In Vitro Biomarker Discovery for Atherosclerosis by Proteomics*

Estelle M. Fach{ddagger}, Leah-Ann Garulacan, Ji Gao, Qing Xiao, Stephen M. Storm, Yves P. Dubaquie, Stanley A. Hefta and Gregory J. Opiteck

From the Department of Clinical Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, NJ 08543


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The purpose of this study was to identify in vitro and then prioritize a tractable set of protein biomarker candidates of atherosclerosis that may eventually be developed to measure the extent, progression, regression, and stability of atherosclerotic lesions. A study was conducted using an in vitro"foam cell" model based on the stimulation of differentiated THP1 cells with oxidized low-density lipoprotein (oxidized LDL) as compared with low-density lipoprotein (LDL). Analysis of the proteins contained in the cell supernatant using proteome scanning technology identified 59 proteins as being increased, 57 with no statistically measurable difference, and 17 decreasing in abundance following treatment with oxidized LDL, as compared with LDL. From the up-regulated list, proteins were prioritized based on their analytical confidence as well as their relevance to atherosclerosis pathways. Within the group of increased abundance, seven families of proteins were of particular interest: fatty acid-binding proteins, chitinase-like enzymes, cyclophilins, cathepsins, proteoglycans, urokinase-type plasminogen activator receptor, and a macrophage scavenger receptor.


Atherosclerosis, and the resulting coronary heart disease and cerebral stroke, is the most common cause of death in industrialized nations (1). Although certain key risk factors have been identified (2), a highly sensitive and specific diagnostic assay that would provide information on the extent, growth, regression, stability, and/or type of atherosclerotic lesions remains to be conclusively identified. Imaging, although effective, is an expensive technique (3, 4). While C-reactive protein (5, 6) and oxidized low-density lipoprotein (oxidized LDL)1 (7, 8) are both gaining attention as independent biomarkers (risk predictors) of atherosclerotic events, neither have been widely embraced and utilized clinically as diagnostics of vessel wall lesions, lesion stability, or lesion load. Therefore, much work remains to be done to find a true pharmacodynamic marker of atherosclerosis (as opposed to hypercholesterolemia) that could be used in clinical trials of anti-atherosclerotic compounds (9).

Cholesterol-laden macrophage models of atherosclerosis have been used previously (1012), because such cells comprise a large percentage of arterial plaques and lesions. LDL is poorly taken up by macrophages in vitro unless it has been modified, because macrophages contain only one native LDL receptor, but several scavenger receptors (1318). These scavenger receptors transport the oxidized LDL from the bloodstream to the subendothelial space, where antioxidant defenses are less prevalent (19). The unregulated uptake of oxidized LDL, and its poor degradation by the macrophages, results in the accumulation of multiple lipid droplets, which is designated as the "foam cell" phenotype (20). Gene expression profiling using microarrays and the foam cell model has proven to be a useful tool in the identification of new genes that may contribute to features of the atherosclerotic lesion (21). However, because a change in gene expression does not necessarily lead to measurable changes in plasma protein levels (22), the use of this model with proteome profiling in lieu of mRNA profiling was deemed to be a worthwhile endeavor.

