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From the Department of Internal Medicine, University of Kentucky Medical Center, Lexington, Kentucky.
ABSTRACT
Objectives— Secretory phospholipase A2 (sPLA2) enzymes hydrolyze the sn-2 fatty acyl ester bond of phospholipids to produce a free fatty acid and a lysophospholid. Group V sPLA2 is expressed in cultured macrophage cells and has high affinity for phosphatidyl choline-containing substrates. The present study assesses the presence of group V sPLA2 in human and mouse atherosclerotic lesions and its activity toward low-density lipoprotein (LDL) particles.
Methods and Results— Group V sPLA2 was detected in human and mouse atherosclerotic lesions by immunohistochemical staining. Electron microscopic analysis showed that mouse group V sPLA2-modified LDL is significantly smaller (mean diameter±SEM=25.3±0.25 nm) than native LDL (mean diameter±SEM=27.7±0.29 nm). Hydrolysis by group V sPLA2 induced spontaneous particle aggregation; the extent of aggregation was directly proportional to the degree of LDL hydrolysis. Group V sPLA2 modification of LDL led to enhanced lipid accumulation in cultured mouse peritoneal macrophage cells.
Conclusions— Group V sPLA2 may play an important role in promoting atherosclerotic lesion development by modifying LDL particles in the arterial wall, thereby enhancing particle aggregation, retention, and macrophage uptake.
Key Words: atherosclerosis ? group V secretory phospholipase A2 ? LDL aggregation ? macrophages
Introduction
A critical event in early atherogenesis is the retention of low-density lipoprotein (LDL) particles in the subendothelium. Accumulating evidence points to LDL aggregation and LDL fusion as key elements of atherogenic lipid accumulation in the artery wall.1 Aggregated lipoproteins that appear to be derived from LDL are prominent in early atherosclerotic lesions.2,3 Aggregated LDL is taken-up by macrophages in vitro at an enhanced rate compared with non-aggregated LDL, leading to macrophage cholesterol accumulation and foam cell formation.4,5 Because native LDL particles do not form aggregates, LDL modification appears to be a prerequisite for aggregation/fusion. Studies in vitro indicate that hydrolysis of LDL by secretory phospholipases A2 (sPLA2) may be linked to LDL aggregation and/or fusion and enhanced retention in the subendothelium.3,6
The sPLA2 family comprises a group of enzymes that hydrolyze the fatty acid esterified at the sn-2 position of glycerophospholipids.7 The secreted enzymes are of low molecular weight (14 kDa), highly enriched in disulfide bonds, and require 1 to 10 mmol/L calcium for activity. The major secreted form present in synovial fluid, termed group IIa, has been proposed to act as a mediator of inflammatory responses. During acute or chronic inflammation, the concentration of group IIa sPLA2 can increase by >100-fold in inflammatory fluids and plasma.8,9 Immunohistochemistry studies have established that group IIa sPLA2 is present in normal arterial tissue and increased in atherosclerotic lesions.10–12 We recently reported that macrophage expression of human group IIa sPLA2 significantly enhances atherosclerotic lesion formation in LDL receptor-deficient mice.13 These studies were performed in the C57BL/6 background, a strain of mice that lack the expression of functional group IIa protein because of a frame shift mutation in exon 3.14
A distinct sPLA2 (group V sPLA2) has been identified in mouse tissues.15 Like the group IIa enzyme, group V sPLA2 is induced by proinflammatory stimuli.15 Group V sPLA2 contains amino acid variations in regions that have been shown to be important for the interfacial binding of other sPLA2.16 Binding studies using phosphatidylcholine (PC)-coated hydrophobic beads demonstrate that human group V sPLA2 binds PC membranes more than 50-times more tightly than human group IIa sPLA2.17,18 In a recent report, group V sPLA2 was shown to be 30-fold more active in hydrolyzing LDL-PC compared with group IIa sPLA2.19
Given the potency of group V sPLA2 in hydrolyzing LDL particles, it has been suggested that this enzyme could promote atherosclerotic lipid accumulation by modifying LDL particles retained in arterial tissues.6,20,21 In the current study, we establish that group V sPLA2 can be detected in human and mouse atherosclerotic lesions. We also demonstrate that LDL particles hydrolyzed by group V sPLA2 are significantly smaller compared with native LDL and are susceptible to spontaneous aggregation. These group V sPLA2-modified particles promote foam cell formation in cultured mouse peritoneal macrophages.
