Published online before print February 27, 2003, doi: 10.1161/01.ATV.0000063614.21233.BF
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© 2003 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Enzymatically Modified LDL Induces Cathepsin H in Human Monocytes
Potential Relevance in Early Atherogenesis
From the Institute of Medical Microbiology and Hygiene and Department for Clinical Chemistry and Laboratory Medicine (M.T.), Johannes Gutenberg-University Mainz, Germany, and Department of Molecular and Preventive Medicine and CREST (S.I.H.), School of Medicine, University of Tokyo, Japan.
Correspondence to M. Husmann, Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-University Mainz, Hochhaus am Augustusplatz, 55131 Mainz, Germany. E-mail MattHusmann{at}web.de
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Abstract |
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Objective— Modification with proteases and cholesterylesterase transforms LDL to a moiety that resembles lipoproteins isolated from atherosclerotic lesions and possesses atherogenic properties. To identify changes in monocyte-derived foam cells laden with enzymatically modified LDL (E-LDL), we compared patterns of the most abundant transcripts in these cells after incubation with LDL or E-LDL.
Methods and Results— Serial analyses of gene expression (SAGE) libraries were constructed from human monocytes after treatment with LDL or E-LDL. Several tags were differentially expressed in LDL-treated versus E-LDL–treated cells, whereby marked selective induction by E-LDL of cathepsin H was conspicuous. We show that cathepsin H is expressed in atherosclerotic lesions in colocalization with E-LDL. Furthermore, we demonstrate that LDL modified with cathepsin H and cholesterylesterase can confer onto LDL the capacity to induce macrophage foam cell formation and to induce cathepsin H.
Conclusions— Cathepsin H could contribute to the transformation of LDL to an atherogenic moiety; the process might involve a self-sustaining amplifying circle.
Key Words: enzymatically modified LDL • monocytes • foam cells • cathepsin H
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Introduction |
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There is wide consensus that atherogenesis is triggered by enhanced entrapment of LDL in the intima, which is followed by its uptake by macrophages that assume the appearance of foam cells.1 Thereby oxidation of LDL is generally believed to be of critical importance, because the lipoprotein will thus be converted to a ligand of scavenger receptors.2 All of the constituents of LDL, including phospholipids, cholesterol, fatty acids, and apolipoprotein, can be subject to oxidation and might contribute to the countless effects that have been observed with oxidized LDL. On the other hand, nonoxidative modifications of LDL can also generate potentially atherogenic molecules, and we have previously demonstrated that LDL modified by trypsin and cholesterylesterase (enzyme-modified LDL [E-LDL]) shares characteristics with lipoproteins isolated from atherosclerotic lesions, including particle size, capacity to bind CRP and to activate complement, and reactivity with a monoclonal antibody raised against the enzymatically modified lipoprotein.3–8 This led us to investigate the biological effects of E-LDL in vitro. Initially, our studies were confined to a selection of candidate factors that are conventionally implicated in the pathogenesis of the disease.7,8 To analyze the transcriptional response of human monocytes in an unbiased fashion and on a large scale, we now ventured to perform a serial analysis of gene expression (SAGE). This method offers several advantages over other strategies for the detection of differentially expressed genes.9–12 Comprehensive SAGE profiles have recently been established for resting and LPS-stimulated monocytes and for colony-stimulating factor–induced macrophages, and these are available for comparative analyses.13,14 We performed SAGE on mRNA populations from LDL-treated and E-LDL–treated monocytes, and this led to the identification of cathepsin H as a candidate protease that may contribute to the enzymatic modification of LDL in vivo.
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Methods |
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Cells and Culture Conditions and LDL Preparations
Buffy coats obtained from healthy donors were provided by the Blood Bank (University of Mainz); monocytes were isolated as described.7 The myelomonocytic THP-1 cell line was obtained from American Type Culture Collection, and culture conditions were as recommended by the supplier.
