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Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism
- Sandy Mosig†1Email author,
- Knut Rennert†1,
- Petra Büttner1,
- Siegfried Krause2,
- Dieter Lütjohann3,
- Muhidien Soufi4,
- Regine Heller2 and
- Harald Funke1
© Mosig et al; licensee BioMed Central Ltd. 2008
Received: 01 April 2008
Accepted: 28 November 2008
Published: 28 November 2008
Elevated plasma cholesterol promotes the formation of atherosclerotic lesions in which monocyte-derived lipid-laden macrophages are frequently found. To analyze, if circulating monocytes already show increased lipid content and differences in lipoprotein metabolism, we compared monocytes from patients with Familial Hypercholesterolemia (FH) with those from healthy individuals.
Cholesterol and oxidized cholesterol metabolite serum levels of FH and of healthy, gender/age matched control subjects were measured by combined gas chromatography – mass spectroscopy. Monocytes from patients with FH and from healthy subjects were isolated by antibody-assisted density centrifugation. Gene expression profiles of isolated monocytes were measured using Affymetrix HG-U 133 Plus 2.0 microarrays. We compared monocyte gene expression profiles from FH patients with healthy controls using a Welch T-test with correction for multiple testing (p < 0.05; Benjamini Hochberg correction, False Discovery Rate = 0.05). The differential expression of FH associated genes was validated at the mRNA level by qRT-PCR and/or at the protein level by Western Blot or flow cytometry. Functional validation of monocyte scavenger receptor activities were done by binding assays and dose/time dependent uptake analysis using native and oxidized LDL.
Using microarray analysis we found in FH patients a significant up-regulation of 1,617 genes and a down-regulation of 701 genes compared to monocytes from healthy individuals. These include genes of proteins that are involved in the uptake, biosynthesis, disposition, and cellular efflux of cholesterol. In addition, plasma from FH patients contains elevated amounts of sterols and oxysterols. An increased uptake of oxidized as well as of native LDL by FH monocytes combined with a down-regulation of NPC1 and ABCA1 explains the lipid accumulation observed in these cells.
Our data demonstrate that circulating FH monocytes show differences in cell physiology that may contribute to the early onset of atherosclerosis in this disease.
Atherosclerosis is the primary cause of coronary heart disease (CHD) and stroke in Western societies . It is characterized by the development of lipid-rich lesions in the arterial wall, in which foam cells, monocyte-derived lipid-laden macrophages, are frequently found.
An important regulator of cellular cholesterol content is the sterol regulatory element binding protein (SREBP) pathway, which controls, by transcriptional regulation, the uptake of cholesterol via LDL-receptor and several steps in the de novo synthesis of cholesterol.
In healthy individuals cells ingest cholesterol by endocytosis of LDL bound to the LDL-receptor (LDLR). After endocytosis, the LDLR uncouples from its ligand and returns to the cell surface, while the LDL is catabolized. Cholesterol accumulation in membranes of the endoplasmatic reticulum (ER) results in a down-regulation of the SREBP pathway and subsequently in the repression of 3-hydroxy-3-methyl-glutaryl-CoA-reductase (Hmgcr), the rate limiting enzyme of the de novo cholesterol biosynthesis . Efflux of excessive cholesterol is mediated by Abca1, the major cholesterol efflux system in macrophages, which transfers cholesterol to apolipoprotein A1 on HDL particles. It is assumed that Npc1 has a pivotal role in cholesterol efflux as it aids in the cellular distribution of lysosomal cholesterol which enables Abca1 dependent efflux [3, 4].
In contrast to native LDL, macrophages utilize scavenger receptors (SR), such as CD36, for the uptake of modified LDL . Several studies have demonstrated that macrophages express high levels of CD36 enabling them to bind and internalize oxidized lipoproteins. It is well established that oxidized LDL (oxLDL) is cytotoxic and that it has the ability to induce apoptosis. As oxLDL is frequently found in atherosclerotic lesions it is assumed that its accumulation contributes to the pathogenesis of atherosclerosis . Therefore, a rapid clearance of oxLDL deposits from the arterial wall via SRs by monocyte derived macrophages is essential for atherosclerosis prevention.
