Published online before print September 7, 2006, doi: 10.1161/01.ATV.0000244681.72738.bc
© 2006 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Lipoprotein-Associated Phospholipase A2 Protein Expression in the Natural Progression of Human Coronary Atherosclerosis
From CVPath Institute, Inc (F.D.K., A.P.B., K.S.S., E.L., R.K., R.V.), Gaithersburg, Md; and the Department of Pathology (A.T.M.), Georgetown University, Washington, DC.
Correspondence to Renu Virmani, MD, Medical Director, CVPath Institute Inc, 19 Firstfield Road, Gaithersburg, MD 20878. E-mail rvirmani{at}cvpath.org
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Abstract |
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Objective— Although lipoprotein-associated phospholipase A2 (Lp-PLA2) has received recent attention as a biomarker of inflammation and risk for acute coronary events, its relative expression in coronary plaque phenotypes, including unstable lesions, has not been established.
Methods and Results— Coronary segments (n=30) were prospectively collected from 25 sudden coronary death patients for immunolocalization of Lp-PLA2. Lesion morphologies were classified as pathologic intimal thickening, fibroatheromas, thin-cap fibroatheromas (fibrous cap thicknesses <65 µm), and rupture. The expression of Lp-PLA2 was detected using a specific monoclonal antibody. Apoptosis was identified by DNA end-labeling using terminal deoxynucleotidyl transferase (TdT). Lp-PLA2 staining in early plaques was absent or minimally detected. In contrast, thin-cap fibroatheromas and ruptured plaques showed intense Lp-PLA2 expression within necrotic cores and surrounding macrophages including those in the fibrous cap. The degree of macrophage apoptosis was greater in thin-cap fibroatheroma and ruptures compared with less advanced plaques with additional double labeling studies showing Lp-PLA2 present in apoptotic cells in regions of high macrophage density.
Conclusions— Lp-PLA2 is strongly expressed within the necrotic core and surrounding macrophages of vulnerable and ruptured plaques, with relatively weak staining in less advanced lesions. These findings together with the association of Lp-PLA2 in apoptotic macrophages suggest a potential role in promoting plaque instability.
The expression of lipoprotein-associated phospholipase A2 (Lp-PLA2) was assessed in early through late, unstable coronary plaques from sudden death victims. Lp-PLA2 was mostly present in plaque ruptures and thin-cap fibroatheromas in necrotic cores and surrounding areas of apoptotic macrophages. The present study suggests Lp-PLA2 may be an important biomarker for plaque instability.
Key Words: lipoprotein-associated phospholipase A2 • sudden coronary death • plaque rupture • apoptosis • cardiovascular risk
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Introduction |
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The natural history of atherosclerosis in humans is a dynamic process involving the progression of early lesions to more complex plaques that are responsible for the majority of acute ischemic coronary and stroke events. Throughout lesion progression, there are transitional plaque phenotypes ranging from early lipid pools to those characterized by a dense fibrous cap of connective tissue and a strong collagen matrix overlying a core of lipids and necrotic debris, and ultimately, to plaques with large necrotic cores and thin fibrous caps invaded by macrophages, referred to as thin-cap fibroatheromas (TCFAs).1 TCFAs are characterized by a thin fibrous cap (<65 µm), a large necrotic core, an abundance of macrophages, and limited luminal narrowing.2 It is widely held that the instability of the TCFA gives rise to the main clinical complications associated with rupture and thrombosis; however, there are important morphological differences between TCFA and ruptured plaques.2 Ruptured plaques demonstrate even thinner fibrous caps (23±19 µm), larger necrotic cores, and greater macrophage infiltrates compared with TCFAs.2 In this context a better understanding of the biology of rupture-prone plaques has the potential to reduce the morbidity and mortality associated with atherothrombotic disease.
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Inflammation plays a primary role in the progression of human atheroma based on the local and systemic inflammatory responses observed throughout the spectrum of atherosclerotic disease—from initial lesion formation to plaque destabilization and rupture.3 The key role of inflammation in atherosclerosis also is evidenced by numerous epidemiology studies indicating an association between inflammatory markers (eg, C-reactive protein [CRP], interleukin [IL]-6) and risk of future cardiovascular events. However, considerably less is known about whether the various inflammatory markers represent similar pathophysiologic processes or unique biological events, and whether they are causally involved in the atherosclerotic process, or merely a marker that correlates with disease progression. In the absence of a convincing animal model of human plaque rupture, studying the expression of inflammatory markers in various plaque morphologies may help focus future research efforts on specific inflammatory markers of greater relevance to clinical events, rather than atherosclerosis per se.
Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a novel inflammatory marker that has been the recent focus of several epidemiology studies involving populations with varying degrees of baseline risk (ie, primary and secondary prevention populations). Most,4–14 but not all,15 studies have indicated that Lp-PLA2 measured in plasma predicts future cardiovascular events, and the association appears to be independent of traditional cardiovascular risk factors and various other novel inflammatory markers. Previous work using combined in situ hybridization and immunocytochemistry, detected Lp-PLA2 mRNA and protein in macrophages in both human and rabbit noncoronary atherosclerotic lesions. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis indicated an increased expression of Lp-PLA2 mRNA in human atherosclerotic lesions.16 As the majority of events related to CV mortality and morbidity are accompanied by the changes in coronary plaque biology, the purpose of the present study was to explore the localization and distribution of Lp-PLA2 protein in human coronary atheroma.
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Materials and Methods |
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Case Selection
Hearts of patients who had died suddenly of coronary causes were obtained as described previously.17 Cases were identified by the presence of an intracoronary thrombus or at least one major epicardial coronary artery with >75% cross-sectional area luminal narrowing, in the absence of a noncoronary cause of death. Sudden deaths were classified by the presumed mechanism of death: acute thrombus attributable to acute plaque rupture, stable plaque (
75% cross-sectional area luminal narrowing) with healed infarct in the absence of an acute thrombus, and stable plaque without evidence of infarction. In most cases, major epicardial arteries were serially sectioned at 3- to 4-mm intervals, and all segments with 50% cross-sectional luminal narrowing were processed for histology. In a limited number of cases, a complete sampling of epicardial coronary arteries was performed, irrespective of the severity of cross-sectional luminal narrowing. A dataset of 72 hearts to include 488 paraffin sections was available with measurements of vessel size, luminal stenosis, necrotic core area, and macrophage density; parts of this data were published previously.18 Immunohistochemical assessment of Lp-PLA2 was performed in 25 prospective cases where 60 frozen (OCT embedded) coronary segments were available. Of these, 30 coronary segments were chosen to match the distribution of the various parameters of the larger sampling of paraffin-embedded tissues. Only cryosections were selected for detection of Lp-PLA2 study because standard techniques of antigen retrieval failed to produce consistent results in paraffin sections.
Classification of Lesions
A simplified scheme developed by our laboratory modified from the current American Heart Association recommendations was used to classify atherosclerotic lesions.1 Coronary plaques were characterized as pathologic intimal thickening, fibroatheromas, thin-cap fibroatheromas, and plaque rupture.
Histological Preparation
Coronary segments at 3- to 4-mm thickness were either fixed in 10% neutral buffered formalin or frozen for cryosectioning. Tissue sections were cut at 6-µm, mounted on charged slides, and stained with hematoxylin and eosin and by the modified Movat pentachrome method. Unstained cyrosections for immunohistochemical markers were cut and stored at –80°C until use.
Immunohistochemistry
Serial cryostat sections were thawed, fixed in 10% neutral buffered formalin, air-dried, and stained with antibodies specific for smooth muscle 
-actin (clone 1A4, Sigma), macrophage marker CD68 (KP-1 clone, Dako, Carpinteria, Calif), and the endothelial marker (von Willebrand factor [vWF]). Immunohistochemical detection of Lp-PLA2 was performed using a monoclonal antibody clone 4B4 (diaDexus, South San Francisco, Calif). The preparation and specificity of the anti–Lp-PLA2 antibody has been described in detail elsewhere.19 All primary antibodies were labeled with a biotinylated link antibody directed against mouse antigen with the use of a peroxidase-based kit (LSAB, Dako) and visualized with the use of a 3-amino-9-ethylcarbazole substrate; the sections were counterstained with Gill hematoxylin (Sigma-Aldrich). Omission of a primary antibody served as a specificity control.
Measurement of Apoptosis
Apoptotic nuclei were visualized by in situ end labeling (ISEL) DNA fragmentation staining using terminal deoxyribonucleotide transferase (TdT)-mediated nick end-labeling (TACS; Trevigen, Gaithersburg, Md) according to previously published methods.20 Only positive nuclei with morphological features of apoptotic cell death (cell shrinkage, aggregation of chromatin into dense masses, and cell fragmentation) were counted. Overall apoptosis data for coronary lesions were obtained by counting total and ISEL-positive nuclei in macrophages (CD-68+)/Lp-PLA2-rich areas in 3 different high power fields (x400 magnification) and related to the total cell number.