Preparation of LDL and E-LDL were as described previously.4 For LDL modification with cathepsin H and cholesterylesterase (CC-LDL), 2.5 µg of purified cathepsin H (Calbiochem) corresponding to 
0.5 mU was added to 1 mL of LDL (3 mg cholesterol/mL) and the mixture was incubated at 37°C for 18 hours. Thereafter, 5 µL of protease inhibitor cocktail (Roche) and 40 µg/mL cholesteryl esterase (Roche) were added into the mixture and incubated at 37°C for another 18 hours at 37°C. As expected, this treatment yielded an electronegative species of LDL, as assessed by agarose gel electrophoresis. E-LDL was then depleted of 50% of free fatty acids by incubation with fatty acid free human serum albumin (Sigma) and subsequent floatation in a sucrose gradient to abrogate cytotoxicity.15 Cells were cultured in serum- and cytokine-free RPMI-1640 for 24 hours in the presence of either lipoprotein at a concentration of 100 µg cholesterol/mL. At this point, ie, 24 hours after isolation, monocytes grow adherent to the plastic dishes.
Serial Analysis of Gene Expression
The basic procedure of SAGE followed the protocol described by Velculescu et al.9 Isolation of total RNA and mRNA and cDNA synthesis was performed with the RNAgents kit from Promega and with the Messagemaker kit and Superscript Choice System (both from Invitrogen, Life Technologies). First-strand synthesis was primed with a gel-purified 5'-biotinylated oligo-dT (Integrated DNA Technologies, Inc). The biotinylated cDNA was extracted with phenol-chloroform, precipitated with ethanol and subsequently digested with NlaIII as the anchoring enzyme.
The biotinylated portion (3'-end) of the digestion products were bound to Dynabeads M-280 Streptavidin (Dynal). Linker-ligation, release of cDNA tags with BsmFI, and blunt ending followed the published protocol, as did the subsequent steps of ditag formation, polymerase chain reaction amplification of ditags, isolation and purification of ditags, concatenation, and cloning. Sequencing was performed on ABI automated sequencers (Applied Biosystems). Data were analyzed using the SAGE2000 software, supplied by K.W. Kinzler, Johns Hopkins Oncology Center, Baltimore, Md.
Western Blot Analyses
Monocytes from 4 healthy donors were isolated and treated with LDL or E-LDL for 24 hours. Cells were detached and washed once in PBS, and the cell pellet was solubilized in 50 µL of SDS-loading buffer and incubated at 60°C for 2 minutes. Proteins (from 1x106 cells) were separated by SDS-PAGE (8%) and transferred to nitrocellulose membranes (Schleicher & Schuell) by semi-dry blotting. Membranes were blocked for 30 minutes in 5% (wt/vol) dry milk powder in PBS and incubated with a rabbit polyclonal antibody against human cathepsin H (20-CR76, Fitzgerald), and blots were developed by enhanced chemiluminescence.
Coronary Artery Specimens and Antibodies
Specimens of coronary arteries were obtained from human hearts at autopsies, fixed in 4% buffered formalin, and then embedded in paraffin. Specimens of early and advanced atherosclerotic lesions16 were selected, and serial transverse sections (4 to 5 µm) were used for immunohistochemistry. The rabbit polyclonal antibody directed against human cathepsin H was also used for the staining of cathepsin H. E-LDL was detected by a murine monoclonal antibody, AIL-3, which recognizes a neoepitope in proteolytically nicked apolipoprotein B.6
Immunohistochemistry
Immunohistochemical staining was performed as described.4,6 In brief, serial sections of deparaffinized slides were treated with 3% H2O2 to block endogenous peroxidase activity and then blocked with host serum of secondary antibody. After the blocking, slides were incubated with primary antibodies of cathepsin H or E-LDL diluted in PBS at 1:500 and 1:5000, respectively, for 1 hour at room temperature, followed by incubation with biotin-conjugated goat anti-rabbit IgG or horse anti-mouse IgG (Vector Laboratories) for 30 minutes. After the incubation with biotin-conjugated secondary antibodies, avidin-biotin peroxidase reagents were incubated for 30 minutes and the reaction was developed with diaminobenzidine tetrachloride (brown color deposits). Finally, the slides were counterstained with hematoxylin and mounted. Normal rabbit serum or irrelevant isotype-matched antibodies were used to control staining specificity.
Staining of Foam Cells and Cholesterol Measurement
Cells were washed twice with PBS and subsequently fixed in 10% formaldehyde in PBS for 30 minutes at room temperature. Intracellular lipid droplets were stained with a saturated solution of oil-red O (Sigma) in 60% isopropanol in PBS for 3 hours. Total cholesterol was quantified using a kit from Roche.