However, only few data exist on the involvement of circulating monocytes in this pathologic process in men . To date, studies of atherosclerosis development have been carried out mostly in pathologic human specimen, cell lines, and primary cell culture systems, as well as in animal models . To study monocyte function in a hyperlipidemic environment in men we examined patients suffering from the monogenic disorder Familial Hypercholesterolemia (FH). FH patients have a defective or missing LDLR which results in dramatically elevated plasma LDL-cholesterol levels and early onset of atherosclerosis. Further, atherosclerosis progression in FH is mainly independent from the presence of additional genetic and environmental risk factors  which makes it a suitable model trait for studying human atherosclerosis.
Homozygous and heterozygous FH patients with documented genetic defects in the LDL receptor (LDLR) gene and healthy volunteers were informed about the aim of the study and gave written informed consent. The study was approved by the Ethics Committee of the Friedrich Schiller University of Jena/Germany. All homozygous FH patients were treated with haemapheresis. In addition, three of eight homozygous patients also received statin treatment. Of the heterozygous patients three received neither statin nor apheresis therapy. Five heterozygous patients received apheresis as well as statin therapy. Those patients who did not receive statin treatment did not show a significant response in their plasma cholesterol levels to the drug treatment. (FH patients: LDLR mutations given in Table 4).
Cell isolation, cryopreservation and cell culture
For microarray analysis and cell culture assays cells were isolated with RosetteSep Monocyte Enrichment Cocktail (Cell Systems, St. Katharinen, Germany) according to the manufacturer's protocol. Cell purity was analyzed by flow cytometry with antibodies directed against CD14 (monocytes), CD3 (T cells), CD235a (erythrocytes), CD19 (B cells), and CD56 (NK cells). All cell preparations had a purity of > 95%. The viability of cells was checked by staining with 7-amino-actinomycin D (7-AAD) (Becton Dickinson, Heidelberg, Germany). The number of 7-AAD positive cells was below 3% in all preparations. In addition, the viability of monocytes was confirmed by their inflammatory response to LPS (Sigma-Aldrich, Munich, Germany) stimulation (data not shown).
For cell culture assays, isolated monocytes were resuspended in 10% DMSO (Sigma-Aldrich, Munich, Germany), 60% IMDM (Invitrogen, Karlsruhe, Germany), and 30% autologous serum. They were then frozen with a temperature decline of -1°C/min to -80°C, and stored in liquid nitrogen. Frozen cells were thawed at 37°C, washed twice with PBS/2 mM EDTA and resuspended in X-VIVO 15 (Cambrex, Walkersville, USA). For all binding, uptake and blocking assays 1 × 105 monocytes per well were cultured in X-VIVO 15 serum free medium with the indicated concentrations of nLDL, oxLDL, and antibodies.
Monocyte to macrophage differentiation was carried out by culturing cryopreserved monocytes for 10 days in X-VIVO 15 medium (Lonza, Walkersville, MD) with addition of 10% of human serum from a healthy, normolipidemic donor.
Freshly isolated cells were lysed in TRIZOL (Invitrogen, Karlsruhe, Germany). RNA was extracted using the RNeasy Micro Kit (Qiagen, Hilden, Germany). RNA was quantified using a Nanodrop photometer (Thermo Scientific, Wilmington, Delaware, USA). RNA quality was measured with an Agilent 2100 Bioanalyzer using the RNA Nano kit (Agilent Technologies, Waldbronn, Germany). Only RNA with a RNA integrity number better than 9.5 was used for microarrays. 1 μg of total RNA was processed with Affymetrix One-Cycle Target Labeling Control reagents and hybridized to Affymetrix HG U133 Plus 2.0 GeneChip Arrays. Raw data were obtained with Affymetrix GCOS 1.3. Quality control of microarray raw data was done with the Bioconductor "affyQCReport" R package including Whisker box plots, density plots, plotting of 3'/5' ratios of GAPDH and beta-actin, scaling factor and background noise, and correlation plots of all microarrays (Supplementary Figure 1). Raw data were imported to GeneSpring GX 7.3.1 (Agilent Technologies) using the RMA (Robust Multi-Array Average) algorithm . They were normalized per chip to the 50th percentile and per gene to the median. Transcripts with a raw data value of 20 and more in at least half of all measured arrays were considered as "expressed". 35.264 transcripts fulfilled this criterion. Functional annotation clustering was performed with DAVID 2008 http://david.abcc.ncifcrf.gov. The resulting group enrichment score represents the negative logarithm of the geometric mean of Fisher's exact test p-values from cluster members [9, 10]. Pathway analysis was done with the Agilent Pathway Architect (Agilent, Waldbronn, Germany).
