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(Circulation. 2004;110:452-459.)
© 2004 American Heart Association, Inc.

Original Articles

Aggregated Low-Density Lipoprotein Uptake Induces Membrane Tissue Factor Procoagulant Activity and Microparticle Release in Human Vascular Smooth Muscle Cells

Vicenta Llorente-Cortés, PhD; Marta Otero-Viñas, PhD; Sandra Camino-López, MS; Oriol Llampayas, MS; Lina Badimon, PhD, FESC

From the Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

Correspondence to Professor Lina Badimon, Cardiovascular Research Center, ICCC/CSIC-Hospital de la Santa Creu i Sant Pau, Jordi i Girona 18-26, 08041 Barcelona, Spain.

Received October 14, 2003; de novo received March 12, 2004; revision received April 22, 2004; accepted May 11, 2004.

	Abstract

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Background— Tissue factor (TF) is the main initiator of the arterial blood coagulation system, and aggregated LDL (agLDL) are found in the arterial intima. Our hypothesis is that agLDL internalization by vascular smooth muscle cells (VSMCs) may trigger TF-procoagulant activity.

Methods and Results— Cultured human VSMCs were obtained from human coronary arteries of explanted hearts during transplant operations. VSMCs were incubated with native LDL (nLDL) or agLDL. TF mRNA was analyzed by real-time polymerase chain reaction, and cellular and released TF protein antigen were analyzed by Western blot. TF microparticle (MP) content was analyzed by flow cytometry and TF activity by a factor Xa generation test. Both nLDL and agLDL strongly and equally increased TF mRNA and cell membrane protein expression, by approximately 5- and 9-fold, respectively. A sustained TF procoagulant activity was induced by agLDL but not by nLDL (agLDL 2.46±0.22 versus nLDL 0.72±0.12 mU/mg protein at 12 hours). AgLDL increased TF antigen release (agLDL 5.64±0.4 versus nLDL 3.28±0.22 AU) and TF MP release (agLDL 89.85±8.51 versus nLDL 19.69±4.59 TF MP/10³ cells). TF activation and release induced by agLDL is not related to apoptosis. Blockade of LDL receptor-related protein, a receptor for agLDL, prevented the agLDL-induced release of TF protein and TF MP.

Conclusions— VSMC-TF expression is upregulated by both nLDL and agLDL. However, only agLDL engagement to LDL receptor-related protein induced cellular TF procoagulant activity and TF release by human VSMCs.

Key Words: tissue factor • cells • lipoproteins • proteins

	Introduction

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Tissue factor (TF) is a transmembrane cell surface glycoprotein of 45 to 50 kDa that forms a complex with activated factor VII, initiating blood coagulation and leading to the focal production of thrombin via the successive activation of factor IX, factor X, and prothrombin.^1,2 TF is highly expressed in atherosclerotic lesions, and its expression at the plaque boundary layer induces thrombus formation.^3,4 In patients with acute coronary syndromes, TF expression is associated with areas of macrophages and vascular smooth muscle cells (VSMC), which suggests a cell-mediated thrombogenicity in the vascular wall.^4,5 Additionally, elevated levels of shed membrane microparticles (MPs) with procoagulant activity have been found in the peripheral circulating blood of patients with acute coronary syndromes⁶ and patients with metabolic syndrome.⁷ Furthermore, both cellular and extracellular TF could play an important role in atherosclerotic plaque thrombogenicity.⁸ Extracellular TF present in the acellular lipid-rich core is highly thrombogenic, and the proximity of TF to the lipid-rich areas might suggest a potential role of LDL in TF expression. In addition to growth factor and cytokines,^9–11 TF expression or activity might be regulated by native LDL (nLDL) and oxidized LDL (oxLDL).^12–14 The retention and aggregation of LDL (agLDL) in the arterial intima, facilitated by the proteoglycans that conform the extracellular matrix, is a key event in atherogenesis.^15,16 We have recently demonstrated that agLDLs induce high intracellular cholesteryl ester accumulation through LDL receptor-related protein (LRP) uptake in human VSMCs.^17–19 Additionally, agLDL upregulates LRP expression in a time- and dose-dependent manner.²⁰ The aim of this study was to analyze the role of agLDL, compared with nLDL, in cellular TF expression, TF release, and TF activity in human VSMCs. We demonstrated that both nLDL and agLDL have the same capacity to increase TF mRNA and cellular membrane TF protein expression in human VSMCs; however, agLDL and not nLDL induced sustained cellular TF activity and TF MP release. These results suggest that lipid deposition in advanced lipid-enriched atherosclerotic plaques could promote cellular TF activation and TF MP release and demonstrate for the first time that LRP-mediated agLDL internalization could be one of the mechanisms that induce the prothrombotic transformation of VSMCs in the atherosclerotic vascular wall.

