Published online before print March 20, 2003, doi: 10.1161/01.ATV.0000067429.46333.7B
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© 2003 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Expression of Human Scavenger Receptor B1 on and in Human Platelets
From the First Department of Internal Medicine, Kagawa Medical University, Kagawa, Japan, and Departments of Medicine and Biochemistry and Molecular Biology (N.C.W.W.), Faculty of Medicine, University of Calgary, Alberta, Canada.
Correspondence to Koji Murao, First Department of Internal Medicine, Central Laboratory, Kagawa Medical University, 1750-1, Miki-cho, Kita-gun, Kagawa, 761-0793, Japan. E-mail mkoji{at}kms.ac.jp
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Abstract |
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Objective— The abundance of HDL particles correlates inversely with the incidence of coronary heart disease. The human scavenger receptor B1 (hSR-B1/CLA-1) is a receptor for HDL. Expression of hSR-B1/CLA-1 mRNA and protein in human platelets was determined using reverse transcriptase–polymerase chain reaction and Western blot, respectively. Presence of the protein on the surface of platelets was shown using flow cytometry.
Methods and Results— Immunochemical staining for hSR-B1/CLA-1 showed that it was expressed in megakaryocytes, the platelet precursors of human bone marrow. These findings prompted us to ask whether hSR-B1/CLA-1 was differentially expressed on platelets obtained from patients with atherosclerotic disease compared with those in control subjects. Our findings showed that abundance of hSR-B1/CLA-1 was significantly reduced on the surface of platelets from patients with atherosclerotic disease. The reduced levels of hSR-B1/CLA-1 were associated with increased cholesterol ester content in platelets from patients with atherosclerotic disease compared with control subjects. A negative correlation existed between hSR-B1/CLA-1 expression and platelet aggregation. In summary, our studies show that the HDL receptor hSR-B1/CLA-1 is expressed in platelets and their precursor, the megakaryocyte. The levels of hSR-B1/CLA-1 expression correlate inversely with cholesterol ester content and platelet aggregation.
Conclusion— These findings suggest that determining the level of hSR-B1/CLA-1 expression on the platelets may be a useful clinical marker for atherosclerotic diseases.
Key Words: CLA-1 • scavenger receptor B1 • platelet • reverse cholesterol transport • HDL
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Introduction |
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Coronary atherosclerotic disease (CAD) is the number one cause of premature death in modern societies. This problem usually arises from the deposition of lipids in vessel walls leading to the formation of atheroma that occludes the vessel lumen. HDL particles play a critical role in cholesterol metabolism, because they mediate a normal physiological process, so-called reverse cholesterol transport (RCT).1,2 In this process, HDL particles shuttle cholesterol from extrahepatic tissues to the liver for additional metabolism and excretion.1 Enhanced RCT lowers total body cholesterol and thereby reduces the risk of developing CAD. Thus, the plasma concentration of HDL is inversely related to the incidence of CAD.3
The mouse scavenger receptor B1 (SR-B1) mediates selective uptake of HDL cholesterol ester into transfected Chinese hamster ovary (CHO) cells. This finding provides an important link between a specific cell-surface receptor and a pathway for the uptake of HDL.4 Overexpression of SR-B1 in CHO cells markedly enhances the bidirectional cholesterol flux in these cells.5,6 Furthermore, SR-B1 mRNA is expressed in the thickened intima of atheromatous aorta, thus suggesting a potential role of SR-B1 as a mediator of cholesterol flux in the arterial wall.7,8 In addition, SR-B1 is a member of a family of proteins that includes CD36.9 This protein is an 88-kDa glycoprotein found on the surface of monocytes, endothelial cells, and platelets. CD36 is a receptor for a modified form of LDL.10 Our previous report shows that human SR-B1 hSR-B1/CLA-1, like mouse SR-B1, functions as a receptor for HDL.11,12 Human SR-B1/CLA-1 is also similar to the mouse homologue, because it also mediates selective uptake of cholesterol ester and is expressed in liver plus steroidogenic tissues. These features suggest that hSR-B1/CLA-1 is functionally related to mouse SR-B1. Previous studies showed that human platelets express CD36, and this factor likely plays an important role in platelet aggregation.10 But there are also other studies that suggest a less important role for this factor in platelet function.13,14 However, whether hSR-B1/CLA-1 is expressed or functions in human platelets is not known. In this study, we probed for expression of hSR-B1/CLA-1 in human platelets and tested its role in cholesterol ester accumulation and platelet aggregation.