The hypothesis being tested herein was that an in vitro proteomics study based on the aforementioned foam cell model would generate a set of promising protein biomarker candidates of the atherosclerosis state. By focusing on only the secreted and/or excreted proteins found in the cell culture media, there would be an intended bias toward those molecules that would have a higher probability of later being found in the blood stream (plasma/serum). Then, using knowledge of the pathogenesis of atherosclerosis (23, 24), the list of candidate biomarker proteins derived during the in vitro experiments would be surveyed to find proteins of interest and that could be easily measured using immunoassays (2527). This triage system would thereby leave only a handful of protein biomarker candidates to be taken forward in future studies where these candidates would then be measured in plasma samples collected from disease state and normal/healthy human populations.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Cell Culture and Treatment—
Human THP-1 cells (10801; American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium containing 10% fetal serum (v/v), 0.45% glutamine (w/v), 10 mm HEPES, 1 mm sodium pyruvate, 1 x 105 m ß-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 µg/ml) and kept at 37 °C in an atmosphere of 5% CO2. The cell medium was replaced every 2 days for the duration of the culture prior to the beginning of cell treatment. Cells were seeded at a density of 1x106/ml in medium containing phorbol myristate acetate (PMA) (Research Biochemical International, Natick, MA) at 1 x 107 m for 72 h. The medium was then replaced by culture medium with 100 µg/ml of vehicle (50 mm Tris, pH 7, 150 mm NaCl, 0.3 mm EDTA), LDL, or oxidized LDL (Intracel, Rockville, MD) (28), which was Cu2+ fully oxidized according to the manufacturer’s certificate of analysis. A marked morphological change was recorded upon PMA treatment, as was a phenotypic change upon oxidized LDL treatment (data not shown), both in agreement with previous reported results on the foam cell model (21). The same number of cells, 3 x 107, was used for each treatment. Both the LDL and oxidized LDL were analyzed by LC-MS/MS to verify that they were free from other proteins, within the limit of detection (data not shown). Exactly 12.0 ml out of 15 ml of total supernatant volume were collected from each treatment after 48 h of incubation and frozen at –80 °C until further analysis. Cell death was assayed by an ELISA (Oncogene Research Products, San Diego, CA), which measured levels of "nuclear matrix protein 41" in the supernatants.

Preparation and Analysis of In Vitro Samples—
To improve the sensitivity of biomarker detection by reducing the dynamic range of the proteins in the cell supernatant, the apolipoproteins introduced during the stimulation step were subsequently removed from the cell-free samples by anion exchange HPLC (SuperQW, 7.5 mm x 7.5 cm; Tosoh, Montgomeryville, PA) on a HPLC system (Model 1100; Agilent, Wilmington, DE). Aliquots of oxidized LDL and LDL were previously analyzed by this method to determine their retention times (data not shown). Fractions containing the secreted/excreted proteins, but not the LDL or oxidized LDL, were reduced, alkylated, and digested as described previously (29). The digests were subjected to size-exclusion chromatography (G2000SWXL; Tosoh) in order to remove undigested protein and excess trypsin (30). Following this step, the peptides were subjected to cation-exchange HPLC (Zorbax 5 µm 300-SCX 4.6 x 50 mm; Agilent). The five fractions having the highest area of UV absorbance at 215 nm were then concentrated and desalted by solid-phase extraction (MicroSpin; The Nest Group, Southborough, MA). The peptides were then sequenced by capillary HPLC coupled to a tandem mass spectrometer (LC-MS/MS), as previously described (31). The proteins identified from each sample were clustered using the BLAST algorithm and compared across treatments using a proprietary ANALYSIS software package to determine relative changes in protein concentration on the basis of peptide "hits" (31, 32).

Immunoassays—
Immunoassays were used to validate the quantitative and qualitative mass spectrometric results on four proteins of interest. Western blots and ELISAs were conducted on aliquots of cell supernatants that had not been manipulated (concentrated, extracted, digested, etc.). Intact aliquots (10 µl) of each treated cell supernatant, which had been generated in triplicate, were withdrawn, diluted in an equal volume of 2x SDS sample buffer under reducing conditions (Nupage LDS; Invitrogen, Carlsbad, CA), boiled for 5 min and separated using 4–12% Bis Tris gels and MES buffer (Invitrogen). Following electrophoresis, the proteins were transferred to a PVDF membrane at 30 V for 1 h, and the blots were subsequently blocked with 5% nonfat dry milk in PBS including 0.05% Tween 20 (Sigma, St. Louis, MO) overnight at 4 °C with gentle shaking. The blots were probed with rabbit anti-cyclophilin B (Bioreagents, 1:2,000). Following incubation with the appropriate HRP-conjugated secondary antibody (anti-rabbit IgG, 1:5,000), the proteins were visualized using ECL detection (Amersham, Piscataway, NJ) and imaged using a cooled charge-coupled device camera (Fluor S-Max; Bio-Rad, Hercules, CA). Densitometry software (Quantity 1; BioRad) was used for analysis of band intensity. An ELISA was used to analyze the level of CATH L (Alexis Biochemicals, San Diego, CA) and the tissue inhibitor of metalloproteinase 1 (TIMP1) (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