	Methods

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VSMC Culture
Primary cultures of human VSMCs were obtained from nonatherosclerotic areas of human coronaries of explanted hearts during transplant operations performed at the Hospital de la Santa Creu i Sant Pau, as described previously.^17,18 Non–LRP-expressing VSMCs were obtained by treatment of the cells with antisense LRP oligodeoxynucleotides (ODNs; 10 µmol/L) as described previously.^17–19

LDL Preparation
Human LDL (d_1.019 to d_1.063 g/mL) was obtained from pooled sera of normocholesterolemic volunteers by sequential ultracentrifugation. AgLDL was prepared as described previously.^17,18 Levels of thiobarbituric acid–reactive substances (<1.2 mmol of malonaldehyde per milligram of protein LDL) remained similar to those in nLDL after LDL aggregation.

Real-Time Polymerase Chain Reaction
Arrested cells were incubated with nLDL or agLDL for 2, 4, 6, 8, 12, 24, and 48 hours. At each time point, cells were washed with cold PBS, and total RNA and protein were isolated with the Tripure isolation reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions. TF mRNA levels were analyzed by real-time polymerase chain reaction (PCR). TaqMan fluorescent real-time PCR primers and probes (6'FAM-MGB) for TF were designed by use of Primer Express software from PE Biosystems and were as follows: TF forward 5'-ttcacaccttacctggagacaaac-3'; TF reverse 5'-aacatcccggaggcttagga-3'; TF probe 5'-caaaagtgaatgtgaccgtag-3'. Assays on demand (Applied Biosystems) were used for Bcl₂ (Hs00153350), BAX (Hs00180269), and CPP32 (Hs 00263337) detection. Human GAPDH (4326317E) was used as endogenous control. TaqMan real-time PCR was performed as described previously.^19,20

Subcellular Fractionation
Subcellular fractionation was performed according to Xiong et al with minor modifications.²¹

Isolation of Cell Culture Medium TF MPs
The culture medium was concentrated on a 60% sucrose cushion by centrifugation at 245 000g as described previously.²² The fraction overlying the 60% sucrose bed that contain MP was collected. One aliquot was dialyzed against 0.01 mol/L NH₄HCO₃, concentrated, and used for Western blot analysis and another fraction for flow cytometry analysis. In a third fraction, the TF-dependent procoagulant activity was determined.

Western Blot Analysis
SDS-PAGE was performed with samples of cellular membranes obtained after cellular subfractionation and with samples of TF MP obtained after sucrose gradient. Blots were incubated with monoclonal antibodies against human TF (American Diagnostica 4501) or against human CPP32 (Transduction Laboratories, clone 19).

Flow Cytometry Analysis of Annexin V– and Tissue Factor–Positive MPs
One aliquot of isolated MPs was incubated with monoclonal anti-human TF (100 µg/mL) for 18 hours at 4°C and then with FITC-conjugated goat anti-mouse IgG for 20 minutes. Another fraction was incubated with annexin V conjugate (Molecular Probes, A-13200) at room temperature for 15 minutes. Total count for TF- and/or annexin V–labeled MPs and annexin V–labeled VSMCs was determined with a Beckman Coulter XL flow cytometer with System II software. TF MPs were defined as elements with a size of 0.2 µm that were positively labeled by specific anti-TF antibody. Particle size was determined by comparison with carboxylate-modified fluorescent microspheres (FluoSpheres, Molecular Probes). Background cell autofluorescence was assessed by omission of the primary antibody.