Apoptosis and Lp-PLA2 Expression
Colocalization of apoptosis in cells expressing Lp-PLA2 was assessed by combined ISEL and immunohistochemistry. Tissue sections were initially stained for DNA fragmentation (as described above) substituting diaminobenzidine (DAB) as the chromogenic substrate with enhancement by nickel salts (brown-black reaction product). Immunostaining of Lp-PLA2 was visualized with a red streptavidin-alkaline phosphatase substrate (Vector, Burlingame, Calif); slides were counterstained with methyl green.
Evaluation of Coronary Lesions
Morphometric measurements of coronary sections were performed using image-processing software (IPLabs, Scanalytics, Rockville, Md) on slides stained with Movat Pentachrome. Quantitative planimetery included areas analysis of the internal elastic lamina (IEL), lumen, and necrotic core size. The percent stenosis was derived from the formula (1-lumen area/IEL area) x 100. In cases with acute plaque rupture, the area of the thrombus was not included for the calculation of percent stenosis. Computer-assisted color image analysis segmentation with background correction was used to quantify immunohistochemical stains of macrophages, smooth muscle cells, and Lp-PLA2 within regions of interest. The percentage of positive staining as a function of total plaque area was determined.
Statistical Analysis
Values are expressed as mean±SD. Mean variables between the various stents were compared with the one-way analysis of variance (ANOVA) (JMP software, Cary, NC) followed by all pairs Tukey-HSD test for all differences among means. A value of P
0.05 was considered statistically significant.
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Results |
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Histomorphometric Analysis
A total of 72 sudden coronary death cases (mean age 53±12 years) with formalin-fixed coronary arteries 488 segments with varying lesions were studied (Table 1). Sudden death secondary to plaque rupture and thrombosis occurred in 55 cases (76%) with the remaining cases (24%) associated with severe luminal narrowing. A summary of histomorphometric measurements including IEL, percent stenosis, and macrophage density in various lesion morphologies is shown in Table 2. Coronary artery cross-sectional area was significantly larger in thin-cap fibroatheromas and acute ruptures relative to lesions with pathologic intimal thickening and fibroatheromas, which were comparable in size. Similarly there were no differences in overall lesion size between plaques with thin caps and ruptures. Together with the change in positive remodeling, both thin-cap fibroatheromas and acute ruptures showed greater percent stenosis, necrotic core area, and overall percentage of lesional macrophages when compared with lesions with pathological intimal thickening and fibroatheromas with macrophage densities highest in ruptured plaques.
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An additional 25 cases with 30 OCT embedded coronary segments were prospectively collected for immunolocalization of Lp-PLA2. The mean patient age was 48.0±10.2 years with 22 men and 5 women (Table 1). The cause of death was attributed to acute plaque rupture in 7 cases with stable plaque the presumed mechanism of death in the remaining 20 patients. The selected lesion morphologies included pathologic intimal thickening (n=7), fibroatheroma (n=8), thin-cap fibroatheroma (n=8), and plaque rupture (n=7). As shown in Tables 2 and 3
, the smaller subset of lesions selected for Lp-PLA2 staining from 25 patients was comparable with that of the larger 72 patient series based on consistent histomorphometric data.
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The expression of Lp-PLA2 was relatively low or absent in early lesions defined as pathological intimal thickening (Table 4 and Figure 1). In particular, lesions of pathologic intimal thickening showed little Lp-PLA2 within lipid pools with only occasional staining of surrounding macrophages. Similarly, Lp-PLA2 staining of fibroatheromas generally showed weak staining of necrotic cores and surrounding macrophages such that the percentage of plaque staining for Lp-PLA2 was <3%. In contrast, thin-cap fibroatheromas and ruptured plaques showed intense Lp-PLA2 expression within the necrotic core and surrounding macrophages including those in the fibrous cap (Table 4 and Figure 1). Further, the percentage of plaque positively stained for Lp-PLA2 was significantly greater in lesions with rupture (15.3±11.1%) compared with other lesion morphologies (TFCA=7.1± 4.2%, FA=1.7±3.1%, and PIT=0.3±0.5%; P<0.002). Although the majority of Lp-PLA2 reactivity was found in macrophages, occasional smooth muscle cells also showed positive staining while endothelial cells were generally negative.