Methods
Immunohistochemistry
Mouse anti-human group V sPLA2 was purchased from Cayman Chemicals (Ann Arbor, Mich). Rabbit anti-mouse group V sPLA2 was raised against residues 9 to 113 expressed in bacteria using the pET system (Novagen, Madison, Wisc). Chicken anti-mouse group V sPLA2 was raised against the synthetic peptide ELKSMIEKVTRKNAFKNY, a region of group V sPLA2 that has no sequence homology with group X sPLA2. For immunostaining, the rabbit and chicken antibodies were eluted from an affinity column conjugated to the corresponding antigen. Anti-human CD68 (macrophage marker) antibody and mouse macrophage-specific antisera were obtained from Dako and Accurate, respectively. Human pathological samples were obtained from subjects in studies approved by the University of Kentucky Institutional Review Board and with informed consent. Formalin-fixed human aortic tissue was embedded in paraffin for sectioning. Sections on glass slides were deparaffinized using EZ-Dewax solution (Biogenex, San Ramon, Calif). Antigen retrieval was performed using an antigen retrieval solution (Dako) and blocked for 30 minutes with 10% mouse serum. Hearts were obtained from female apoE-deficient or male LDL receptor-deficient mice (C57BL/6 background, Jackson Laboratory) fed a diet enriched with 21% fat and 0.15% cholesterol (wt/wt) or 7.5% fat and 1.25% cholesterol, respectively, for 15 weeks. Immunohistochemistry was performed on acetone-fixed, frozen sections of the aortic root as described previously.13 All animal procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee.
Expression of Mouse Group V sPLA2 by Adenoviral Vector
A replication-defective adenoviral vector encoding mouse group V sPLA2 (AdmGV) was generated using the AdEasy system.22 To express group V sPLA2 in vitro, COS-7 cells were incubated with AdmGV at a multiplicity of infection of 4000 particles per cell. To prepare partially purified enzyme, conditioned media was collected 48 hours after infection and sequentially chromatographed on Hi-Trap SP ion exchange and Hi-Trap heparin columns (Amersham Biosciences) as previously described for group IIa sPLA2.23
Phospholipase Assays Using Synthetic Micelles as Substrate
Phospholipase activity was measured using a colorimetric assay.24 Mixed micelles were prepared by warming to 37°C 7 mg of either 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) (Avanti Chemicals, Alabaster, Ala) with a 0.2 mL mixture of 4.0% (wt/vol) Nonidet-40 and 2.0% sodium deoxycholate, and then adding 1.8 mL warm assay buffer (0.12 mol/L Tris-HCl pH 8, 12 mmol/L CaCl2, 0.1 mmol/L EDTA). For enzyme assays, 10 μL of mouse group V sPLA2 (diluted in 0.1 mol/L HEPES, 0.1 mol/L NaCl, 1 mmol/L CaCl2, 1 mg/mL fatty acid-free BSA) was added to 50 μL of substrate solution in a 96-well plate pre-coated with gelatin. After incubating at 37°C, the amount of free fatty acids (FFA) released was quantified using a NEFA-C kit (Wako Chemicals). Phospholipase activity of conditioned media from AdmGV-treated COS-7 cells and partially purified group V sPLA2 were 500 to 750 U/mL and 2200 to 10 000 U/mL, respectively (1 unit generates 1 nmol of FFA in 20 minutes using POPG as substrate).
LDL Hydrolysis by Mouse Group V sPLA2
LDL (d=1.019 to 1.063 g/mL) was isolated from human plasma by density gradient ultracentrifugation. For some experiments, group V sPLA2 hydrolysis of LDL was performed by adding LDL to cultures of COS-7 cells 24 hours after treatment with AdmGV. Alternatively, LDL was incubated for 4 to 24 hours at 37°C with varying amounts of partially purified enzyme in 1X LDL hydrolysis buffer (6 mmol/L HEPES, 6 mmol/L CaCl2, 84 mmol/L NaCl, 20 mg/mL BSA, and 2.4 mmol/L MgCl2). Reaction mixtures were stopped by the addition of EDTA (10 mmol/L final concentration). The extent of phospholipid hydrolysis was determined by measuring the amount of FFA liberated in the reaction by colorimetric assay (Wako Chemicals). The degree of aggregation/fusion of LDL particles was determined by separating unfiltered samples of LDL (75 μg) on a Superose 6 column (Pharmacia LKB Biotechnology Inc.). For electron microscopy, LDL preparations were stained with 2% uranyl acetate solution and then viewed and photographed in a Philips Tecnai 12 TEM at the Electron Microscopy and Imaging Facility, University of Kentucky.