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Methods |
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Antibodies
To create an antibody directed against the extracellular domain (residues 185 to 300) of CLA-1,9 the corresponding cDNA fragment was amplified from human monocyte-derived THP-1 cells (American Type Culture Collection) cDNA using polymerase chain reaction (PCR). The product of this reaction was inserted into a pGEX-2T vector (Pharmacia). The nucleotide sequence was verified and the peptide was expressed in Escherichia coli. The resulting fusion peptide fused to GST was isolated using glutathione-Sepharose 4B beads (Pharmacia). The bound material was used to generate an antiserum in guinea pigs. The IgG fraction from immunized animals was purified before use in Western blot and FACS analyses. Western blot analysis of proteins extracted from the cells stably expressing CLA-1 showed that the antibody directed against an extracellular portion of the protein recognized a single band with an estimated molecular mass of 83 kDa, as previously described.11 A second antibody, either an HRP-conjugated or a FITC-conjugated goat anti–guinea pig IgG (Sigma), was used in Western blot or flow cytometric analysis, respectively.
Cell Culture
HepG2 cells (obtained from RIKEN CELL BANK, Ibaragi, Japan) were grown in DMEM (Life Technologies) with 10% FCS. Mo7e cells were grown in RPMI 1640 medium (Life Technologies) supplemented with 20% FCS and 5 ng/mL human recombinant GM-CSF (Life Technologies) in a humidified atmosphere containing 5% CO2. Human embryonic kidney HEK 293 cells (American Type Culture Collection) were cultured in DMEM with 10% FCS. HEK 293 cells were transfected with 5 µg of linearized plasmid DNA including hSR-B1/CLA1 cDNA. Stable transfectants were selected by their resistance to G418 sulfate (0.8 mg of active drug per mL), as described previously.11
Isolation of Human Platelets
Platelets were isolated from 11 age- and sex-matched healthy volunteers (average age±SEM, 57.5±7.4; 6 male, 5 female; none of the controls were taking medication) and 11 patients with atherosclerotic disease, including cerebral infarction, ischemic heart disease, and arteriosclerosis obliterans (Table). The study was approved by the institutional review board of the Kagawa Medical University, and informed consent was obtained from all participants before sample collection. Blood samples were collected by venipuncture into plastic tubes containing the anticoagulant acid-citrate-dextrose buffer/sodium EDTA. The platelet-rich plasma was fractionated by centrifugation at 1000g for 10 minutes at room temperature. The platelets were isolated by additional centrifugation of the platelet-rich plasma at 2000g for 10 minutes, and the pellet was washed twice with PBS at 4°C.
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Amplification of CLA-1 cDNA
Total RNA was extracted from isolated platelets or HepG2 cells using a single-step acid guanidinium thiocyanate-phenol-chloroform technique, as described previously.15 The primer sequences for RT-PCR amplification of hSR-B1/CLA-1 mRNA16 were sense primer 5'-ATGATCGTGATGGTGCCGTC-3' and antisense primer 5'-ACTGAACCTGCAGGTGCTGA-3'. Reverse transcriptase (RT)-PCR amplification of ß-actin mRNA was analyzed under identical conditions using the appropriate set of primers.17 CLA-1 expression was determined by PCR analysis of the reverse-transcribed RNA, as described previously.18
Western Blot Analysis
Cells were washed in PBS and lysed in PIRA buffer (10 mmol/L Tris-HCl [pH 7.4], 1% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 mol/L NaCl, 1 mmol/L EDTA, and 10 µg/mL aprotinin). The proteins were resuspended under reducing conditions, and 15 µg was fractionated by size on 7.5% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes for immunoblotting.19 The membranes were blocked overnight at room temperature with 0.1% Tween 20 in PBS (PBS-T) containing anti-hSR-B1/CLA-1 antibody (diluted 1/3000 from whole antiserum)11 or anti–cycliphilin A antibody (Biomol Research, Plymouth Meeting, Pa; diluted 1/1000). These membranes were washed with PBS-T, incubated for 1 hour at room temperature in PBS-T containing horseradish peroxidase–linked anti–guinea pig IgG (diluted 1/3000), and rinsed in PBS-T, and antibody binding was visualized by chemiluminescence detection (ECL, Amersham Corp).