The immunoassay data were not normalized to the total amount of protein in each supernatant, as it varied as a result of the lipoprotein treatment. Instead, the data are proportional to the total supernatant volume, which were each carefully controlled during cell culture and harvest.

Statistical Analysis—
Pairwise Student t tests (p ≤ 0.2) were used to compare the number of MS peptide hits to each protein and establish up- or down-regulation. This atypical 0.2 p value was used for statistical significance in keeping with survey-type nature of proteome profiling and the reliance on follow up assays to corroborate the results. Wilcoxon two-sample t tests (p ≤ 0.1) were used for comparing the levels of cyclophilin B and cathepsin L produced in vitro by cell treatment with oxidized LDL versus LDL in the immunoassays. This test was preferred over Student t tests in this case because of the small sample set and uncertainty over the normalcy of distribution.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
In all, 133 proteins were identified and measured in the cell supernatants in the course of the in vitro profiling experiments. To describe the change in protein concentration in the supernatant following treatment, and because this proteome profiling method was semi-quantitative, the proteins identified were classified into three broad categories: up-regulated, undetermined/unchanged, or down-regulated (each relative to the LDL treatment). These three categories were based on the peptide hits to each proteins producing a statistically significant result (p ≤ 0.2) in a Student t test. In the case of equal variance, this corresponded to a minimum change of 3-fold. The 133 proteins identified fell into groups of 59 up-regulated proteins (Table I), 57 proteins of undetermined change (Table II), and 17 down-regulated proteins (Table III). To reflect the analytical confidence, proteins were ranked according to their p value and sequence coverage. Several proteins were very poorly annotated in the available databases, garnering an "XP" prefix within the dataset.


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TABLE I The 59 proteins found up-regulated in the supernatant of macrophages following treatment with oxidized LDL, as compared with LDL, based on the results of two experiments

The proteins are listed in the order of their analytical confidence, obtained from their p values (column 4), and their sequence coverage (column 5). NA applies to instances when the variance was equal to zero.

 

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TABLE II The 57 proteins for which no statistically difference was observed in the supernatant of macrophages following treatment with oxidized LDL, as compared with LDL, based on the results of two experiments

The proteins are listed in the order of their analytical confidence, obtained from their sequence coverage (column 4). All p values obtained with a pairwise t test comparing oxidized LDL and LDL in this table were higher than 0.2.

 

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TABLE III The 17 proteins found down-regulated in the supernatant of macrophages following treatment with oxidized LDL, as compared with LDL, based on the results of two experiments

The proteins are listed in the order of their analytical confidence, obtained from their p values (column 4), and their sequence coverage (column 5). NA applies to instances when the variance was equal to zero.

 
Considering that proteome profiling is a relatively new technology, and in order to validate the proteomic technique through the use of an orthogonal measurement, it was decided to further explore four proteins of biological interest and for which reagents were immediately available. To this end, immunoassays were conducted on unadulterated cell supernatants to measure the levels of cyclophilin B, cathepsin L, TIMP1, and matrix metalloproteinase 9 (MMP9). From the proteome profiling data (Table I), and within the statistical p value we chose, cyclophilin B and cathepsin L were believed to both increase with statistical certainty (p = 0.11 for cyclophilin B and p = 0.17 for cathepsin L). The results from the correspondings immunoassays supported the profiling results. For cyclophilin B, the Western blot results showed an increase of 84% (p = 0.009) in the oxidized LDL versus the LDL/vehicle-treated cell supernatants (Fig. 1). Similarly, the levels of cathepsin L in the cell supernatants (Fig. 2), as measured by an ELISA, were 5.6 ± 1.8, 5.6 ± 1.3, and 8.1 ± 2.1 ng/ml for the vehicle-, LDL-, and oxidized LDL-treated cells, respectively (p = 0.1 for LDL versus oxidized LDL).



