxidized free fatty acids from oxidation of low-density lipoprotein (LDL).1 Plaque rupture and subsequent thrombus formation is the most important mechanism leading to acute coronary syndrome (ACS).2 Lp-PLA2 has been found to be upregulated in atherosclerotic lesions, and some studies have found that inhibition of Lp-PLA2 reduced complex coronary atherosclerotic plaque development.3,4 A wealth of studies have proven that increased Lp-PLA2 activity in circulation is positively related to coronary events in patients with coronary artery disease (CAD).5-7 Data are sparse concerning the potential association between lipoprotein-associated phospholipase A2 and human coronary atherosclerotic plaque. Therefore, we conducted this study to test whether plasma Lp-PLA2 activity correlates with plaque rupture in patients with CAD.
The study enrolled 146 consecutive patients with CAD who underwent clinically-indicated coronary angiography and preinterventional intravascular ultrasound (IVUS) at our institution between January 2009 and February 2010. Patients with the following criteria were excluded: (1) adequate IVUS images of the culprit site could not be obtained; (2) ostial lesion or bifurcated lesion; (3) serious calcification lesion; (4) diffuse long lesion; (5) chronic total occlusion lesion; (6) restenosis in a single vessel after percutaneous coronary intervention; (7) any known inflammatory or infectious disease; (8) malignancy; (9) treatment with steroids, immunosuppressive drugs or nonsteroidal anti-inflammatory drugs except for aspirin; (10) severe hepatic or renal diseases. The protocols of this study were approved by our institutional ethics review board and conformed to the Declaration of Helsinki. All patients provided written informed consent before enrollment in the study.
ST segment elevation myocardial infarction (STEMI) was determined by ≥ 30 minutes of continuous chest pain, a new ST-segment elevation on at least two contiguous electrocardiographic leads or pathologic Q waves, and elevation of troponin I, troponin T or creatine kinase greater than the upper limit of normal. Non-ST segment elevation myocardial infarction (NSTEMI) was diagnosed by chest pain and a positive cardiac necrosis marker without new ST-segment elevation. Unstable angina pectoris (UAP) was diagnosed if the patient's chest pain, which was accompanied by ST-T segment changes, was either new or worse in frequency, severity or duration, superimposed on a preexisting pattern of anginal pain. Stable angina pectoris (SAP) was defined as no change in frequency, duration, or intensity of chest pain more than 2 months before the intervention. A myocardial infarction more than one month before the IVUS measurement was regarded as old myocardial infarction (OMI). Individuals with STEMI, NSTEMI and UAP were categorized as ACS.
Blood collection and biochemical analysis
The fasting venous blood was collected after admission to hospital for the measurement of Lp-PLA2 activity, using a vacuum blood collection tube coated with ethylenediaminetetraacetic acid (EDTA) K2 (Becton Dickinson, Franklin Lakes, NJ, USA). Plasma was separated by centrifugation for 10 minutes at 4°C and stored at -70°C until analysed.
Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), glucose and creatinine were measured using standard enzymatic methods. High-sensitive C-reactive protein (hs-CRP) was measured using immunonephelometry. All these measurements were conducted on a Hitachi 7600 automatic analyzer (Hitachi Ltd, Tokyo, Japan).
Lp-PLA2 activity measurement
Lp-PLA2 activity was determined by the colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA) using 2-thio PAF as a substrate and expressed as nmol PAF hydrolyzed per min per ml of plasma. The intra-assay and inter-assay coefficient of variation were 5.8% and 8.1%, respectively. All plasma samples were tested in duplicate and performed by personnel who did not know which group the samples were from.
Two experienced interventional cardiologists reviewed all angiographic data to decide the extent of CAD, TIMI flow grade and culprit lesions. When there was a disagreement, the difference was resolved by a further joint analysis. The extent of CAD was defined as one-vessel disease, two-vessel disease, or three-vessel disease according to the number of major coronary vessels with luminal stenosis ≥50%. TIMI criteria were employed to evaluate coronary flow.8 Culprit lesion was identified on the basis of the association of angiographic lesion appearance, electrocardiogram before and during event, and left ventricle wall motion abnormalities as previously reported.9
IVUS imaging and analysis
IVUS examinations and analyses were performed in accordance with the American College of Cardiology's clinical expert consensus document on IVUS.10 All IVUS examinations were performed before any intervention and after intracoronary administration of 100 to 200 μg of nitroglycerin using a commercially available IVUS system (Atlantis SR Pro 2.5F, 40-MHz; Boston Scientific, Natick, Massachusetts, USA). The IVUS catheter was carefully advanced distal to the target lesion, and imaging was performed retrograde back to the aorto-ostial junction with an automatic pullback at 0.5 mm/s.