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Results |
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Abundant Transcripts in Monocytes After Incubation With LDL or E-LDL
Cloned concatemers from cDNA libraries derived from E-LDL–treated or LDL-treated monocytes were analyzed by automated sequencing. After exclusion of irrelevant sequences (linker, incorrect length, etc), a total of 11 694 (LDL) and 11 494 (E-LDL) tags were identified, corresponding to 6288 individual transcripts in the LDL library and 6326 in the E-LDL library. Of these, 1374 and 1350 different tags, respectively, were found
2 times in the LDL- and E-LDL library, thereby passing the usual criteria for additional analysis. Table 1 shows the 50 most abundant tags identified from the libraries. At the top of each list is a tag representing mRNA coding for ferritin heavy polypeptide. This tag alone accounted for 1.23% and 1.32%, respectively, of all tags in the 2 libraries; these figures precisely matched the reported frequency of the tag in unstimulated human monocytes.17 Ferritin heavy chain was followed by tags derived from ß2-microglobulin mRNA, ferritin light chain, and cathepsin D, which also occurred in normal monocytes at similar frequencies as found in the present investigation.17 The sixth most frequent tag in both E-LDL and LDL profiles was that of fatty acid binding protein 4 (FABP4), which is not expressed in normal monocytes (Dr S.I. Hashimoto, unpublished data, 1999). Furthermore, psoriasis-associated fatty acid binding protein (FABP5), which is structurally and functionally related to FABP4, ranked among the top 50 tags in the LDL and E-LDL libraries (position 21). This transcript is also absent in normal monocytes. Tag number 24 in the list (aattaaatta) was conspicuous, because it was increased 3-fold in E-LDL–treated versus LDL-treated cells (0.26 versus 0.09%), whereas it is reportedly downregulated in LPS-treated monocytes (from 0.05% to undetectable). This tag is not unique to a single transcript, so additional analyses are needed to identify the relevant gene product in the present case.
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Differentially Expressed Tags in E-LDL–Treated Versus LDL-Treated Monocytes
Tags and the corresponding gene products that were found at frequencies differing 6-fold in monocytes treated with E-LDL or LDL are listed in Table 2 (upregulated by E-LDL) and Table 3 (downregulated by E-LDL). Apart from several translation-related genes, the most conspicuous upregulation by E-LDL was found for tags representing transcripts of cathepsin H and phospholipase A2 (PAF-acetylhydrolase). The transcripts most downregulated by E-LDL have not been described to be associated with atherosclerosis.
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In addition to these, a large array of tags was detected at different frequencies in LDL- and E-LDL libraries, but with a lower total number of tags. The total data set can be obtained from the authors on request.
Cathepsin H Protein Expression in Monocytes Is Increased by E-LDL
Cathepsins have been implicated in the remodelling of the vessel wall,1,18,19 and so the observed upregulation of cathepsin H was of high interest. The ratio of the tag in the LDL versus E-LDL library was 1:10, rendering the probability P for a chance observation of this difference to be <0.006. To ascertain that the SAGE result had a genuine biological correlate, monocytes were treated with lipoprotein preparations and analyzed by Western blot using a specific antibody directed against cathepsin H. Expression of cathepsin H was enhanced in E-LDL–treated cells compared with LDL-treated cells, a result that was confirmed in 4 donors. Figure 1A depicts a representative blot; oxidized LDL (oxLDL) was additionally used for comparison. A strong band corresponding to mature 28-kDa cathepsin H was seen in E-LDL–treated cells (Figure 1A). In addition, a second band running at 13 kDa was also detected. The intensity of this band correlated with that of the corresponding 28-kDa band in each lane; most likely it represents the pro-piece cleaved off from the 41-kDa precursor. A very faint band of 41 kDa could also be discerned in 2 of the 4 E-LDL–treated samples (not shown), suggesting incomplete precursor processing in these samples.