Immunolocalization
Bone marrow was aspirated at the posterior iliac crest from healthy volunteers under local anesthesia using a protocol approved by the institutional review board of the Kagawa Medical University. The cellular component of the sample was obtained by centrifugation, and the cells imbedded in a paraffin block. The slices from the block were fixed in 4% paraformaldehyde, deparaffinized in xylene, and then rehydrated through graded ethanol solutions. To inhibit endogenous peroxidase activity, the processed slices were incubated at room temperature for 15 minutes with methanol containing 3% hydrogen peroxide. Sections were blocked for 60 minutes in 10% normal goat serum (NGS) in PBS and incubated for 2 hours with a guinea pig antibody directed against hSR-B1/CLA-111,20 in 4% NGS in PBS or with preimmune antibody under identical conditions. Each section was rinsed in PBS, incubated for 30 minutes with a biotinylated goat anti–guinea pig IgG (Vectastain Elite Kit, Vector Laboratories, Burlingame, Calif) in 1% NGS in PBS, rinsed in PBS, and incubated with an avidin-biotinylated peroxidase complex (Vectastain Elite Kit) in PBS as suggested by the manufacturer. Antibody binding was visualized with the diaminobenzidine reaction, and sections were counterstained with Mayer’s hematoxylin.
Flow Cytometric Analysis
The washed platelets were resuspended in FACS buffer (0.1% BSA in PBS) at a concentration of 1x107 platelets/mL in the same buffer. These cells were incubated with anti-SR-B1/CLA-1 guinea pig antibody (5:100 dilution) on ice for 1 hour followed by exposure to fluoresce in isothiocyanate (FITC)-conjugated anti–guinea pig IgG antibody (2:100 dilution, IMMUNOTECH, Marseille, France) on ice for 1 hour. Cells were then washed twice with analysis buffer and resuspended in the same buffer. For controls, cells were incubated with FITC-conjugated mouse IgG (DAKO Japan Co. Ltd, Kyoto, Japan). Platelets were also incubated with phycoerythrin (PE) conjugated anti-CD41 antibody (DAKO). Fluorescent-positive cells were analyzed on a FACScan (Becton Dickinson).
Platelet Aggregometry
Venous blood was collected in tubes containing sodium citrate and then centrifuged at 1500g for 10 minutes to obtain platelet-rich plasma (PRP). PRP aggregation was simultaneously determined by measuring the maximum percent decrease in optical density (OD) and laser light scatter (LS) intensity using an aggregometer, PA-200 (Kowa). ADP 100 nmol/L was used as an agonist for platelet aggregation and added to PRP 60 seconds after the start of measurement. The principles of the LS method have been described previously.21 This method is based on the fact that the intensity of scattered light emitted from a particle increases in proportion to the square of its diameter. Particles with an intensity of 25 to 400 mV represented small aggregates (9 to 25 µm), those with an intensity of 400 to 1000 mV represented medium aggregates (25 to 50 µm), and those with an intensity of 1000 to 2048 mV represented large aggregates (50 to 70 µm). Small aggregates usually contain approximately 70 to 1400 platelets. Generally, aggregates smaller than 10 µm are found in the early phase of aggregation.19 Quantitative estimation was performed by determining the area under the curve from the sum of 30 measurements of the LS intensity. Data arising from this analysis cannot detect the any aggregation in response to 10 nmol/L ADP in normal or patient-derived platelets.
Other Assays
Cellular cholesterol was measured using a highly sensitive fluorometric method, as described previously.22 Protein was determined by the method of Lowry et al.23
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Results |
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Presence of hSRB1/CLA-1 Transcript in the Megakaryocytic Cell Line and Human Platelets
Previous studies showed that CD36 is found on the surface of platelets,10 and its presence affects platelet function. CD36 belongs to a family of proteins including hSR-B1/CLA-1. Whether hSR-B1/CLA-1 is present on platelets is not known. To answer this question, we probed for expression of hSR-B1/CLA-1 in human platelets and the megakaryocytic cell line, Mo7e. RT-PCR was used to probe for hSR-B1/CLA-1 mRNA in total RNA extracted from human platelets, Mo7e, and the positive control Hep G2 cells.24 The results revealed a single RT-PCR product of the expected size of 930 bp (Figure 1A).