Oil Red O Staining
Cells were harvested from the peritoneal cavity of C57BL/6 mice 4 days after intraperitoneal injection of 1% Biogel polyacrylamide beads (1 mL). The cells were incubated for 16 hours with DMEM supplemented with 10% fetal bovine serum and 1000 IU/mL M-CSF before incubation with LDL. For preparation of group V sPLA2-modified LDL, 2 mg LDL was incubated for 24 hours with 1000 U of partially purified enzyme in 1 mL 1X LDL hydrolysis buffer. For control, LDL was incubated similarly without added enzyme. To prepare aggregated LDL produced by vortexing, LDL in 1X LDL hydrolysis buffer (2 mg/mL) was placed in a polystyrene tube and vortexed at maximum speed for 1 minute. Peritoneal macrophage cells were incubated with 200 μg/mL LDL for 48 hours at 37°C, washed with PBS, fixed with 10% formalin for 10 minutes, and then washed with 60% isopropanol. Cells were stained with Oil Red O for 30 minutes and then washed with 60% isopropanol.
Results
Immunohistochemical Staining of Group V sPLA2 in Human and Mouse Atherosclerotic Lesions
Previous studies have established that group IIa and group X sPLA2 are present in human and/or mouse atherosclerotic lesions.10–12,25 In this study, we assessed whether group V sPLA2 could also be detected in human and mouse lesions. Immunohistochemical analysis of advanced human atherosclerotic aortic lesions and aortic root sections from apoE-deficient mice fed an atherogenic diet demonstrated the presence of group V sPLA2 (Figure 1B and 1D). The sections stained with control antibodies (mouse- and rabbit-irrelevant IgG) were negative (Figure 1A and 1C). Immunostaining of apoE-deficient mouse lesions with affinity-purified chicken anti-mouse group V sPLA2 IgY produced results similar to those depicted in Figure 1D (data not shown). To more clearly see the distribution of group V sPLA2 in human lesions, please see Figure I (available online at http://atvb. ahajournals.org), which shows staining of the sections with antisera specific for smooth muscle cell actin, macrophages, or group V sPLA2 at lower magnifications. Group V sPLA2 was also detected in lesions of LDL receptor-deficient mice stained with affinity-purified chicken anti-mouse group V sPLA2 (Figure II, available online at http://atvb.ahajournals.org).
Figure 1. Immunohistochemical detection of group V sPLA2 in atherosclerotic lesions. Sections from a human aortic plaque were stained with mouse anti-human IgG (A) or mouse anti-human group V sPLA2 (B). Sections from the aortic sinus region of an atherogenic diet-fed apoE-/- mouse were stained with rabbit anti-mouse IgG (C) or rabbit anti-mouse group V sPLA2 (D). Immunoreactivity was visualized using the brown chromogen diaminobenzidine tetrahydrochloride (human) or the red chromogen aminomethyl carbazole (mouse). Sections were counterstained with hematoxylin. Magnification x100 (human) or x200 (mouse).