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Cathepsin H Supports Generation of Atherogenic LDL
Enzymatic transformation of LDL to atherogenic E-LDL requires combined treatment with the protease and cholesterylesterase. We have been using trypsin, which is effective but unphysiological, and it was of interest to discern whether cathepsin H could replace trypsin in the reaction mix. Treatment of LDL with a mixture of purified cathepsin H and cholesterylesterase generated E-LDL that was electronegative, similar to trypsin-generated E-LDL (data not shown). This modification, which we term CC-LDL (cathepsin H/cholesterylesterase-modified LDL), induced cathepsin H in monocytes (Figure 1B) and foam cell formation as revealed by oil-red O staining (Figure 2). Quantification of cellular cholesterol revealed a 5-fold increase of sterol content after incubation of the cells with 100 µg/mL TC-LDL or CC-LDL compared with an equal amount of LDL.
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Cathepsin H Colocalizes With E-LDL in Human Atherosclerotic Lesions
Next we sought evidence for cathepsin H expression in human coronary atherosclerotic lesions. To this end, tissue sections were stained for cathepsin H using rabbit polyclonal antibody, 20-CR76, and compared with E-LDL staining, which is already known to be present in vivo.6 The anti–E-LDL antibody AIL-3, which was derived against LDL modified by trypsin/cholesterylesterase treatment, also reacted with E-LDL generated by sequential incubation with cathepsin H and cholesterylesterase as demonstrated by ELISA (not shown). As shown in Figure 3A (right panel), the predominant manifestation of E-LDL deposits in an early lesion was a diffuse deposition in the deep fibroelastic and fibromuscular layers of the intima adjacent to the media, as described previously.4,6 There was a close association and definite overlapping of E-LDL and cathepsin H epitopes within the deeper portion of the intima. In a more advanced atherosclerotic lesion (Figure 3B), cathepsin H (left panel) also colocalized with the E-LDL epitopes (right panel) more closely. Both cathepsin H and E-LDL epitopes were diffusely scattered among the extracellular components of the lipid cores of lesions, predominantly in the central part (left panel). In addition, massive intracellular staining for cathepsin H of foam cells was evident. As reported previously, these cells did not stain positively for E-LDL, which was likely attributable to destruction of the neoepitope in the course of extensive proteolysis, as has been demonstrated in vitro.6 Control staining performed with normal rabbit serum or the irrelevant isotype-matched antibodies yielded negative results with all tissue specimens (data not shown).
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Discussion |
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SAGE was used to investigate transcriptional changes in monocytes in response to E-LDL because it represents an unbiased, open screening method and because emphasis is inherently on abundant transcripts, which are therefore likely to be of relevance. After commencement of the project, a large-scale transcript profile of monocytic cells was published in which PMA-prestimulated THP-1 cells were analyzed after incubation with oxLDL using microarrays with 6800 genes20; to our knowledge, this is the only available large-scale gene expression analysis of human macrophage foam cells. Despite their usefulness as a model for many macrophage functions, however, PMA-stimulated THP-1 cells likely differ from primary cells in their transcript profiles, and we consequently elected to use unstimulated blood monocytes in our study.
SAGE analyses of unstimulated human monocytes, LPS-treated monocytes, and colony-stimulating factor–induced macrophages have been published,13,14 and they provided excellent references for this investigation. Tag frequencies for major abundant transcripts were found to be remarkably similar to the published reports, which demonstrated the high degree of reproducibility of the method and lent credence to the differences observed. One was the high abundance of tags for fatty acid–binding proteins 4 and 5 in lipoprotein-treated cells. FABPs probably serve as intracellular transporters of fatty acids21 and may also assume regulatory functions.22 Increased expression of FABPs likely represents an adaptive response of cells to the enhanced uptake of fatty acids. Although FABP4 was reported to be enhanced in THP-1 cells after treatment with oxLDL compared with native LDL,23 we discerned no differences in either FABP4 or FABP5 tags in LDL-treated versus E-LDL–treated monocytes.