Microarray data samples were deposited in the Gene Expression Omnibus database http://www.ncbi.nlm.nih.gov/geo. They are accessible under the series GSE6054.
Quantitative Real-time PCR
Quantitative Real-time PCR (qRT-PCR) was performed using the Eppendorf RealPlex 2S PCR (Eppendorf, Hamburg, Germany) and QuantiTect Primer Assays (Qiagen, Hilden, Germany). Quantification was done using standards with known copy numbers and normalization to ubiquitin C (UBC).
LDL-isolation, preparation and labelling
LDL were isolated from fresh serum of normolipidemic donors by sequential ultracentrifugation  and stored in 0.02% NaN3, Na2EDTA (0.24 mM) at 4°C overlaid with nitrogen. Preparations were used within two weeks. Oxidation of LDL was achieved by a 6 hour incubation of native LDL with 10 μM CuSO4 at 37°C. Oxidized LDL was desalted by PD-10 Desalting columns from GE Healthcare (Uppsala, Sweden) and eluted in PBS.
nLDL and oxLDL were labelled using the Alexa-Fluor488- and Alexa-Fluor647-Protein labelling kits (Molecular Probes, Leiden, Netherlands) according to the manufacturer's protocols. All LDL forms were endotoxin-low (< 0.02 EU/ml), tested by QCL-1000 Chromogenic LAL (Cambrex, Walkersville, USA). The OxLDL ELISA-Kit was obtained from Immundiagnostik AG (Bensheim, Germany). LDL protein content was measured by a modified Lowry protein assay (Biorad, Hercules, USA).
Oil red O staining
105 monocytes/well were seeded and adhered to coverslips (Nalge Nunc International, Rochester, USA) in X-VIVO 15 for 4 hours. After adhesion, cells were washed twice with PBS/2 mM EDTA and once with PBS. They were then fixed using 4% paraformaldehyd for 10 minutes, rinsed with 60% isopropanol, incubated for 10 minutes with Oil Red O, rinsed again with isopropanol, and washed with aqua dest. After hematoxylin-incubation and -oxidation coverslips were rinsed again and were imbedded in aqueous mounting medium. Images have been taken on Zeiss AxioVert200M (Carl Zeiss, Jena, Germany).
Plasma-concentrations of sterols
10 μg/ml butylhydroxytoluol (Sigma Aldrich, Munich, Germany) were added to fresh plasma of donors which was subsequently frozen and stored at -80°C until analysis. Serum concentrations of cholesterol were measured by gas chromatography-flame ionization detection using 5alpha-cholestane as internal standard and cholesterol precursors and plant sterols by gas chromatography-mass spectrometry with epicoprostanol as internal standard. Oxidized cholesterol metabolites (7α-, 24S- and 27-hydroxycholesterol) were analyzed using an isotope dilution method and quantified by selected ion monitoring gas chromatography-mass spectrometry .
Time and dose dependency, binding and blocking experiments
For uptake studies monocytes were washed as described above and incubated with LDL for the indicated times at 37°C, 5% CO2. Binding studies were performed at 4°C. CD36 and Lrp1 blocking was done with 10 μg/ml murine IgG (MOPC-21) (Abcam, Cambridge, UK), rabbit IgG (Innovative Research, Southfield, USA), anti-human CD36 (FA6-152) (Abcam, Cambridge, UK) or anti-human Lrp1 (Biomac, Leipzig, Germany) for 30 minutes at 37°C, 5% CO2 and incubated with the indicated LDL forms for 4 hours at 37°C, 5% CO2. All antibodies used were endotoxin-low and azide-free.
Purity control antibodies, isotype control antibodies and anti-CD36 were from ImmunoTools (Friesoythe, Germany). Anti-CD91 was from BD Biosciences (Heidelberg, Germany). Flow cytometry has been performed on a FACSCalibur (Becton Dickinson, Heidelberg, Germany) and data analyzed with FlowJo 7.2.2. (TreeStar, Ashland, OR, USA).