Flow Cytometry Detection of Annexin V–Positive VSMCs
Arrested VSMCs were incubated in the absence (negative control) or presence of nLDL and agLDL (100 µg/mL) for 18 hours. As a positive control of apoptosis, VSMCs were treated with H₂O₂. VSMCs were exhaustively washed with PBS and trypsinized, and the pellet was resuspended in 1 mL of annexin binding buffer (10 mmol/L HEPES, 140 mmol/L NaCl, and 2.5 mmol/L CaCl₂, pH 7.4). One aliquot of 200 µL of cell suspension was incubated with 20 µL of annexin V conjugate and 10 µL of propidium iodide (cell-death indicator) at room temperature for 15 minutes. Then, 400 µL of annexin binding buffer was added. Stained VSMCs were detected by flow cytometry. Experiments were performed with an Epics XL flow cytometer (Coulter Corporation).

High-Performance Thin-Layer Chromatography Analysis of Phospholipid Composition of VSMC Membrane and Cell-Derived MPs
Arrested VSMCs were incubated in the absence or presence of nLDL and agLDL (100 µg/mL) for 18 hours. Cells were then exhaustively washed and harvested into 1 mL of 0.10 N NaOH. The culture medium was concentrated on a 60% sucrose cushion by centrifugation at 245 000g as described previously.²² One aliquot of 200 µL was used for lipid extraction. Lipid extraction was performed as described previously.^17,18 The chromatographic conditions were as previously described,²³ with minor modifications.

Factor Xa Generation Test in Cells and Their Culture Medium
Previously described techniques with minor modifications were followed.^24,25 VSMCs collected in TBS-EDTA buffer (0.1 mol/L NaCl, 0.05 mol/L Tris, EDTA 0.1 mol/L, pH 7.5) and their culture medium, prepared as previously explained, were frozen at –80°C. TF procoagulant activity (PCA) was measured by a factor Xa generation test.

Data Analysis
Data are expressed as mean± SEM. A Statview (Abacus Concepts) statistical package for the Macintosh computer system was used for all analyses. Multiple groups were compared by ANOVA or Wilcoxon test as needed. Statistical significance was considered at P<0.05.

	Results

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Effect of nLDL and agLDL on TF mRNA and Protein Expression and TF Activity
As shown in Figure 1A, both nLDL and agLDL (100 µg/mL) significantly increased TF mRNA expression by approximately 5.7-fold over the levels of untreated VSMCs after 2 hours of incubation. The increased TF mRNA accumulation induced by LDL declined below basal levels between 4 and 12 hours.

View larger version (21K): [in this window] [in a new window]   Figure 1. TF mRNA and protein expression quantification. A, Time course of TF mRNA to nLDL and agLDL. VSMCs were incubated in presence of nLDL (squares) or agLDL (circles; 100 µg/mL) for increasing times. B, Western blot analysis of cellular membrane TF antigen. VSMCs were incubated in absence or presence of nLDL or agLDL (100 µg/mL) for 12 hours. Results are expressed as arbitrary units and are shown as mean±SEM (n=3).

We determined membrane TF protein expression by Western blot analysis after cellular subfractionation. As shown in Figure 1B, untreated VSMCs express low levels of TF on their surface (47 kDa). Cell-surface TF antigen was strongly increased both by nLDL (9.43-fold) and by agLDL (9.6-fold) after 12 hours of LDL exposure.

Time-course analysis of TF PCA (Figure 2A) indicated that after 2 hours of LDL incubation, both nLDL and agLDL significantly increased cellular TF PCA over the levels found in VSMCs incubated in the absence of LDL (nLDL 1.57±0.22, agLDL 2.15±0.32 versus no LDL 0.85±0.09 mU/mg protein; P<0.05). However, only TF PCA induced by agLDL exposure was maintained throughout the time (agLDL 1.88±0.12 versus nLDL 0.13±0.06 mU/mg protein at 12 hours; P<0.05), which started to decline at 24 hours. As shown in Figure 2B, agLDL (12 hours) induced the highest PCA (1.90±0.11 mU/mg protein) at the lowest tested concentration (100 µg/mL). The area under the curve was significantly higher in agLDL-incubated VSMCs than in nLDL-incubated VSMCs both in the time-course analysis (agLDL 16.52±2.3 versus nLDL 2.64±0.66 hours x mU/mg protein; P<0.05) and the dose-response analysis (agLDL 3.5±0.23 versus nLDL 0.31±0.06 µg/mL LDL x mU/mg protein; P<0.05).