Stimulation of monocytes with LPS or differentiation with colony-stimulating factors leads to marked upregulation of apolipoprotein C-1 and apolipoprotein E,14,17 but this was not observed in lipoprotein-treated cells. On the other hand, lipoprotein-treated cells exhibited several changes that have been observed during differentiation of monocytes to macrophages. For example, MRP-14, the most abundant transcript in monocytes, disappears on granulocyte-macrophage colony-stimulating factor stimulation and was also absent in both of our libraries. Conversely, NMB, a transmembrane glycoprotein that is expressed in macrophages but absent in monocytes, is abundant in our libraries. A second relevant aspect relates to the absence of markers characteristic of the inflammatory macrophage phenotype in lipoprotein-treated cells. For example, MIP1
, by far the most abundant transcript in LPS-stimulated monocytes, was not detected in our libraries from both LDL-treated and E-LDL–treated cells. Similarly, interleukin-8, which is reportedly stimulated by oxLDL,24 was markedly upregulated by LPS, whereas the frequency of this transcript is >10-fold lower in our libraries. Collectively, these observations add to the realization that E-LDL induces responses in monocytes that deviate fundamentally from patterns that have been observed with proinflammatory agents, growth factors, and oxLDL.
The principal goal of this study was to identify genes differentially regulated in monocytes treated with native versus enzymatically modified LDL, and several candidate transcripts were identified. Cathepsin H ranked second among the genes upregulated by E-LDL. Satisfactorily, Western blot analyses confirmed the SAGE data and showed enhanced cellular amounts of cathepsin H in monocytes from all 4 tested donors after E-LDL treatment. Cathepsins are cysteine proteases25 usually located in lysosomes. However, these enzymes are not strictly confined to this compartment; cathepsin B and L have been detected in the cytoplasm and nuclei of apoptotic macrophages within human atheroma.26 For cathepsin H, it has been shown that substantial concentrations of the enzyme circulate in the blood.27 Together with stromelysin and metalloproteases, cathepsins have been suspected to contribute to the destruction of the extracellular matrix and are implicated in the evolution of the unstable plaque.1 This is supported by the finding that cathepsin B activity, as detected by in vivo imaging, is associated with the vulnerability of plaques.28 Here we detected cathepsin H in extracellular compartments of plaques, a finding that concurs very nicely with the report that tissues from abdominal aneurysmata express 30-fold higher levels of cathepsin H mRNA than normal aorta.29 Indeed, cathepsin H mRNA led the list of enhanced transcripts in that study.
The question then arises of how cathepsin H expression in the atherosclerotic plaque is triggered. As shown here, at least one possibility is the induction of cathepsin H by enzymatically modified LDL. The observed colocalization of E-LDL and cathepsin H is in accord with this possibility. In some contrast, cathepsin H upregulation was not mentioned in the microarray study of THP-1 cells loaded with oxLDL.20
Although the pathogenic potential of cathepsin H in the development of the late, unstable plaque is quite evident, the possibility now emerges that this protease may already play a role in early atherogenesis. Cathepsin H is one of the few noncomplement proteases that cleaves native C5 to generate the potent chemotaxin C5a.30 Furthermore, we have found that cathepsin H supports the formation of E-LDL. Cholesterylesterase, the second enzyme needed to generate E-LDL, might be cosecreted from infiltrating macrophages in the lesion. Cathepsin H thus emerges as an agent that could propel events leading to foam cell formation via several mechanisms.
The present analysis also indicated that E-LDL induces phospholipase A2 (PAF acetyl-hydrolase).31 PAF acetylhydrolase has been detected by immunochemistry in macrophages of atherosclerotic lesions,32 but the data on its role in the disease are somewhat conflicting: because PAF acetylhydrolase attacks oxidized phospholipids,33 it could additionally contribute to the accumulation of fatty acids in the lesion and thereby drive atherogenesis. On the other hand, expression of PAF acetylhydrolase in the apolipoprotein E-/- mouse was shown to protect against neointima formation.34 Possibly the spatiotemporal coordination of expression is critical for the net effect.
The ATP-binding cassette protein A1 (ABCA1), although reportedly upregulated by modified LDL,35 was not represented in our libraries. This may simply have been attributable to the low copy number: ABCA1 has been found to be a rather rare transcript in all accessible SAGE libraries (1:50 000) and, therefore, the analyses of a much larger number of clones would have been required to detect even a 10-fold upregulation of this or other nonabundant candidate genes.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft, grant Bh 2/3–1, and the Verband der Chemischen Industrie. Dr Suriyaphol received partial support from Mahidol University, Bangkok. We thank Rosemarie Schweigert for expert technical assistance.
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Footnotes |
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*These authors contributed equally to this work.
Received December 17, 2002; accepted January 20, 2003.
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