Expression of Group V sPLA2 in Cultured Cells
To study group V sPLA2 activity in vitro, a replication-defective adenoviral vector encoding mouse group V sPLA2 (AdmGV) was used to express the enzyme in COS-7 cells. Western blot analysis showed group V sPLA2 expression in cells 48 hours after treatment with AdmGV but not cells treated with a control adenoviral vector (Figure 2A). A single immunoreactive band that migrated similarly with an apparent molecular weight of 14 kDa was also detected in supernatants from AdmGV-treated, but not control, COS-7 cells (not shown). Phospholipase activity secreted by AdmGV-treated cells was quantified using mixed micelles containing either POPC or POPG as substrate. In these assays, phospholipase activity in supernatants collected from control COS-7 cells was <5% of the activity present in supernatants from AdmGV-treated cells. The results from this analysis showed 2-fold difference in specific activity toward these 2 phospholipid substrates (Figure 2B). This contrasts to group IIa sPLA2, which has been shown to be more than 100-fold more active in hydrolyzing PG compared with PC.18
Figure 2. Expression of mouse group V sPLA2 in COS-7 cells by adenoviral vector. Cells and media were collected 48 hours after addition of AdmGV or control virus, Adnull. A, Aliquots of cell lysates (10 μg protein) were separated by SDS-PAGE (15% acrylamide), immunoblotted with rabbit anti-mouse group V sPLA2, and visualized by chemiluminescence detection. The migration of molecular weight standards and 13.6-kDa group V sPLA2 is indicated. B, The indicated amount of partially purified mouse group V sPLA2 was incubated with mixed micelles containing either POPC or POPG. After 4-hour incubations at 37°C, the amount of FFA released was quantified. The results shown are the mean of triplicate determinations (±SD) and are representative of 3 separate experiments.
Group V sPLA2 Hydrolysis of LDL
The ability of group V sPLA2 to hydrolyze PC is notable, given that PC is the major phospholipid component of LDL. Thus, it was of interest to determine whether mouse group V sPLA2-expressing cells have the capacity to hydrolyze LDL. Accordingly, COS-7 cells were treated with AdmGV or control virus, Adnull. Twenty-four hours after adenovirus treatments, cells were incubated for an additional 24 hours with media containing 0.2 mg/mL LDL (0.4 μmol/L), and the extent of LDL hydrolysis was assessed by measuring the FFA content of media. As shown in Figure 3A, a significant amount of FFA was liberated when LDL was added to group V sPLA2-expressing cells but not control cells. The data indicates that 85 molecules of FFA were generated per particle of LDL added. Group V sPLA2 hydrolysis of LDL particles was further analyzed using partially purified enzyme, prepared as described in Methods. At maximal hydrolysis, group V sPLA2 generated 1 nmol FFA per μg LDL (Figure 3B). Assuming 1.4 nmol phospholipid per μg LDL, this represents 70% hydrolysis by group V sPLA2.
Figure 3. Hydrolysis of LDL by mouse group V sPLA2. A, Twenty-four hours after treatment with AdmGV or Adnull, COS-7 cells were incubated an additional 24 hours in the absence or presence of 0.2 mg/mL LDL (0.4 μmol/L), and the FFA content of media was determined. Values shown represent the mean (±SD) of 4 to 5 different experiments. The phospholipase activity in the media at the end of the experiments was 500 to 750 U/mL. B, LDL (300 μg) was incubated for 20 hours at 37°C with the indicated amount of partially purified mouse group V sPLA2, and the amount of FFA liberated in the 600 μL reactions was determined. The data shown are the mean of duplicate determinants and are representative of 3 experiments.
To assess whether modification by group V sPLA2 alters LDL particle size, hydrolyzed particles were analyzed by electron microscopy after negative staining. As shown in Figure 4, moderately hydrolyzed LDL particles (38% phospholipids hydrolyzed) were significantly smaller (mean diameter±SEM=25.3±0.25 nm) compared with native LDL (mean diameter±SEM=27.7±0.29 nm). This finding is significant, because small dense LDL particles have increased affinity for proteoglycans and may be more effectively retained in the vessel wall subendothelium.26,27
Figure 4. Size distribution of LDL particles before and after group V sPLA2 hydrolysis. LDL was incubated with COS-7 cells expressing group V sPLA2 and then re-isolated by density gradient ultracentrifugation (d<1.063). The diameter of >200 randomly selected lipoprotein particles were measured from the electron micrographs using Scion Image software (Scion Corporation, Frederick, Md). Mean diameters of group V sPLA2-modified LDLs were significantly smaller than unmodified LDLs (P<0.001)
Interestingly, electron microscopic analysis of extensively hydrolyzed LDL particles (68% phospholipids hydrolyzed) showed evidence of particle aggregation (Figure 5A, inset). To analyze the extent of aggregation quantitatively, group V sPLA2-modified LDLs were separated by size exclusion chromatography. Aggregated/fused particles elute in the void volume of a Superose 6 gel filtration column. The analysis of maximally modified LDL showed that a large portion (>50%) of the modified LDL was in an aggregated form. To examine the relationship between phospholipid hydrolysis and LDL aggregation, LDLs hydrolyzed to various extents were separated by gel filtration, and the percent of particles that had undergone spontaneous aggregation (defined as the amount of lipoprotein cholesterol eluting in the void volume relative to the total amount of cholesterol eluting from the column) was determined. Correlation analysis showed a significant relationship (r=0.75, P<0.001) between the degree of particle aggregation and the percent of phospholipids hydrolyzed (Figure 5B). Analysis of group V sPLA2-modified LDLs (48% hydrolyzed, 50% aggregated) by SDS-PAGE showed no evidence of apoB-100 degradation (data not shown).