View larger version (13K): [in this window] [in a new window]   Figure 2. Factor Xa generation test of TF activity. A, Time course of TF PCA to nLDL and agLDL. VSMCs were incubated in absence (rhomboid) or presence of nLDL (squares) or agLDL (circles) (100 µg/mL) for increasing times. B, Dose response of TF activity to increasing concentrations of nLDL or agLDL for 12 hours. Results are expressed as mU/mg protein and are shown as mean±SEM of 2 independent experiments performed in triplicate.

Effect of nLDL and agLDL on TF Release by Human VSMCs
Western blot analysis of TF protein in the culture medium from VSMCs (Figure 3A) revealed that secreted TF was larger (52 kDa) than cellular TF (47 KDa). VSMCs incubated with agLDL for 24 or 48 hours released higher amounts of TF antigen than VSMCs incubated with nLDL or incubated in absence of LDL (agLDL 5.64±0.4 versus nLDL 3.28±0.22 or no LDL 3.49±0.35 AU after 24 hours; P<0.05). However, agLDL-induced TF antigen release (5.83±0.4 AU) was completely reduced (97±4% at 10 µmol/L) in antisense LRP-ODN–treated VSMCs but not in sense LRP-ODN–treated VSMCs (Figure 3B).

View larger version (26K): [in this window] [in a new window]   Figure 3. Western blot analysis of released TF antigen. A, Medium was harvested from untreated VSMCs or VSMCs incubated in absence (rhomboid) or presence of nLDL (squares) or agLDL (circles) (100 µg/mL) for increasing times. B, Medium was harvested from untreated VSMCs (gray bars), antisense (black bars), and sense (white bars) LRP-ODN–treated VSMCs (10 µmol/L) incubated in absence or presence of nLDL or agLDL (100 µg/mL). Results are expressed as arbitrary units and are shown as mean±SEM (n=3). AS indicates antisense; S, sense.

The culture medium from VSMCs contained MPs of a similar size to microspheres of 0.2 µm used as a control. As shown in Figure 4A, the number of TF MPs was much higher in the culture medium from VSMCs incubated with agLDL than in the medium from cells incubated with nLDL (100 µg/mL, 24 hours) or from those incubated in the absence of LDL (agLDL 89.85±8.51 versus nLDL 19.69±4.59 or no LDL 17.5±2.5 MP/10³ cells; P<0.05). However, agLDL, in contrast to H₂O₂ treatment that induced a large increase in the number of annexin V–positive MPs (annexin V-MP), did not induce any alteration in the number of annexin V-MPs against control VSMCs (Figure 4B).

View larger version (51K): [in this window] [in a new window]   Figure 4. Fluorescent cytometric analysis of medium TF. A, Representative image showing TF-positive MP (TF MP). B, Representative image of annexin V–positive MP. C, Bar graphs showing quantification of TF MP in medium of untreated VSMCs (gray bars), antisense (black bars), and sense (white bars) LRP-ODN–treated VSMCs (10 µmol/L) incubated in absence or presence of nLDL or agLDL (100 µg/mL). Results are expressed as TF MP/103 cells and are shown as mean±SEM of 3 independent experiments performed in duplicate.

AgLDL-induced TF MP release (86.65±1.4 MP/10³ cells) was completely reduced (99±2% at 10 µmol/L) in antisense LRP-ODN–treated VSMCs but not in sense LRP-ODN–treated VSMCs (Figure 4C). Factor Xa generation test showed no significant TF PCA in MPs from any tested culture medium.

Apoptosis has been proposed as one of the main mechanisms for MP release in several cell types.^25,26 However, nLDL or agLDL did not alter the mRNA Bcl₂/BAX ratio of VSMCs at any tested time (until 48 hours; Figure 5A). In agreement, in the present study, nLDL or agLDL did not significantly alter CPP32 mRNA (data not shown) or protein (Figure 5B). Neither nLDL nor agLDL, in contrast to H₂O₂, which induced an important increase in apoptotic (20±2%) and necrotic (78±1.3%) VSMCs, induced any significant difference in the percentage of apoptotic or necrotic VSMCs against control VSMCs (Figure 5C).