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Next we used Western blot analysis to determine whether hSR-B1/CLA-1 is present in extract from cells of interest and control cells. Cell extract probed with the antibody directed against hSR-B1/CLA-1 revealed a single band in all cells examined. Extract from human platelets and Mo7e cells had a single band of approximately 83 kDa. The mass of this protein matched the expected MW of the hSR-B1/CLA-1 protein (Figure 1B). However, mock transfected HEK 293 cells also contained detectable hSR-B1/CLA-1 protein, but this signal was much lower than that in the same cells stably transfected with the hSR-B1/CLA-1 cDNA. As expected, the positive control Hep G2 cells had abundant expression of hSR-B1/CLA-1. These findings indicate that both hSR-B1/CLA-1 mRNA and protein are present in human platelets and Mo7e cells.
HSR-B1/CLA-1 Is Present in Megakaryocytes
The distribution of hSR-B1/CLA-1 protein in adult human bone marrow cells was determined by immunostaining. Results arising from use of the hSR-B1/CLA-1 antibody showed that immunoreactivity was highest in megakaryocytes (Figures 2A and 2B). That this finding was specific is supported by lack of signal in megakaryocytes after use of nonspecific IgG to stain the cells (Figures 2C and 2D). Additionally, all other hematopoietic cells in the marrow showed some reactivity to the hSR-B1/CLA-1 antibody. Together, these data suggest that expression of hSR-B1/CLA-1 is highest in megakaryocytes, with low levels evident in other cells of the bone marrow.
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Expression of hSR-B1/CLA-1 Protein on the Surface of Human Platelets
The antibody against hSR-B1/CLA-1 was used to examine whether hSR-B1/CLA-1 protein could be detected on the surface of the platelets. For these studies, we used multiparameter flow cytometric analysis. Freshly isolated platelets were doubly labeled with a PE-anti-CD41 monoclonal antibody and FITC-anti–guinea pig IgG monoclonal antibody to detect anti-hSR-B1/CLA-1 antibody. Results showed that hSR-B1/CLA-1 was expressed in approximately 30% of CD41-positive platelets (Figure 3).
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Next we probed for differential levels of hSR-B1/CLA-1 on platelets from patients with atherosclerotic disease compared with controls. A representative profile of hSR-B1/CLA-1 surface expression on the platelets from a healthy individual was compared with that of a patient with atherosclerotic disease (Figure 4A). The presence of hSR-B1/CLA-1 on the surface of platelets from the patient was clearly decreased compared with that of a subject without disease. This finding was confirmed using Western blot analysis. Results in Figure 4B show that abundance of hSR-B1/CLA-1 protein from the patient was decreased compared with that in a control patient. Together, these studies show that hSR-B1/CLA-1 expression in platelets is decreased in patients with atherosclerotic disease compared with the control group (Figure 5).
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, healthy control subjects (n=11); •, patients with atherosclerotic disease (n=11). Each bar and large circle show the mean±SE of each group. *Difference as defined by P<0.005.
Abundance of hSR-B1/CLA-1 Correlates With Cholesterol Ester Content and Platelet Aggregation
The preceding results show the presence of hSR-B1/CLA-1 in human platelets, but the functional significance of this observation is not known. Therefore, we asked whether the presence of hSR-B1/CLA-1 correlated with its function in cholesterol metabolism and platelet aggregation. Platelets were isolated from healthy volunteers and patients with atherosclerotic disease. The abundance of hSR-B1/CLA-1 protein was correlated with plasma levels of HDL (Figure 6).
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Next, we measured the content of cholesterol ester in platelets using an ultrasensitive method, as described previously.22 Results showed that abundance of hSR-B1/CLA-1 on the surface of the platelets correlated negatively with accumulation of cholesterol ester in the platelets (Figure 6). In patients with atherosclerotic disease, the content of cholesterol ester in platelet cholesterol ester/platelet total cholesterol (PCE/PTC) (control, 16.1±7.1%; patient, 7.9±15%; mean±SE; P<0.0001) and abundance of hSR-B1/CLA-1 on the surface of the platelets (Figure 5) was significantly increased and decreased, respectively.