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In situ end labeling (ISEL) as a marker of apoptotic cell death was present to a greater degree in lesions classified as thin-cap fibroatheroma or ruptured plaques compared with fibroatheromas or arteries with pathologic intimal thickening (Table 3 and Figure 2). Quantitative analysis of cell types undergoing apoptosis revealed an increased susceptibility of macrophages to apoptosis, whereas detection of apoptotic nuclei in smooth muscle cells was generally low in all lesions types; occasional cells were negative for both markers. Apoptotic indices were significantly greater in plaque ruptures (30.6±15.7%) compared with lipid pool lesions referred to as pathologic intimal thickening (8.4±4.4, P<0.02). Further, double-labeling studies revealed colocalization of Lp-PLA2 in cells undergoing apoptosis in regions showing a relatively high density of macrophages (Figure 2).
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Discussion |
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The present findings indicate an association of Lp-PLA2 expression in advanced ruptured and rupture-prone lesions we designate as thin-cap fibroatheromas. Immunostaining of lesions defined as pathologic intimal thickening or fibroatheromas showed only minimal reactivity to Lp-PLA2 and when present, was mostly localized to the lipid pool or necrotic core, respectively. In contrast, thin-cap fibroatheromas and ruptured plaques showed extensive Lp-PLA2 accumulation closely associated within the areas of the necrotic core and surrounding macrophages including those in the fibrous cap. Although Lp-PLA2 was prominent in macrophages of advanced lesion, there was minimal expression in smooth muscle cells. Double staining experiments showed localization of Lp-PLA2 within apoptotic macrophages suggesting that its products either represent a marker of apoptosis or they might play a causal role in the induction of cell death. Together, these findings suggest that Lp-PLA2 may be closely linked with the progression and vulnerability of human coronary atheroma. Our findings are an important extension of an earlier study,16 in which the expression of Lp-PLA2 (mRNA and protein) was investigated in human aortic lesions. As in the present study, Lp-PLA2 protein expression was greatest in advanced lesions, and simultaneous in situ hybridization and immunostaining identified macrophages as the primary source of Lp-PLA2 secretion within these plaques.
The biologic role of Lp-PLA2 in human atherosclerosis has not yet been definitively established, which is attributable, in part, to the proposed differences in atherogenic potential between the substrate and products of the enzymatic activity of this phospholipase. Initial reports ascribed atheroprotective characteristics to Lp-PLA2, proposing that the enzyme is involved in hydrolyzing, and thereby inactivating, noxious polar phospholipids (ie, oxidized phosphatidylcholines in modified LDL) that may be responsible for promoting inflammation and atherosclerosis.21–23 More recently, it has been proposed that the hydrolysis of polar phospholipids by Lp-PLA2 generates large amounts of two downstream inflammatory mediators—lysophosphatidylcholine (lysoPC) and oxidized fatty acids—which elicit proinflammatory responses (ie, activation) from various cell types involved in atherosclerosis.24 The specific mechanism that predominates in human atherosclerosis is not fully known; however, the findings from the present study add to accumulating epidemiology data supporting a proatherogenic role for Lp-PLA2.
Recently, there has been considerable interest in identifying new cardiovascular risk markers to help refine risk assessment and guide treatment decisions prior to those subjects experiencing ischemia or necrosis from acute coronary syndromes. An interesting question that requires additional research is whether differences exist between specific markers of plaque vulnerability and general markers of systemic inflammation. Although subtle, the differences may lie in the biologic plausibility (ie, causally involved) and specificity to the vascular inflammatory processes (versus systemic response). Examples of biomarkers with specificity toward vulnerable plaque include CRP, matrix metalloproteinases (MMPs), zinc peptidases (eg, pregnancy-associated plasma protein A [PAPP-A]), CD40 ligand, and myeloperoxidase. CD40 signaling, MMP-9, myeloperoxidase, and PAPP-A are plausible markers of plaque vulnerability because of their relationship with macrophages and fibrous cap integrity.25–29 However, some (eg, myeloperoxidase, soluble CD40 ligand) are not specific to the vascular inflammatory processes. For other risk markers, such as CRP—arguably the most extensively studied of the inflammatory cardiovascular risk markers—its role in plaque vulnerability is less clear. Although numerous studies consistently indicate that adverse cardiovascular outcomes are associated with elevated plasma CRP levels, there is debate as to whether CRP is an active mediator of inflammation and atherosclerosis within the arterial vascular wall.30 CRP has been detected in macrophages and within the lipid core in advanced coronary atheroma31–33; however, whether these findings reflect systemic (ie, liver) production and local uptake versus local production is not clear.34
