Chemical Analysis of C-Reactive Protein Synthesized by Human Aortic Endothelial
Cells Under Oxidative Stress
Ming-Hua Tsai †, Chia-Liang Chang †, Yu-San Yu †, Ting-Yu Lin †, Chin-Pong Chong †,
You-Sian Lin †, Mei-Yu Su †, Jian-Ying Yang †, Ting-Yu Shu †, Xuhai Lu ‡, Chu-Huang
Chen *†‡§, and Mine-Yine Liu *†
† Department of Chemistry, National Changhua University of Education, Changhua, Taiwan 50058 ‡ Vascular and Medicinal Research, Texas Heart Institute, Houston, Texas 77030, United States § Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan 40402 L5 Research Center, China Medical University Hospital, Taichung, Taiwan 40447
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, United States
Abstract
C-Reactive protein (CRP) is a clinical biomarker of inflammation, and high levels of
CRP correlate with cardiovascular disease. The objectives of this study were to test
our hypothesis that oxidized low-density lipoprotein (ox-LDL) induces the release of
CRP from human aortic endothelial cells (HAECs) and to optimize several analytical
methods to identify CRP released from cultured cells in a model of atherogenic
stress. HAECs were incubated with copper-oxidized LDL, and the supernatant was
subsequently purified by diethylaminoethyl chromatography and analyzed by liquid
chromatography-tandem mass spectrometry (LC-MS/MS). We identified an optimal
buffer for the elution of CRP, which contained 0.05 M sodium phosphate and 2.0 M
NaCl (pH 4.5). Purified CRP was digested with trypsin and subjected to
high-performance LC with an optimal mobile phase of acetonitrile–water containing 0.1%
formic acid (50:50, v/v) and an optimal mobile phase flow rate of 0.2 mL/min. We
identified optimal parameters for MS/MS analysis of CRP, including sheath gas
pressure (80 psi), capillary temperature (275 °C), collision energy (25%), tube lens
offset (−5 V), auxiliary gas pressure (0 psi), and isolation width of parent ion (m/z
value = 3). Characterization of CRP was based on the extracted ion chromatograms
and selected multiple-reaction monitoring spectra of three peptides (peptide-1, -2,
and -3) derived from trypsin-digested intact CRP standard. CRP peptide-2 and
peptide-3 were identified in the supernatant of ox-LDL-treated HAECs. Confirmation
of CRP was based on LC-MS/MS and enzyme-linked immunosorbent assay analysis of
CRP in purified HAEC supernatant, as well as real-time PCR analysis of CRP mRNA
levels in HAECs.
C-Reactive protein (CRP) is a well-known clinical biomarker of acute and chronic
inflammation. Many studies have indicated that a high concentration of CRP closely
correlates with cardiovascular disease.1.10 CRP is a member of the pentraxin family
of plasma proteins and is composed of five identical subunits (molecular weight of 23
kDa per subunit); each subunit noncovalently interacts with the neighboring one to
form a disk. CRP can dissociate into monomers in response to heat, urea, low pH, and
the absence of calcium ions. Each CRP monomer has two calcium ion-binding sites.
One side of the disk binds the phosphate group of phosphorylcholine through two
calcium ions, and the other side of the disk binds to complement C1q protein, which
triggers the classical complement pathway and leads to the clearance of microbes
and apoptotic cells.11.18 Whether CRP directly participates in atherosclerosis is not
understood.19.21 However, CRP has been shown to positively correlate with oxidized
LDL (ox-LDL) antibody levels in plaque, thickness of the intima-media, and incidence
of plaque.22 Ox-LDL contributes to the pathogenesis of cardiovascular disease.