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FIG. 1. Densitometry data of Western blots for cyclophilin B, as a result of macrophage treatment with vehicle, LDL, or oxidized LDL, based on the average of five experiments. Cyclophilin B was found to be up-regulated by LC-MS/MS, and this result was corroborated via the data shown here. The mean ± SD from densitometry analysis of the blots were 100 (which was the assigned value for normalization purposes across experiments) for the vehicle treatment, 90 (±20) for the LDL treatment, and 166 (±87) for the oxidized LDL treatment. Based on these values, the change in the level of cyclophilin B in the cell supernatants between LDL and oxidized LDL treatments was +84% (p = 0.008).

 


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FIG. 2. Levels of cathepsin L (CATH L) as determined by ELISA as a result of macrophage treatment with vehicle, LDL, or oxidized LDL, based on the average of four experiments. CATH L was found up-regulated by LC-MS/MS, and this result was corroborated via the data shown here. The mean ± SD were 5.6 ± 1.3 ng/ml for the vehicle treatment, 5.6 ± 1.8 ng/ml for LDL, and 8.1 ± 2.1 ng/ml for oxidized LDL-treated cells. Based on these values, the change in the level of cathepsin L in the cell supernatants between LDL and oxidized LDL treatments was + 69% (p = 0.11).

 
According to the proteome profiling data, both TIMP1 and MMP9 fell in the undetermined category (Table II). The levels of TIMP1 (Fig. 3), as determined by an ELISA, were found to be lower following the oxidized LDL treatment compared with the LDL (p = 0.1; 155 ± 70, 149 ± 42, and 42 ± 14 ng/ml of supernatant for vehicle-, LDL-, and oxidized LDL-treated cells, respectively). MMP9, however, showed no difference in the Western data (Fig. 4): the mean ± SD from densitometry analysis of the blots were 100 (which was the assigned value for normalization purposes across experiments) for the vehicle treatment, 107 (±6) for the LDL treatment, and 101 (±3) for the oxidized LDL treatment. The results obtained with these two proteins underline the necessity for caution in the interpretation of the results from the "undetermined category."



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FIG. 3. Levels of TIMP1 as determined by ELISA as a result of macrophage treatment with vehicle, LDL, or oxidized LDL, based on the average of three experiments. The mean ± SD were 155 ± 70 ng/ml for the vehicle treatment, 149 ± 42 ng/ml for LDL, and 42 ± 14 ng/ml for oxidized LDL-treated cells. Based on these values, the change in the level of TIMP1 in the cell supernatants between LDL and oxidized LDL treatments was –122% (p = 0.1)

 


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FIG. 4. Densitometry data of Western blots for MMP9, as a result of macrophage treatment with vehicle, LDL, or oxidized LDL, based on the average of three experiments. MMP9 was found to be unchanged by LC-MS/MS, and this result was corroborated via the data shown here. The mean ± SD from densitometry analysis of the blots were 100 (which was the assigned value for normalization purposes across experiments) for the vehicle treatment, 107 (±6) for the LDL treatment, and 101 (±3) for the oxidized LDL treatment. Based on these values, the change in the level of MMP9 in the cell supernatants between LDL and oxidized LDL treatments was –6%, which was not statistically significant (p = 0.66).