Whether differences of hSR-B1/CLA-1 on the surface of the platelets correlated with function was tested using laser LS, a measure of platelet aggregation. A representative profile of platelet aggregation using LS appears in Figure 4C. Platelet aggregation over time leads to a change in both OD and laser light scattering intensity. This technique enables the detection of small, medium, and large aggregates of platelets. In the absence of ADP, aggregation of the platelets from both patients with atherosclerotic disease and controls was the same. In contrast, formation of small aggregates after stimulation with 100 nmol/L ADP was significantly higher in patients with atherosclerotic disease compared with the controls (P<0.05). Another way to view this finding is that aggregation of platelets with low levels of hSR-B1/CLA-1 was high, thus underlining the inverse relationship between hSR-B1/CLA-1 expression and function of the platelet. However, accumulation of cholesterol ester and platelet aggregation did not significantly correlate with plasma cholesterol, HDL, LDL, or triglycerides (data not shown). These results suggest that abundance of hSR-B1/CLA-1 protein correlates inversely with both cholesterol ester content in and ADP-stimulated aggregation of the platelets, two parameters that reflect function of the platelets.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this study, we examined the expression of hSR-B1/CLA-1 in human platelets and megakaryocytes. The results of our studies show that platelets and its precursor, the megakaryocyte, expressed hSR-B1/CLA-1 mRNA and the protein. Abundance of the hSR-B1/CLA-1 protein on human platelets correlated with serum levels of HDL (Figure 6). In contrast, hSR-B1/CLA-1 expression was negatively correlated with cholesterol ester content in the platelets (Figure 6). The significance of this inverse relationship was evident in patients with atherosclerotic disease. In these patients, decreased expression of hSR-B1/CLA-1 correlated inversely with the content of cholesterol ester in platelets and their ability to aggregate. These two parameters were significantly increased (Figure 6) in platelets with low levels of hSR-B1/CLA-1. Together, the findings reported here suggest an important relationship between hSR-B1/CLA-1 expression and the function of human platelets. The possibility that a deficiency in the platelet levels of CLA-1 may arise from a generalized decrease in the levels of this protein cannot be excluded at this point. Many factors, including hormones (ACTH and estrogen), lipids (cholesterol, oxidized LDL, and polyunsaturated fatty acids), and other agents (PPAR agonist and vitamin E) modulate the expression of hSR-B1/CLA-1.25 These findings limit our ability to make general comments, because a few study patients were treated with medications that may affect expression. Expanded clinical studies will be needed to clarify the role of hSR-B1/CLA-1 in platelets.
How do we relate the changes in hSR-B1/CLA-1 expression with cholesterol metabolism? The present belief is that the protective effect of HDL comes from its participation in RCT. This is a normal physiological process whereby cholesterol from cells in the arterial wall may be transported on HDL particles and shuttled to the liver for additional metabolism and disposal.1,2 Efflux of cellular free cholesterol from peripheral cells to the acceptor HDL particles is the first step of this process. Several reports have suggested that rodent SR-B1, a protein that is functionally related to hSR-B1/CLA-1, is an attractive candidate receptor that selectively takes up HDL cholesterol ester.4–6 SR-B1 is believed to play an important role as a docking receptor for HDL in connection with selective uptake of cholesterol esters.26,27 It is tempting to speculate that decreased levels of hSR-B1/CLA-1 in platelets, isolated from patients with atherosclerotic disease, reflect a decrease in RCT and thus enhance their risk for developing the disease.