Given the epidemiologic association between plasma Lp-PLA2 and cardiovascular events in several studies and the findings from the present study, Lp-PLA2 may be another important marker, and possibly a mediator, of plaque progression and vulnerability. The observation that Lp-PLA2 staining in this study was most intense in regions that are abundant in lipids and oxidation products (ie, necrotic core) is consistent with the putative role of this enzyme. The expansion of the necrotic core is considered to be an important step in the progression toward plaque vulnerability and may be correlated with macrophage apoptosis. Oxidized LDL (ie, the substrate for Lp-PLA2 hydrolytic activity) and lysoPC (ie, the product of Lp-PLA2 hydrolytic activity) both have been associated with proapoptotic effects on macrophages.35,36 Interestingly, in studies involving cultured human leukocytes, the addition of selective Lp-PLA2 inhibitors to LDL before copper-induced oxidation decreased lysoPC production, reduced proinflammatory and cytotoxic effects elicited by oxidized LDL.35,37 Although the precise signaling mechanism(s) capable of exerting proapoptotic effects of lysoPC are unclear, the naturally occurring Fas-FasL induction of caspase-3 cleavage may represent a viable pathway.38
In humans, Lp-PLA2 in circulation is bound predominantly to low-density lipoprotein particles (
80%), with the rest distributed among high-density lipoprotein and remnant-lipoproteins.39,40 However, Lp-PLA2 is also produced de novo within the atheroma by the same inflammatory infiltrate that is considered to be responsible for driving the inflammatory responses (ie, macrophages, T-lymphocytes, etc).41,42 Regardless of how the enzyme reaches the intima (via LDL or de novo production), its substrates (ie, polar phospholipids) are abundant within the atheroma, either from modified LDL or from apoptotic/necrotic macrophages.43 In fact, in advanced lesions, the toxic effects of lysoPC on macrophage apoptosis may ultimately result in a feedback cycle of increased lysoPC generation and apoptosis.44 Another finding suggestive of a causal role for Lp-PLA2 in atherosclerosis is that fibrous cap thickness is determined, in part, by the extent of macrophage infiltrate within the fibrous cap region. The products of Lp-PLA2 activity (ie, lysoPC and oxidized fatty acids) are chemoattractants for circulating monocytes, and they are also involved in macrophage activation.24,36,37 The potential role of macrophages in fibrous cap thickness and necrotic core expansion, together with relationship between macrophages and Lp-PLA2 expression in the fibrous cap region may indicate that Lp-PLA2 is involved in plaque vulnerability, particularly in the progression from TCFA to plaque rupture. As such, Lp-PLA2 hydrolytic activity may represent an important biologic pathway confirming that TCFAs are indeed the precursor lesion to ruptured plaques. Although the findings from the present study are intriguing and suggest biologic plausibility and specificity to vascular inflammation, definitively demonstrating a causal role of Lp-PLA2 for the progression atherosclerosis and plaque vulnerability will likely require additional research beyond necropsy studies.
In summary, this is the first study to characterize the expression of Lp-PLA2 protein within human coronary atheroma of various morphologies or phenotypes. Lp-PLA2 was expressed by macrophages within the fibrous cap region of rupture-prone and ruptured lesions, and Lp-PLA2 staining colocalized with apoptotic macrophages. In advanced lesions, Lp-PLA2 staining also was intense in regions abundant in lipids and oxidation products (eg, necrotic core). Based on these findings, Lp-PLA2 and the products of its enzymatic activity may play an expanded role in promoting plaque instability, and additional studies are warranted to explore the potential causal role for Lp-PLA2 in plaque progression and vulnerability.
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Acknowledgments |
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We thank Lila Adams (CVPath, International Registry of Pathology) and Dr Wenhui Liu (diaDexus) for their valuable technical assistance.
Sources of Funding
This work was supported in part by a research grant from the National Institutes of Health (R01 HL61799-02, to R.V.) and through funds from diaDexus.
Disclosures
R.M. received company sponsored research support from Medtronic AVE, Guidant, Abbott, W.L. Gore, General Electric Health, diaDexus, GlaxoSmithKline, Takeda, Atrium Medical Corporation, Invatec, ev3, TopSpin Medical (Israel) Ltd, Boston Scientific, NDC Cordis Corporation, Paracor Medical Inc, C.R. Bard Inc, Orbus Medical Technologies Inc, and Alchimer S.A. R.M. is a consultant for Atrium Medical Corporation, Medtronic AVE, Guidant, Abbott Laboratories, W.L. Gore, Terumo, TopSpin Medical (Israel) Ltd, InFlow Diagnostic, Prescient Medical, CryoVascular Systems Inc, and Volcano Therapeutics Inc.
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Footnotes |
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Original received July 7, 2006; final version accepted August 25, 2006.
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