Figure 5. Extent of aggregation of group V sPLA2-modified LDL. A, Aliquots containing 75 μg unmodified LDL or LDL treated with group V sPLA2 (70% phospholipids hydrolyzed) were separated on a Superose 6 column, and the cholesterol content of 0.5 mL fractions was determined. Inset, Negative staining and electron microscopy of group V sPLA2-modified LDL particles. B, Individual preparations of group V sPLA2-hydrolyzed LDLs were analyzed to determine extent of phospholipid hydrolysis and aggregation as described in Methods.
Macrophage Uptake of Group V sPLA2-Modified LDL
Previous studies have shown that aggregated LDL is a potent inducer of macrophage foam cells.4,5 Thus, it was of interest to determine whether group V sPLA2-modified LDL particles that are susceptible to spontaneous aggregation promote foam cell formation in vitro. LDL was incubated at 37°C in the presence or absence of group V sPLA2. Peritoneal macrophages from C57BL/6 mice were incubated with the group V sPLA2-modified LDL (30% hydrolyzed) or "mock" hydrolyzed LDL as described in Methods. For comparison, cells were also incubated with aggregated LDL produced by vortexing. After 48-hour incubations, cells were stained with Oil Red O and analyzed by light microscopy. This analysis clearly showed that cells incubated with vortexed LDL or group V sPLA2-modified LDL accumulated more neutral lipid compared with control cells (Figure 6).
Figure 6. Oil Red O staining of peritoneal macrophages incubated with unmodified and modified LDLs. Magnification x400.
Discussion
In humans, circulating levels of group IIa sPLA2 are an independent risk factor for coronary artery disease and a predictor of cardiovascular events.28 The possibility that sPLA2 plays a role in the pathophysiology of atherosclerosis (and is not merely a predictor of events) is substantiated by the fact that transgenic C57BL/6 mice expressing human group IIa sPLA2 have significantly larger aortic lesions compared with non-transgenic littermates.29 In a recent study using a bone marrow transplantation approach, we determined that macrophage expression of human group IIa sPLA2 significantly enhanced atherosclerotic lesion formation in LDL receptor-deficient mice.13 This finding points to a local pro-atherogenic effect of sPLA2 in the microenvironment of a developing lesion. It is now recognized that other related sPLA2 isozymes in addition to group IIa sPLA2 may play a role in cardiovascular disease.6,20,21,25 In this study, we investigated another member of the sPLA2 family, group V sPLA2. Our data show that this enzyme is present in human and mouse atherosclerotic lesions in regions of lipid accumulation. The source of group V sPLA2 in lesions has not been determined. Group V sPLA2 is expressed by a murine macrophage cell line,30 and it is possible that lesional macrophage cells produce group V sPLA2. We cannot rule out the possibility that other cell types within the vessel wall produce and secrete group V sPLA2. Alternatively, sPLA2 associated with lipoprotein particles may enter the subendothelium and be deposited in the vessel wall at sites of lipid accumulation. Irrespective of the cellular source of group V sPLA2, our data indicate that hydrolysis of LDL by group V sPLA2 promotes spontaneous particle aggregation, a modification that has been associated with atherogenic lipid accumulation in the artery wall.3 In assays in vitro, we demonstrate that group V sPLA2 modification of LDL leads to enhanced lipid accumulation in macrophages. Taken together, our data suggest that group V sPLA2 in the arterial wall may lead to the localized production of aggregated lipoprotein particles, which consequently leads to foam cell formation and enhanced atherosclerosis.