View larger version (36K): [in this window] [in a new window]   Figure 5. Effect of nLDL and agLDL on VSMC apoptosis. A, Bcl2/BAX mRNA ratio of VSMCs incubated in absence (rhomboid) or presence of nLDL (squares) or agLDL (circles; 100 µg/mL) for increasing times (n =3). B, CPP32 protein expression levels of VSMCs incubated in absence or presence of nLDL and agLDL (100 µg/mL) for 24 hours (n=2). C, Flow cytometric detection of PS exposure in VSMCs exposed to nLDL or agLDL compared with H2O2-treated VSMCs. Percentage of annexin V–positive cells in each sample is shown. R13 indicates necrotic; R14, alive; R15, apoptotic. Data are representative of at least 3 independent experiments.

Effect of nLDL and agLDL on Phospholipid Composition of VSMC Membrane and VSMC-Derived MPs
We analyzed the ability of nLDL and agLDL to modify the phospholipid composition of VSMC membrane and VSMC-derived MPs by high-performance thin-layer chromatography (HPTLC). HPTLC from control VSMCs (Figure 6A) revealed that sphingomyelin is the main phospholipid of VSMC membrane (0.6±0.04 µg/mg protein), together with phosphatidylethanolamine (0.19±0.05 µg/mg protein), phosphatidylcholine (0.15±0.06 µg/mg protein), and phosphatidylserine (PS; 0.08±0.01 µg/mg protein). However, whereas agLDL did not exert any effect on phosphatidylethanolamine, phosphatidylcholine, or PS content, agLDL but not nLDL increased sphingomyelin content to 1.16±0.005 µg/mg protein.

View larger version (44K): [in this window] [in a new window]   Figure 6. Phospholipid composition of VSMCs and VSMC-derived MPs. A, Representative HPTLC of VSMCs and bar graph showing quantification of phospholipid bands. B, Representative HPTLC of VSMC-derived MPs and bar graph showing quantification of phospholipid bands. Results are expressed as micrograms of phospholipid per milligram of protein for VSMCs and as micrograms of phospholipid per milliliter of medium for VSMC-derived MPs. Results are shown as mean±SEM of 2 independent samples performed in triplicate. L-PC indicates lysophosphatidylcholine; SM, sphingomyelin (black bars); PC, phosphatidylcholine (hatched bars); PS, phosphatidylserine (gray bars); PI, phosphatidylinositol; PE, phosphatidylethanolamine (white bars). *P<0.05 vs control cells.

HPTLC from VSMC-derived MPs (Figure 6B) indicated that MPs from control VSMCs only have detectable levels of PS (0.18±0.01 µg/mg protein). However, MPs derived from nLDL/agLDL-incubated VSMCs have an increased amount of PS (nLDL 0.33±0.04, agLDL 0.34±0.09 µg/mg protein) and high levels of phosphatidylcholine (nLDL 0.37±0.02, agLDL 0.35±0.01 µg/mg protein) and sphingomyelin (nLDL 0.18±0.01, agLDL 0.20±0.01 µg/mg protein). No statistically significant differences were found between the effect of nLDL and agLDL on the phospholipid composition of VSMC-derived MPs.

	Discussion

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LRP-mediated agLDL uptake has proved to be one of the main mechanisms for intracellular lipid accumulation in human VSMCs,^17–20 cells that have the capacity to secrete TF MP.^22,27 In the present study, we hypothesized that agLDLs were able to induce cellular TF expression and activity. We found that both nLDL and agLDL induced TF mRNA after 2 hours of LDL exposure to the same level, in agreement with previous studies showing that nLDL and oxidized LDL induce TF mRNA in different cell types,^12–14 which is likely unrelated to the receptor involved in LDL uptake. The increase in mRNA transcription levels induced by nLDL or agLDL did not require new protein synthesis but required a functional transcriptional mechanism. The increase in mRNA led to a strong increase in membrane-associated cellular TF protein expression. With regard to TF activity, the effect of agLDL on TF PCA, in contrast to a shorter time effect of nLDL, was maintained for 24 hours. The ability of nLDL to induce TF expression but not TF activity beyond 2 hours is in agreement with previous results¹² and confirms the idea that TF expression and TF procoagulant activation are independent processes. It has been demonstrated that most of TF is in an encrypted-inactivated state in the cell membrane.²⁸ Like nLDL, agLDL contributes to increase the pool of membrane TF, but it is also able to increase TF activity in a prolonged manner. Additionally, agLDL but not nLDL is able to increase TF release to the extracellular medium. The secreted TF has a higher molecular weight than cellular TF, and it is mainly associated with MPs of 0.2 µm, as reported previously.²²