 
Seven proteins or protein families from the list of 59 up-regulated proteins were of particular interest based on their analytical confidence as well as their previously reported links to atherosclerosis: macrophage scavenger receptor 1 (MSR 1), cyclophilin B, urokinase-type plasminogen activator receptor (UPAR), chitinase-like enzymes, and cathepsins.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The PMA-differentiated THP-1 cells had previously been found to be a suitable macrophage model system (33) in atherosclerosis-related studies (1012, 34, 35) and employed previously with mRNA-based expression profiling technology (21). The use of an immortalized cell line, rather than ex vivo-derived macrophages, was also necessary because of the number of cells required to achieve the high level of total protein required for the various processing steps leading up to MS while remaining above the lower limit of detection. A previous study had demonstrated the ability of gel-free multidimensional LC, coupled to MS/MS, to rapidly and effectively analyze cellular supernatants for proteins that change in abundance as a result of treatment (29).

This technology proved here to be straightforward and successful in identifying proteins that changed as a result of treatment, as corroborated by the immunoassays performed on the cell supernatants. However, a non-negligible number of proteins, as shown in the case of TIMP1 and MMP9, although successfully sequenced, could not be precisely quantified. A key aspect of the interpretation was to not confuse absence of statistical difference with absence of change. Furthermore, because of limitations inherent to the technique, i.e. peptide hydrophobicity and ionization, not all proteins were identified with excellent peptide coverage. The sequence coverage in column 5 depends on various factors including protein abundance, peptide ionization efficiency, but also and possibly the integrity of the protein we detected. The results, though, supported the hypothesis that the combination of the THP-1 cell line and the multidimensional LC-MS were a relevant system for the discovery of biomarkers of atherosclerosis.

A number of the up-regulated proteins identified were well-known components of inflammatory pathways. Examples of these included CD14, ß2 microglobulin, rho- and rab-GDP dissociation inhibitors ß, IL-1 receptor antagonist, nitric oxide synthase, manganese superoxide dismutase, and NAD-dependent malate dehydrogenase.

Many of the proteins found in the supernatant are known to be secreted or possess a secretion signal sequence, as assessed with the SignalP Server web site (www.cbs.dtu.dk/services/SignalP). However, a number of proteins identified are nuclear (histones, nucleolin, ribonucleoprotein, calcium/calmodulin-dependent protein kinase, etc.) or plasma membrane proteins (MSR, glypicans, etc.) with no known secretion sequence, and therefore are not expected in the extracellular medium of healthy cells. The nuclear matrix protein 41 assays indicated occurrence of cell death, which would provide an explanation for the presence of such proteins; we did not, however, observe differences in cell deaths among treatments. It has been stated that oxidized LDL induces damage to the macrophage lysosomal membranes, with ensuing leakage of lysosomal contents and macrophage apoptosis (36), which would provide an explanation for the up-regulation of such proteins. It is also possible that, while cell death is constant among treatments, the increase of nuclear or membrane proteins in the supernatant is a result of increased protein translation.

TIMP1 is relevant to plaque remodeling via its action on MMPs (37, 38) and showed a clear decrease upon oxidized LDL stimulation by ELISA, which was consistent with previous reports (39, 40). We were surprised to not observe an up-regulation of MMP9 upon oxidized LDL treatment, as was reported before with monocyte-derived macrophages (39). This may originate from the choice of the THP1 cell line, as the necessary stimulation of these cells by phorbol ester is known to itself induce release of MMP9. Various reports have presented recently presented MMP9 as a solid potential atherosclerosis biomarker (4143).

The large number of candidates differentially expressed in the in vitro study, akin to previous mRNA-based expression profiling studies (21, 44), necessitated a prioritization toward proteins that displayed the highest analytical confidence as well as previously reported links to atherosclerosis. Fatty acid-binding proteins (FABPs), chitinase-like enzymes, cyclophilins, cathepsins, proteoglycans, the UPAR, and MSRs will thus be discussed in details below.

Two cytoplasmic FABPs, namely FABP4 and FABP5, were identified and quantified with excellent analytical confidence. These proteins transport fatty acids between cellular compartments, thereby modulating intracellular lipid metabolism, and most likely affecting gene expression (45). In fact, a dose-dependent increase in both transcription and translation of FABPs has been reported as a result of THP-1 cell stimulation with oxidized LDL (46). These findings are consistent with our results, as we observed an increase in the extracellular levels of FABP 4, 5, and prostaglandin D2 synthase (Table I). Two of these proteins, FABP 4 and prostaglandin D2 synthase, have already been proposed as a possible biomarkers for atherosclerosis-related diseases: acute coronary syndrome (47) and stable angina (48), respectively.