1 × 106 monocytes were lysed in lysis buffer (50 mM TrisCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, protease inhibitors). Protein content was measured using BCA-Protein-Assay Kit (Pierce, Rockford, USA). 10 μg protein/individual were subjected to SDS-PAGE and Western Blotting. Primary antibodies against β-Actin (Cell Signaling, Danvers, USA), ABCA1 (Novus Biologicals, Littleton, USA), SCAP, NPC1 (Santa Cruz Biotechnology, Heidelberg, Germany), CD36 (FA6-152) (Abcam, Cambridge, UK) and Lrp1 (R2629, kindly provided by DK Strickland) were used. Secondary horseradish-peroxidase conjugated goat anti-mouse, rabbit anti-goat and goat anti-rabbit antibodies were purchased from KPL (Gaithersburg, USA). ECL was from Perkin Elmer (Boston, USA).
Statistical analyses were performed with SPSS 14.0 (SPSS, Chicago, USA) using two-sided Students t-Test, if not otherwise indicated. Error bars indicate the standard deviation.
Baseline characteristics and lipid levels of the study group.
female n = 6
female n = 5
female n = 16
male n = 2
male n = 3
male n = 10
30.2 ± 11.1
28.8 ± 7.5
33.8 ± 11.2
413.82 ± 92.33 (***)
220.64 ± 27.81 (***)
107.03 ± 17.78
60.33 ± 8.09
54.17 ± 3.71
61.17 ± 6.60
141.31 ± 39.89
233.61 ± 132.80
107.63 ± 33.04
676.67 ± 347.06
442.73 ± 420.11
187.53 ± 153.59
7.64 ± 0.95
5.45 ± 0.57
6.38 ± 0.8
253.12 ± 22.2
277.39 ± 37.51
258.12 ± 21.97
Plasma concentrations of sterols and sterol to plasma-cholesterol ratio of homozygous and heterozygous FH patients and controls.
homozygous FH (n = 5)
heterozygous FH (n = 5)
Controls (n = 8)
0.608 ± 0.075 (***)
1,60*10-3 : 1
0.442 ± 0.062 (*)
1,73*10-3 : 1
0.278 ± 0.038
1,59*10-3 : 1
0.725 ± 0.226
1,78*10-3 : 1
0.563 ± 0.182
1,85*10-3 : 1
0.335 ± 0.121
1,61*10-3 : 1
0.692 ± 0.17 (**)
1,77*10-3 : 1
0.651 ± 0.243 (*)
2.58*10-3 : 1
0.267 ± 0.031
1.40*10-3 : 1
4.773 ± 1.096 (**)
1,23*10-5 : 1
5.237 ± 1.402 (**)
2.09*10-5 : 1
2.151 ± 0.253
1.37*10-5 : 1
24.805 ± 7.533 (**)
6.54*10-5 : 1
19.163 ± 6.081 (*)
7.79*10-5 : 1
7.772 ± 1.84
4.25*10-5 : 1
0.522 ± 0.103 (**)
1.37*10-3 : 1
0.519 ± 0.161 (*)
2.08*10-3 : 1
0.184 ± 0.035
1.06*10-3 : 1
8.597 ± 2.603 (*)
2.13*10-5 : 1
7.206 ± 2.261 (*)
2.76*10-5 : 1
2.933 ± 0.625
1.96*10-5 : 1
84.004 ± 17.864 (**)
2.18*10-4 : 1
80.706 ± 26.213 (*)
3.15*10-4 : 1
29.173 ± 2.612
1.88*10-4 : 1
39.812 ± 8.347
1.01*10-4 : 1 (**)
34.568 ± 12.712
1.23*10-4 : 1
31.123 ± 5.523
1.79*10-4 : 1
0.359 ± 0.045 (**)
9.37*10-4 : 1
0.254 ± 0.046
9.54*10-4 : 1
0.167 ± 0.034
9.62*10-4 : 1
87.820 ± 18.094 (*)
2.26*10-7 : 1
74.078 ± 9.541
2.90*10-7 : 1
50.585 ± 9.936
3.02*10-7 : 1
196.463 ± 27.528 (**)
5.12*10-7 : 1
179.638 ± 19. 015 (*)
6.98*10-7 : 1
49.95 ± 9.533
7.75*10-7 : 1
142.908 ± 49.165 (*)
3.95*10-7 : 1
63.429 ± 10.802
2.43*10-7 : 1
3.02*10-7 : 1
385,748 ± 50.41 (***)
260.55 ± 22.070 (*)
165.669 ± 31.263
Functional cluster analysis of differentially regulated genes in monocytes between FH individuals and controls.