Two independent observers who were blind to the angiographic and clinical findings assessed all IVUS images qualitatively and quantitatively. Discordance with qualitative analysis between the observers was adjusted by assessing together to obtain a consensus. Equipping with planimetry software (Echoplaque 3, INDEC Systems Inc, California, USA), lesion and proximal and distal references were analyzed as follows: external elastic membrane cross-sectional area (EEM CSA, in millimeters squared), lumen CSA (in millimeters squared), and plaque and media CSA (P&M CSA=EEM CSA minus lumen CSA, in millimeters squared). Plaque burden (percent) was calculated as P&M divided by EEM CSA. Reference segments were the most normal-looking cross sections within 10 mm proximal and distal to the lesion but before any side branch. A remodeling index was calculated as the lesion divided by the mean reference EEM. Positive remodeling was defined as a remodeling index >1.0.
Calcium was brighter than the adventitia with acoustic shadowing. Calcified plaque had an arc >90°. Fibrous plaque was as bright as or brighter than the adventitia without shadowing. Soft plaque was less bright than the adventitia. When there was no dominant plaque composition, the plaque was considered “mixed”. A ruptured plaque contained a cavity that communicated with the lumen with an overlying residual fibrous cap fragment.
Statistical analysis was performed with SPSS 13.0 (SPSS Inc, Chicago, IL, USA). Continuous variables with normal or skewed distribution were presented as mean ± standard deviation (SD) or median, respectively. Continuous variables were compared using the unpaired Student's t test or the Kruskal-Wallis test according to the distribution of data. Categorical variables were assessed using the chi-square test. Multiple Logistic regression models were used to estimate the independent factors associated with plaque rupture in patients with CAD. P <0.05 was considered to indicate statistical significance.
Among all the 146 patients, 63 patients were excluded from the analysis based on the exclusion criteria. Finally, 83 patients were included in the study; the mean age was (62.0±11.6) years, and there were 65 (78.3%) men. Among the 83 culprit lesions assessed by IVUS, 60 (72.3%) were from ACS patients and 23 (27.7%) from SAP patients. Thrombolysis was not performed for any patient. All interventional procedures were well tolerated by all patients, with no major complications or adverse events. Table 1 shows the clinical characteristics between patients with and without plaque rupture. There were no significant differences in clinical characteristics between the two groups, except for smoking, plasma hs-CRP levels and Lp-PLA2 activity (all P <0.05).
Angiographic findings are showed in Table 2. Number of diseased vessels and culprit lesion locations were statistically similar between patients with and without plaque rupture. Fifteen patients with TIMI flow grade ≤1 were AMI and aspiration thrombectomy was performed by an aspiration catheter (ZEEK catheter, ZEON Medical, Tokyo, Japan) before intracoronary imaging, but predilation with a balloon catheter was not allowed. After reperfusion with TIMI flow grade 3, the culprit lesion was observed by IVUS as described above.
Qualitative and quantitative analyses of IVUS images are presented in Table 3. Plaque rupture at culprit lesion occurred in 39 (47.0%) patients, with 34 (87.2%) from ACS patients and 5 (22.8%) from SAP patients. Patients with plaques rupture had more frequent positive remodeling (74.4% vs. 43.2%, P=0.004) and soft plaques (64.1% vs. 36.4%, P=0.012) than patients without plaque rupture. In addition, remodeling index in patients with plaque rupture was higher than that in patients without plaque rupture (1.13±0.16 vs. 0.99±0.11, P=0.041). No essential differences were found between patients with and without plaque rupture in other IVUS parameters.
Multivariate Logistic regression analysis indicated that plasma Lp-PLA2 activity was independently associated with plaque rupture at culprit lesions after adjusting for positive remodeling, smoking and soft plaque (Model 1 : odds ratio (OR) 1.13, 95% confidence interval (CI): 1.06-1.20), or adjusting for hs-CRP, positive remodeling, smoking and soft plaque (Model 2: OR 1.11, 95% CI: 1.04-1.19).