Chitotriosidase and a related enzyme, chitinase 3-like 1, were found elevated with good analytical confidence as well (Table I). These secreted enzymes, first discovered for their potential to degrade chitin-containing pathogens (49), have recently sparked interest. Patients suffering from lysosomal storage disorders display increased levels of chitotriosidase (50, 51), leading researchers to hypothesize that this enzyme is a marker of lipid accumulation in macrophages. Both chitotriosidase and chitinase 3-like 1 have been linked to atherosclerosis through large increases in their levels of expression in atherosclerotic plaques (52). In the same study, the enzymatic activity of chitotriosidase was found elevated 55-fold in extracts of atherosclerotic tissues and correlated with the dramatically increased expression of chitotriosidase mRNA (52). More recently, chitotriosidase was shown to be elevated in the plasma of individuals suffering from atherosclerosis (53), making it an attractive candidate for further evaluation in the clinical plasma samples described here. In light of the results, and because macrophages are the major source of chitotriosidase secretion, it would seem likely that foam cells have a significant contribution to the release of this enzyme in the blood stream and therefore warrants further clinical evaluation.

Based on their analytical confidence, cyclophilin A and B rank respectively 4th and 11th (Table I). Cyclophilins are a family of peptidyl-prolyl cis-trans isomerases and involved in protein folding and trafficking. In particular, cyclophilin B is considered a secreted oxidative stress-induced protein (54). Although the atherosclerosis literature precedent is scarce, the MS data, the corroborating immunoassay, the link to oxidative stress, and its documented presence in plasma (55) combine to make cyclophilin B in particular an appealing candidate as a plasma-based biomarker of atherosclerosis.

The data also suggested an increase in the release of five lysosomal enzymes, namely cathepsins D, B, L, S, and X in order of confidence (Table I). Li et al. have reported evidence of the inactivation of cathepsins B and L and a subsequent relocation from the lysosome to the cytoplasm of macrophages of the human atheroma (56, 57). Once in the cytoplasm, the release in the extracellular medium, documented in atheromatous plaques (58), might be a result of secretion, or from the programmed death of the lipid-overloaded cells (59); there is also evidence that macrophages can extrude some of their lysosomal contents (59). Cathepsin L is of particular interest because it is easily detectable in plasma. Although our data suggests an implication of the foam cells contribution in the extracellular release of cathepsins, it is still unclear if their major source in circulating blood is the activated endothelium in the entire vascular bed or the macrophages in atheroma. However, cathepsin L, and by inference other cathepsin family members, may be attractive candidates for expanded biomarker research.

Proteoglycan 1 and glypican 4 and 6 showed elevated levels following oxidized LDL treatment as well. Proteoglycans bind apolipoproteins (60), and the resulting lipoprotein-proteoglycan complexes are colony-stimulating factors (61) leading to cholesterol ester accumulation in smooth muscle cells (62), where they modulate the response of the lysozymes (62). The uptake of oxidized LDL, but not acetylated LDL, by peritoneal macrophages can be mediated by proteoglycans (63), whereas the binding of proteoglycan 1 to the MSRs has been suspected to inhibit the binding of modified LDL by peritoneal macrophages (64). Our in vitro observation of an up-regulation of proteoglycans, in addition to the accumulation of proteoglycans observed in vivo in atherosclerotic regions (65), could make this family of proteins interesting biomarkers of atherosclerosis.