Levels of ox-LDL in human plasma were found to positively correlate with
inflammation of the arteries. Evidence has also indicated that ox-LDL aggregates
within human arterial plaque.23.27 CRP binds to ox-LDL and apoptotic cell
membranes via the recognition of oxidized phosphatidylcholine, suggesting that CRP
functions in the innate immune response to oxidized phosphatidylcholine of ox-LDL
particles and apoptotic cells.28 In addition, Li and colleagues29 found that CRP
enhances the expression of LOX-1 (lectin-like ox-LDL receptor-1), which is the cell
receptor for ox-LDL in human aortic endothelial cells (HAECs). The uptake of ox-LDL
through this receptor is unregulated and causes endothelial dysfunction, a vital step
in atherosclerosis.29 Therefore, we hypothesized that the endothelial dysfunction
caused by ox-LDL may also enhance endothelial CRP expression mediated by LOX-1.
Because the uptake of ox-
LDL through LOX-1 is unregulated, it is possible that ox-LDL increases the synthesis and release of CRP, which in turn increases both expression of LOX-1 and uptake of ox-LDL.The conventional method used for detecting CRP is the enzyme-linked
immunosorbent assay (ELISA). Several new methods for the detection of CRP have also been reported. Lee and colleagues30 have measured CRP by using resonant frequency shift in the monolithic thin film cantilever of PTZ
[Pb(Zr0.52Ti0.48)O3]. Kriz and colleagues31 have used magnetic permeability detection and a two-site heterogeneous immunoassay for detecting CRP. In addition, CRP has also been detected
by using a competitive immunoassay that applies magnetic nanoparticles under magnetic fields.32 Furthermore, an RNA aptamer-based sandwich format has
been used for the detection of CRP.33 Recently, due to technologic
advancements, mass spectrometry (MS) has been shown to be a powerful tool for the identification of proteins in a complex matrix. Several researchers have detected and quantified CRP in human sera34.39 or in rat urine40 using multiple reaction monitoring (MRM) MS.
However, the analytical power of using MRM to identify proteins in a complex matrix needs further exploration. The purpose of this study was to test our hypothesis that ox- LDL treated HAECs release CRP and to optimize analytical methods, including diethylaminoethyl (DEAE) chromatography and liquid chromatography (LC)-MS/MS, for characterizing CRP generated from HAECs. ■ EXPERIMENTAL SECTION
Preparation and Oxidation of LDL by Copper Ions. The isolation and oxidation of LDL was performed as previously described41 (see Supporting Information for details). Capillary electrophoresis (CE) was then performed to examine the oxidation of LDL as previously described.41 Subsequently, ox-LDL was used to stimulate HAECs. Cell Culture. HAECs (Lonza, Walkersville, MD) were used at passages 4.6. The cells were cultured in an EGM-2 (microvascular endothelial cell growth medium) bullet kit (Lonza) containing EBM-2 (endothelial basal medium) and GM-2 SingleQuots (supplements and growth factor) kits. For subculturing, cells were seeded at a density of 2500.5000 cells/cm2 per 10-cm dish. The cultures were grown at 37 °C with a gas mixture of 5% CO2 and 95% air. To examine the effects of ox-LDL, cells grown to 80% confluence were seeded at a density of 6000 cells/cm2 per 6-cm dish and were treated with 600 μL of medium, PBS, native LDL (3.3 mg/mL protein), or ox-LDL (33.4 μM malondialdehyde). LDL protein concentration was measured by using the Modified Lowry Protein Assay kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. The
malondialdehyde concentration of ox-LDL was measured by using the TBARS Assay kit (Abnova, Walnut, CA) according to the manufacturer’s instructions. After 24 h of stimulation, cell culture supernatant was collected for further analysis. Diethylaminoethyl (DEAE) Chromatography for the Purification of CRP. Anion-exchange chromatography (Hi-Trap DEAE FF column; GE Healthcare BioSciences AB, Uppsala, Sweden) with DEAE resin was used to purify CRP from ox-LDL