Although Lp-PLA2 is emerging as a relatively specific marker of vascular and plaque inflammation, few studies have addressed the direct relationship between plaque rupture and Lp-PLA2 in humans. By means of IVUS, the present study shows that increased plasma Lp-PLA2 activity is independently related to plaque rupture in CAD patients, after adjustment for traditional CAD risk factors, plasma hs-CRP level and IVUS parameters.
Inflammation plays an important role in plaque progression and vulnerability. One of the major characteristics of vulnerable plaque is the high propensity to rupture. In the past decade, it has been uncovered that plaque rupture accounts for 60% to 70% coronary events in CAD patients.11 The key characteristics of rupture-prone plaque are thin fibrous cap, large lipid core and wide infiltration by inflammation cells including macrophages and T cells.12 The inflammation cells at rupture-prone plaque are activated, and then secrete some enzymes and proinflammatory mediators to degrade extracellular matrix and amplify inflammation cascade. As a result, the fibrous cap is thinned and weakened, leading to plaque vulnerability with subsequent rupture and thrombosis.
Lp-PLA2 is synthesized and secreted largely by monocytes/macrophages and T lymphocytes which are the fundamental cells at atherosclerotic plaque.1 In humans, Lp-PLA2 is bound mainly to LDL particles in circulation, where it remains latent until the LDL particles are subjected to oxidative modification. Lp-PLA2 acts on oxidized phospholipids on oxidized LDL particles which mainly located at atherosclerotic lesion to generate two key proinflammatory mediators - lysophosphatidylcholine and oxidized nonesterified fatty acids.1 These two mediators may exert multiple biological effects related to plaque disruption.1 Häkkinen et al13 found that Lp-PLA2 was expressed in macrophages from both human and rabbit atherosclerotic lesion, and Lp-PLA2 activity in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits was about 6-fold higher than that of normal aortas from control rabbits. Kolodgie et al3 reported that Lp-PLA2 was significantly upregulated in apoptotic macrophages within necrotic core and fibrous cap of ruptured and rupture-prone plaques, but not within less-advanced plaques. In addition, the demonstration of darapladib, a potent inhibitor of human Lp-PLA2, notably retarded the progression of atherosclerotic lesions to advanced phenotypes in animal and human studies, provided more powerful evidence to support the notion that Lp-PLA2 may be crucial in plaque progression and vulnerability.4,14
Extensive clinical studies demonstrated that increased Lp-PLA2 activity in circulation is associated with the high risk of coronary events. In the Bruneck study, elevated plasma Lp-PLA2 activity amplified the risk of 10-years cardiovascular diseases (CVD) event mediated by oxidized phospholipids/apolipoprotein B.15 PROVE IT-TIMI 22 indicated that plasma Lp-PLA2 activity was associated with an increased risk of cardiovascular events independent of CRP and LDL-C levels.16 In THROMBO study, Lp-PLA2 was a significant and independent predictor of recurrent coronary events in postinfarction patients.17 Recently, the Bruneck study also found that increased plasma Lp-PLA2 activity correlated with incident fatal and non-fatal CVD, but not with non-CVD mortality.18
There were several limitations in this study. Firstly, the inclusion criteria in this study require preinterventional IVUS, but IVUS measurement is only performed for some selective patients in our center. Therefore, the sample size of patients is relatively small. In addition, plaque rupture may occur in multiple atherosclerotic lesions of CAD patients. Unfortunately, this study only detected plaque rupture by IVUS at culprit lesion. Lastly, the study may also have missed some ruptured plaques in which the cavity was extremely small, filled by thrombus, or sealed by reattachment of the flap.
In summary, plaque rupture is the most common cause of coronary thrombosis, in this prospective study, we found Lp-PLA2 was associated with plaque rupture, which demonstrated Lp-PLA2 may be a risk marker for vulnerable plaque.
1. Zalewski A, Macphee C. Role of lipoprotein-associated phospholipase A2
in atherosclerosis biology, epidemiology, and possible therapeutic target. Arterioscler Thromb Vase Biol 2005; 25: 923-931.
2. Waxman S, Ishibashi F, Muller JE. Detection and treatment of vulnerable plaques and vulnerable patients: novel approaches to prevention of coronary events. Circulation 2006; 114:2390-2411.