Although lipid-loading induces TF release through apoptotic mechanisms in macrophages,^29,30 agLDL-lipid loading did not induce apoptosis of VSMCs, in agreement with the lack of apoptosis previously found in agLDL-loaded macrophages and VSMCs.³¹ The present results are in agreement with those of Schecter et al,²² who proposed that release of TF-containing MPs from VSMCs represents a constitutive property of VSMCs not related to apoptosis. In fact, the marked discrepancies in TF localization between VSMCs (in caveolae) and macrophages (clathrin-coated pits)³² could explain in part the different mechanisms involved in TF activation and TF release between VSMCs and macrophages.

The increase in the number of TF MPs released by agLDL lipid-loaded cells was not associated with an increase in TF PCA. It is not completely known where or how TF MP activity is modulated, but the exposure of PS appears to contribute to TF activity. However, the present results clearly demonstrate that MPs released from agLDL-incubated VSMCs, as opposed to those released by apoptotic VSMCs, have no PS exposure on their external surface. Indeed, they are annexin V negative. Although MPs derived from LDL-incubated VSMCs have a different phospholipid composition than MPs derived from control VSMCs, no differences were found between MPs from nLDL- or agLDL-incubated VSMCs, in contrast to the differential sphingomyelin content of the plasmatic membrane induced by agLDL but not by nLDL. Although some authors have found PCA in circulating MPs,^5,22 others have not.⁶ Secreted TF might become active under certain unknown conditions, contributing to thrombosis, angiogenesis, or transcellular signaling.³³

The specific ability of agLDL to induce TF MP release appears to be associated with LRP-mediated agLDL uptake, because no increase in TF release induced by agLDL was observed in non–LRP-expressing VSMCs. LRP-mediated agLDL uptake could induce TF MP release by different mechanisms. For one thing, LRP is not only involved in endocytosis but also in signal transduction³⁴ and in alterations in intracellular calcium concentrations, which leads to cell activation.³⁵ Cell activation has been described as one of the mechanisms responsible for MP release.³⁶ On the other hand, LRP and inactive TF are localized in certain patches of the membrane, named caveolae.^37,38 The relevance of LRP localization in caveolae for TF MP release and TF activation is supported by our demonstration that the binding of agLDL to LRP could influence the enrichment of released MPs with inactive TF, which is mainly in caveolae in the encrypted form.²⁸ Additionally, as we have demonstrated, agLDL induces the enrichment of the plasmatic membrane of VSMCs with sphingomyelin, one of the main phospholipids in the structure of caveolae. Interestingly, phospholipid membrane composition can influence the topological organization of proteins and their activity.³⁹ Furthermore, a sphingomyelin increase in caveolae induced by agLDL likely participates in TF activation. It has been reported that oxLDL induces TF activation through cellular lipid peroxidation,⁴⁰ which could cause changes in TF structure. Both oxLDL and agLDL, although different mechanisms and receptors, appear to induce cellular membrane perturbations that cause TF activation.

In this work, we demonstrate for the first time that LRP-mediated agLDL-lipid loading of VSMCs is one of the mechanisms that induces TF activation and TF MP release. In lipid-enriched advanced atherosclerotic lesions, in which agLDL is one of the main modifications of the LDL in the arterial intima, agLDL-lipid loading could be an important mechanism in increasing the prothrombotic potential of VSMCs.

	Acknowledgments

 
This study was funded in part by a Merck Sharp Dohme unrestricted grant, SAF2003–03187, and Fundación Investigación Cardiovascular-Catalana Occidente. We thank the heart transplant team of the Division of Cardiology and Cardiac Surgery, Blood Bank of the Vall d’Hebron Hospital. Dr Otero is a postdoctoral fellow of Departament D’Universitats Recerca i Societat de la Informació. S. Camino and O. Llampayas are predoctoral fellows of the Fundació d’Investigació Cardiovascular. The authors thank Vanessa Martín for her technical assistance. Flow cytometric analyses were performed at the Central Services of the University of Barcelona.

	References

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