Welch T-Test (p < 0.05, Benjamini Hochberg Testing Correction FDR = 0.05)
Functional GO cluster
Group Enrichment Score
2,318 transcripts regulated
protein transport, establishment of protein localization, intracellular protein transport
clathrin coated vesicle, trans Golgi network transport vesicle, Golgi associated vesicle membrane, transport vesicle membrane
vacuole, lysosome, lytic vacuole
regulation of JNK activity activation of JNK activity,, positive activation of JNK activity, activation of MAPK activity
clathrin coated endocytotic vesicle, AP-2 adaptor complex, endocytotic vesicle membrane
Individual LDL-R characteristics of each FH patient those monocytes where analyzed by microarray.
C88R (FH Münster 1), D333G (FH Münster 2); < 5% LDL-R activity, binding defect
W556R; < 5% receptor activity (class 2A)
W556R; < 5% receptor activity (class 2A)
W556R; < 5% receptor activity (class 2A)
LDL-R binding defect demonstrated by fibroblast LDL binding assay
Diagnosis based on clinical parameters according to the Simon Broome Register Group criteria (xanthomas, lipid profile, familial history)
promoter defect -135 bp C->G, 5–15% activity
C88R (FH Münster 1), 15–30% receptor activity binding defect
D333G (FH Münster 2), 15–30% receptor activity binding defect
C88R (FH Münster 1), 15–30% receptor activity binding defect
LDL-R binding defect demonstrated by fibroblast LDL binding assay
W556R; < 5% receptor activity (class 2A)
insertion of G at 588 bp -> STOP at codon 178
insertion of G at 588 bp -> STOP at codon 178
We also found the intracellular levels of lanosterol, a precursor in the de-novo cholesterol biosynthesis, reduced by about 50% in monocytes from homozygous FH patients (control monocytes: 0.019887 μg lanosterol/mg cellular dry weight; FH monocytes: 0.0100980 μg lanosterol/mg cellular dry weight; p < 0.05). Taken together, these data argue for a down-regulation of the SREBP-pathway due to an elevated uptake of nLDL by an unknown mechanism and oxLDL via CD36 resulting in a cellular cholesterol overload of FH monocytes.
An important role of macrophages in arterial wall metabolism and in the pathogenesis of atherosclerosis is well documented . We were interested to see, if already their precursors in the bloodstream, the monocytes possess an increased lipid content. Oil Red O staining of freshly isolated monocytes revealed that FH monocytes have higher lipid content than control monocytes. They also show an increased uptake of nLDL and oxLDL. This and the three to fourfold higher plasma concentrations of oxLDL and nLDL in FH patients can explain the observed higher number of lipid-laden monocytes in FH. Our data is in agreement with recent reports from LDLR deficient mice which have an increased intracellular cholesterol content in macrophages .
To assess the consequences of the intracellular lipid enrichment in vivo we analyzed gene expression profiles from freshly isolated FH patient monocytes and from control monocytes using whole genome cRNA microarrays. The gene expression profiles of FH monocytes were clearly distinct from those of healthy individuals as demonstrated by unsupervised principal component analysis. Further, GO enrichment analyses pointed to an altered vesicle transport from the plasma membrane to the lysosome. We identified two scavenger receptors, CD36 and Lrp1 to be overexpressed in FH monocytes. CD36 has been identified as one of the principal receptors for oxLDL uptake by macrophages in mice [17, 21]. The expression of this receptor is largely restricted to lymphoid and haematopoietic lineages, including monocytes, macrophages, platelets and endothelial cells . Blocking of CD36 uptake with an antibody resulted in a reduction of oxLDL uptake by more than 50% which identified CD36 as the major receptor for oxLDL in circulating monocytes from FH patients and healthy controls. However, the total amount of oxLDL uptake via CD36 clearly distinguishes FH monocytes from control monocytes.