The UPAR ranks 34th in our prioritized list of candidates (Table I), but extensive literature supports its possible role in atherosclerotic events. It is believed that the binding of the MSRs to apolipoprotein ligands leads to an up-regulation of the urokinase-type plasminogen activator (UPA) expression via protein tyrosine phosphorylation and an increase protein kinase activity (66). This hypothesis was supported by the work of Ganne and coworkers, who observed increases in both UPA and its receptor UPAR production by monocytes upon exposure to oxidized LDL (67). The data described herein complements Ganne’s findings and correlates with the results from a similar transcriptional profiling experiment using the THP-1 model (21). The contribution to the circulating UPAR levels from the atherosclerotic plaque may be minor compared with the contribution from smooth muscle cell. Nonetheless, there is more than adequate data to hypothesize that the circulating levels of UPAR would be elevated in patients with significant levels of atherosclerotic plaques and lesions.

Finally, although its sequence coverage was small, the data indicated an increase in the extracellular levels of MSR 1 following oxidized LDL treatment (Table I). The uptake of oxidized LDL starts with its interaction with the MSRs, implicated in both cholesterol uptake and metabolism during atherogenesis (68), which bind and internalize oxidized LDL, but not unmodified LDL (69). Our data was consistent with a report describing an increase in the transcription of MSR 1 following treatment of murine macrophages with oxidized LDL, and in peritoneal macrophages isolated from mice following injection of lipoproteins (70). While transcriptional and translational increases in MSR 1 could be rationalized as a simple response to the loading of oxidized LDL, the release of the receptor into the extracellular space could not be so readily explained. However, the MSR 1 peptides identified from the cellular supernatants originated exclusively from the extracellular domain of the protein, and not the cytoplasmic or transmembrane domains (Fig. 5). This may be caused by a splice variant form of the protein being translated and secreted to the extracellular space. Alternatively, proteolytic fragments may simply originate from the cell surface, through the action of a yet unidentified protease, as a cellular defense mechanism to the sudden onslaught of oxidized LDL. Both of these are untested hypotheses, but in either case, MSR 1 makes an attractive candidate in itself as a possible biomarker of atherosclerosis.



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FIG. 5. The single letter amino acid sequence of MSR 1. The letters in italic indicate the cytoplasmic and transmembrane regions. The bold and underlined letters indicate the specific peptides identified in the course of the MS experiment. Note that all of the peptides observed originated from the extracellular region.

 

    CONCLUSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
The purpose of this study was to identify and subsequently evaluate a set of candidate biomarkers of atherosclerosis. Proteomic profiling obviated the need for individual immunoassays and generated a number of valid candidates. Within these 59 candidates, seven proteins or families of proteins were of particular interest: FABPs, chitinase-like enzymes, cyclophilins, cathepsins, proteoglycans, UPAR, and a MSR. A large and controlled examination of these proteins in a well-defined clinical population would appear to have a more than reasonable chance of successfully evaluating their true positive predictive value.


    ACKNOWLEDGMENTS
 
We appreciate the aid of Jeffrey D. Hulmes, Keith Ho, Mark Friedrichs, Li-An Xu, Andrew Shenker, and Cort S. Madsen for their contributions to these experiments. We are particularly indebted to the help and insights provided by John Krupinski with regard to MSR 1 in particular, and the in vitro experiments in general.


    FOOTNOTES
 
Received, October 12, 2004

Published, MCP Papers in Press, October 20, 2004, DOI 10.1074/mcp.M400160-MCP200

1 The abbreviations used are: LDL, low-density lipoprotein; PMA, phorbol myristate acetate; TIMP1, tissue inhibitor of metalloproteinase 1; MMP9, matrix metalloproteinase 9; MSR, macrophage scavenger receptor; UPAR, urokinase-type plasminogen activator receptor; FABP, fatty acid-binding proteins. Back

* The costs of publication of this article were defrayed in partby thepayment of page charges.This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Bristol-Myers Squibb Company, Post Office Box 5400, Princeton, NJ 08543-5400. Tel.: 609-818-6008; Fax: 609-818-6057; E-mail: estelle.fach{at}bms.com


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 MATERIALS AND METHODS
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 DISCUSSION
 CONCLUSION
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