Our analysis of recombinant mouse group V sPLA2 demonstrates that this enzyme, like human group V sPLA2,17,18 hydrolyzes PC-containing substrates, including human LDL. Additional studies have demonstrated that mouse group V sPLA2 is similarly potent in hydrolyzing phospholipids on LDL isolated from LDL receptor-deficient mice and very-low-density lipoprotein isolated from apoE-deficient mice (data not shown). Group V sPLA2 was approximately 2-fold more potent in hydrolyzing PG compared with PC. In contrast, group IIa sPLA2 has been shown to be more than 100-fold more active in hydrolyzing PG compared with PC18 and has relatively low enzymatic activity toward LDL. This difference in activity between the 2 enzymes has been attributed to the presence of tryptophan residues in the interfacial binding region of group V sPLA2 that are absent in group IIa.31
Our data indicate that group V sPLA2-modified LDL undergoes structural alterations that could lead to atherogenic lipid accumulation in the vessel wall. Moderately hydrolyzed LDL particles were significantly smaller compared with native LDL. Because small dense LDL particles have increased affinity for proteoglycans,26,27 group V sPLA2 modification could enhance LDL retention in the vessel wall subendothelium. We also show that hydrolysis of 15% to 20% of the phospholipids on the LDL particle is sufficient to induce particle aggregation, and the extent of aggregation is proportional to the degree of LDL hydrolysis. We are not aware of any other published report showing that hydrolysis by a mammalian sPLA2 leads directly to LDL aggregation. Although human group IIa sPLA2 has been implicated in LDL aggregation/fusion, this activity appears to require LDL binding to proteoglycans, which may itself promote LDL aggregation.32 In a recent study, human group X sPLA2 was shown to have the capacity to hydrolyze virtually all PC molecules on LDL.25 Interestingly, extensive modification by group X sPLA2 was not reported to induce LDL aggregation. This may be caused by the fact that LDL hydrolysis was performed in the presence of 0.0125% BSA. In the absence of lipid-binding proteins, such as albumin, LDL hydrolysis by sPLA2 generates lyso-PC and FFA that accumulate in the LDL particle. In the presence of physiological albumin concentrations, such as in our study, most of the FFA and some of the lyso-PC molecules are transferred from LDL to albumin.33 This can lead to conformational changes in apoB-100 and reorganization of lipids that induce particle aggregation.33,34
Analysis by electron microscopy indicated that group V sPLA2-modified LDL does not undergo a substantial amount of particle fusion. It is possible, however, that within the microenvironment of the vessel wall, group V sPLA2 lipolysis could promote subsequent LDL modification leading to fusion. The extracellular matrix (ECM) may play an important role, both by mediating the retention of LDL particles and by co-localizing group V sPLA2 and its substrate LDL, as has been suggested for group IIa sPLA2.32 Group V sPLA2 exhibits high-affinity binding to proteoglycans that is mediated by a cluster of cationic residues near the C-terminal end.35 Group V sPLA2 may also act in concert with secretory sphingomyelinase (s-SMase) in the vessel wall to produce pro-atherogenic LDL. Treatment of LDL with SMase in vitro induces aggregation and fusion of particles and enhances binding to human aortic proteoglycans.36,37 s-SMase has been detected by immunocytochemistry in human atherosclerotic lesions in association with the ECM.38 Although native LDL in plasma is not hydrolyzed by s-SMase at neutral pH, group IIa sPLA2 hydrolysis of LDL can confer susceptibility to this enzyme at neutral pH.39 Interestingly, in vitro data suggest that SMase modification can alter the susceptibility of LDL particles to group V sPLA2 because progressive depletion of LDL sphingomyelin results in a proportional increase in phospholipid hydrolysis by group V sPLA2.19 Thus, both group IIa and group V sPLA2, together with s-SMase and ECM, may synergistically promote atherosclerosis by enhancing LDL retention, LDL aggregation/fusion, and, ultimately, foam cell formation.
Acknowledgments
The authors thank Dr P. Moreno for providing human aortic tissue sections and Dr A. Daugherty for providing apoE-deficient mouse aortic tissue sections. This work was supported by National Institutes of Health grant HL071098 (to N. R. Webb) and an Atorvastatin Research Award (to N. R. Webb).
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