Although none of the homozygous FH patients who took part in this study had a residual LDL-R activity of more than 5 percent and LDL-R expression was slightly down-regulated in these individuals (data not shown), we observed a 50% higher nLDL uptake in FH monocytes. This finding was surprising, as LDLR defective fibroblasts do not take up nLDL. It indicates that FH monocytes have an alternative uptake mechanism for nLDL not present in FH fibroblasts. Although Lrp1 was shown to be a receptor for the binding and uptake of native LDL in peritoneal and J774 macrophages [23, 24], we found no participation of this receptor in the uptake of nLDL by FH monocytes. Llorente-Cortes et al. recently reported that in monocyte-derived macrophages Lrp1-expression is regulated by Srebf2, whereupon a down-regulation of Srebf2, due to elevated intracellular cholesterol, results in a down-regulation of the LDL receptor and in an up-regulation of Lrp1 which is in good concordance with our findings [25, 26]. Although we cannot explain the mechanism for nLDL uptake, macropinocytosis could be one possible explanation, as it has recently been shown that this process contributes to foam cell formation by the uptake of nLDL in macrophages .
To prevent a cholesterol/lipid overload macrophages are able to release excessive cholesterol via Npc1 mediated transport to intracellular destinations and the subsequent efflux of cholesterol to apo A1 via Abca1 . Both proteins were found down-regulated in FH monocytes, which further contributes to the lipid overload of FH monocytes. Studies regarding the function of oxLDL with respect to ABCA1 transcription have indicated that in macrophages oxysterols lead to the induction of ABCA1-expression via LXR [28, 29] which is different from our observation in FH monocytes where expression of LXRα and LXRβ is unchanged. However, Zhou et al recently reported that LDLR deficiency impairs ABCA1 expression in macrophages via a SREBP dependent mechanism . ABCA1 expression was also inhibited under oxysterol treatment and its regulation was independent from LXRα and LXRβ expression. They reported that LDLR is crucial in the regulation of ABCA1 expression by inhibiting SREBP1 proteolysis. Lack of LDLR led to abnormal SREBP response which resulted in both, reduced ABCA1 expression and cholesterol efflux . This may explain the reduced ABCA1 expression observed in FH monocytes. Our observation of an impaired ABCA1 gene expression in response to human LDL receptor deficiency during differentiation of FH monocytes into macrophages suggests that deranged reverse cholesterol transport is a major contributor to foam cell formation in FH. Furthermore, the lipid overload of FH monocytes is likely to cause the observed down-regulation of the cholesterol sensitive SREBP2, which is a positive regulator of ABCA1 transcription  and thus can aid to further enhance ABCA1 repression. It has been reported that ABCA1 is absent in cells of atherosclerotic lesions , which demonstrates that the tight regulation of this efflux system is critical to atherosclerosis development. Interestingly, INSIG2 and SCAP, which are also members of the SREBP pathway that regulates intracellular cholesterol content , and the rate limiting enzyme of the cholesterol de novo synthesis, HMGCR, are down-regulated in FH monocytes as well. In addition, intracellular lanosterol, a precursor of cholesterol in the de novo biosynthesis pathway, was found to be decreased in FH monocytes, further arguing for a cellular lipid overload caused by elevated lipid influx and reduced efflux.
In conclusion, we have demonstrated significant differences between circulating monocytes from patients with FH and those from control persons. FH monocytes contain larger amounts of lipids as evidenced by Oil Red O staining. This finding can be explained by the observed increased uptake of nLDL and oxLDL, which is accompanied by a down-regulation of the SREBP pathway and its target genes, and by a reduced level of intracellular lanosterol. Moreover, a down-regulation of key proteins of cholesterol transport and cholesterol efflux was observed. The increased uptake of oxLDL into FH monocytes was found to be largely mediated by the scavenger receptor CD36. We have identified processes that substantially alter the metabolism of circulating monocytes in an extreme hypercholesterolemic environment such as the blood compartment in FH. These changes that lead to a lipid overload of monocytes already in the circulation may promote the formation of local inflammatory sites that favour the onset of atherosclerosis. The large numbers of genes that are significantly altered in FH monocytes suggest, however, that many as yet unidentified genes also contribute to this process.
We thank all blood donors for donating blood for this study and the help of Dr. T. Schreiner, Prof. Dr. K. Oette, Prof. Dr. H. Borberg (German Hemapheresis Center Cologne), Prof. JR. Schäfer and Prof. G. Klaus (University Marburg-Giessen). We are further grateful to Barbara Kühn for technical assistance. The authors wish to thank D. K. Strickland (Department of Surgery, University of Maryland, USA) for providing the Lrp1-antibody. We are also thankful to F. Brew, M. Pruess, and Affymetrix for supporting this project. This work was supported by a grant from BMBF (project 01ZZ0105).
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