3. Kolodgie FD, Burke AP, Skorija KS, Ladich E, Kutys R, Makuria AT, et al. Lipoprotein-associated phospholipase A2
protein expression in the natural progression of human coronary atherosclerosis. Arterioscler Thromb Vase Biol 2006; 26: 2523-2529.
4. Wilensky RL, Shi Y, Mohler ER 3rd, Hamamdzic D, Burgert ME, Li J, et al. Inhibition of lipoprotein-associated phospholipase A2
reduces complex coronary atherosclerotic plaque
development. Nat Med 2008; 14: 1059-1066.
5. Corson MA, Jones PH, Davidson MH. Review of the evidence for the clinical utility of lipoprotein-associated phospholipase A2
as a cardiovascular risk marker. Am J Cardiol 2008; 101 (12A): 41F-50F.
6. Koenig W, Khuseyinova N. Lipoprotein-associated and secretory phospholipase A2
in cardiovascular disease: the epidemiological evidence. Cardiovasc Drugs Ther 2009; 23: 85-92.
7. Anderson JL. Lipoprotein-associated phospholipase A2
: an independent predictor of coronary artery disease
events in primary and secondary prevention. Am J Cardiol 2008; 101 (12A): 23F-33F.
8. The TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial. N Engl J Med 1985; 312: 932-936.
9. Fujii K, Kobayashi Y, Mintz GS, Takebayashi H, Dangas G, Moussa I, et al. Intravascular ultrasound
assessment of ulcerated ruptured plaques: a comparison of culprit and nonculprit lesions of patients with acute coronary syndromes and lesions in patients without acute coronary syndromes. Circulation 2003; 108: 2473-2478.
10. Mintz GS, Nissen SE, Anderson WD, Bailey SR, Erbel R, Fitzgerald PJ, et al. American College of Cardiology clinical expert consensus document on standards for acquisition, measurement and reporting of intravascular ultrasound
studies (IVUS). A report of the American College of Cardiology Task Force on Clinical Expert Consensus Documents. J Am Coll Cardiol 2001; 37: 1478-1492.
11. Casscells W, Naghavi M, Willerson JT. Vulnerable atherosclerotic plaque
: A multifocal disease. Circulation 2003; 107: 2072-2075.
12. Naghavi M, Libby P, Falk E, Casscells SW, Litovsky S, Rumberger J, et al. From vulnerable plaque
to vulnerable patient: A call for new definitions and risk assessment strategies: Part I. Circulation 2003; 108; 1664-1672.
13. Häkkinen T, Luoma JS, Hiltunen MO, Macphee CH, Milliner KJ, Patel L, et al. Lipoprotein-associated phospholipase A2
, platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions. Arterioscler Thromb Vase Biol 1999; 19: 2909-2917.
14. Serruys PW, García-García HM, Buszman P, Erne P, Verheye S, Aschermann M, et al. Effects of the direct lipoprotein-associated phospholipase A2
inhibitor darapladib on human coronary atherosclerotic plaque
. Circulation 2008; 118: 1172-1182.
15. Kiechl S, Willeit J, Mayr M, Viehweider B, Oberhollenzer M, Kronenberg F, et al. Oxidized phospholipids, lipoprotein(a), lipoprotein-associated phospholipase A2
activity, and 10-year cardiovascular outcomes: prospective results from the Bruneck study. Arterioscler Thromb Vase Biol 2007; 27: 1788-1795.
16. O'Donoghue M, Morrow DA, Sabatine MS, Murphy SA, McCabe CH, Cannon CP, et al. Lipoprotein-associated phospholipase A2
and its association with cardiovascular outcomes in patients with acute coronary syndromes in the PROVE IT-TIMI 22 (PRavastatin Or atorVastatin Evaluation and Infection Therapy-Thrombolysis In Myocardial Infarction) trial. Circulation 2006; 113: 1745-1752.
17. Corsetti JP, Rainwater DL, Moss AJ, Zareba W, Sparks CE. High lipoprotein-associated phospholipase A2
is a risk factor for recurrent coronary events in postinfarction patients. Clin Chem 2006; 52: 1331-1338.
18. Tsimikas S, Willeit J, Knoflach M, Mayr M, Egger G, Notdurfter M, et al. Lipoprotein-associated phospholipase A2
activity, ferritin levels, metabolic syndrome, and 10-year cardiovascular and non-cardiovascular mortality: results from the Bruneck study. Eur Heart J 2009; 30: 107-115.