The cloning of hSR-B1/CLA-1 was facilitated by amino acid sequence homologies that were highly conserved among CD36 and LIMPII.9 CD36 is present in megakaryocytes, monocytes, capillary endothelium, and also platelets.10 The interaction of CD36 with the fibrinogen-liganded form of GP IIb/IIIa is postulated to stabilize platelet aggregation. This interaction enables the completion of the platelet activation process.10,28 Several studies have confirmed the possible importance of anti-CD36 antibodies in various thrombotic disorders, including thrombotic thrombocytopenic purpura and idiopathic thrombocytopenic purpura.29,30
The above discussion relates hSR-B1/CLA-1 expression to cholesterol metabolism but does not address the changes in platelet aggregation. Our interest in this parameter led us to use laser light scattering, a technique for measuring changes in ability of the platelets to aggregate. This method is capable of detecting clumps of approximately 70 to 1400 platelets that are formed at an early stage of platelet aggregation.31 Increased platelet aggregation is one of the more important processes in the development of cardiovascular diseases, including acute myocardial32 and cerebral infarction.33 Sagel et al34 reported that platelets are sensitive to aggregating agents (ADP and collagen) and this feature is most marked in frank diabetics, intermediate in latent diabetics, and least in prediabetics. These observations suggest that platelet aggregation may be increased early in diabetes and may be involved in the genesis of diabetic microangiopathy.
The results of our study show that the platelet aggregation correlated negatively with the level of hSR-B1/CLA-1 expression. Because CD36 and hSR-B1/CLA-1 belong to the same family, a potential clue that hSR-B1/CLA-1 may affect platelet function was reported by Volf et al.35 They noted that CD36 was responsible for oxidized LDL binding to human platelets.35 We have previously reported that hSR-B1/CLA-1 had the ability to bind to a modified form of LDL.11 Therefore, it is possible that hSR-B1/CLA-1 as well as CD36 might play similar important roles on platelet aggregation and mediating lipoprotein metabolism in human platelets.
Overexpression of SR-B1 in the liver results in a significant increase in biliary cholesterol content,5 an observation that is consistent with gene-targeting studies6,36,37 that point to an important role for SR-B1 in RCT. In vitro studies of SR-B1 showed that this receptor might also mediate efflux of unesterified cholesterol from cells to HDL.7 Hirano et al38 reported that hSR-B1/CLA-1 was also detected in atherosclerotic lesions. The expression of hSR-B1/CLA-1 in atheromatous lesions raises the possibility that HDL may dock to this receptor and thus promote cholesterol efflux from atheromatous arterial walls. A recent report indicated that inhibition of cholesterol synthesis with simvastatin results in a 3- to 4-fold increase in both SR-BI mRNA and protein levels. Conversely, the addition of 25-hydroxycholesterol suppressed SR-BI levels by approximately 50% in keratinocytes.39 Consistent with this observation, our findings show a negative correlation between hSR-B1/CLA-1 expression and cholesterol ester accumulation in human platelets, raising the possibility that cholesterol content of the platelet may regulate expression of hSR-B1/CLA-1.
The preceding observation suggests that hSR-B1/CLA-1 expression on human platelets might shed light on RCT activity in peripheral tissues. This hypothesis is supported by data showing that in SR-B1/apolipoprotein E double-homozygous knockout mice, SR-B1 can protect against the early onset of atherosclerosis.37 Potential causes of the dramatically accelerated atherosclerosis in double-knockout mice include changes in the relative amounts of cholesterol that increase proatherogenic and decrease antiatherosclerotic lipoproteins. This altered flux of cholesterol into or out of the aortic wall may perhaps be directly related to the SR-B1–mediated efflux7,37 resulting in decreased RCT. The antiatherosclerotic effect of SR-B1 expression in apolipoprotein E knockout mice suggests that pharmacologic stimulation of endogenous SR-B1 activity may be antiatherogenic, possibly related to its role in RCT.6,37 Recently, ATP-binding cassette transporter 1 gene (ABC1) was shown to mediate cholesterol efflux from cells to HDL.40 However, Chen et al41 reported that SR-BI inhibited ABC1-mediated cholesterol efflux even at low SR-BI expression level. This finding suggests that SR-B1 and ABC1 seem to have distinct but competing roles in mediating cholesterol flux between HDL and macrophages. The role of hSR-B1/CLA-1 in cholesterol metabolism, as well as ABC1 on platelets, serves as a target for future investigations.
In summary, the expression levels of hSR-B1/CLA-1 correlate positively with plasma HDL but negatively with cholesterol content in and aggregation of platelets. These findings raise the possibility that a measurement of the hSR-B1/CLA-1 expression on human platelets may provide a valuable insight that reflects the status of RCT in patients with atherosclerosis.
Received December 27, 2002; accepted March 4, 2003.
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