stimulated HAECs. Characteristics of the column were as follows: column volume, 1 mL; column dimensions, 0.7 cm × 2.5 cm; total ionic capacity, 0.11.0.16 mmol Cl./Ml medium; dynamic binding capacity, 110 mg HSA/mL medium; and working pH stability, 2.9. First, intact CRP standard (1 mg/mL; EMD Chemicals, San Diego, CA), BSA (1 mg/mL; Sigma Chemical, St. Louis, MO), and cell culture medium
(EGM-2) were used for optimization of DEAE conditions. A 200-μL volume of each sample was loaded onto the column. Binding between the sample and
anionexchange resin was achieved by keeping the column at room temperature for 1 h. The column was washed with 5 mL of washing buffer (0.05 M sodium phosphate, pH 8.1) and then eluted with 5 mL of elution buffer (0.05 M sodium phosphate, pH 4.5, with 0, 0.5, 1, or 2.0M NaCl; 0.05 M sodium phosphate, pH 6.5, with 2.0 M NaCl; or 0.05 M sodium phosphate, pH 8.1, with 2.0 M NaCl); 10
fractions, each with 0.5 mL eluent, were collected. The procedure was a modified version of a previously described protocol.42
After cells were treated with ox-LDL, 5 mL of supernatant was collected for each 6-cm dish and concentrated to 100 μL by using a 10-kDa ultrafiltration filter
(Amicon, Micron Centrifugal Filter Devices, Ultracel YM-100; MW cutoff: 100 000, Millipore, Bedford, MA). Molecules were separated by molecular weight by using a 100-kDa ultrafiltration filter. Tris-HCl buffer (200 μL; 20 mM, pH 7.8) was added to wash the sample remaining on the filter. The procedure was repeated three times, and the filtrate (molecules with lower molecular weight) was collected. The total volume of the filtrate was concentrated to 100 μL by using a 10-kDa ultrafiltration filter. Tris-HCl buffer (200 μL; 20 mM, pH 7.8) was added to exchange the buffer, and the sample was reconstituted to a final volume of 200 μL. The sample was then separated by DEAE chromatography by using optimal conditions. The volume (0.5 mL) of each DEAE fraction was concentrated to 50 μL by using a 10-kDa ultrafiltration filter. Tris-HCl buffer (200 μL; 20 mM, pH 7.8) was added to
exchange the buffer. This procedure was repeated two times, and the sample was reconstituted to a final volume of 100 μL. Then, 10% (v/v) SDS was added to a final concentration of 0.1% (v/v), and the solution was incubated in a 95 °C water bath for 10 min. The sample was filtered by using a 10-kDa ultrafiltration filter. Tris-HCl buffer (200 μL; 20 mM, pH 7.8) was added to exchange the buffer. The procedure was repeated three times, and the final volume of sample was reconstituted to 100 μL, followed by trypsin digestion.
ELISA for the Identification of CRP. After DEAE separation, CRP in each fraction was measured by using the AssayMax Human CRP ELISA kit (Assaypro, St. Charles, MO). The procedures were performed according to the manufacturer’s
instructions. RNA Extraction and Real-time PCR. After HAECs were treated with ox-LDL, total RNA was extracted from cells, and real-time PCR was performed to examine CRP mRNA levels in ox-LDL-stimulated HAECs (see Supporting Information for details).
Trypsin Digestion of CRP. CRP from human ascites (EMD Chemicals, San Diego, CA) was used as protein standard for the identification of CRP. The trypsin digestion of
CRP standard was performed according to standard procedures (see Supporting Information for details). For the trypsin digestion of CRP from cell culture
supernatant, trypsin (2 μL; 1 mg/mL) was added to the purified sample, and the solution was incubated in a 37 °C water bath for 24 h. To determine the optimal method of sample clean up after the trypsin digestion of CRP protein standard, we compared the performance of 3-kDa and 10-kDa ultrafiltration filters (Amicon), a C18 macro spin column (Harvard Apparatus, Holliston, MA), and a C18 Zip Tip (Millipore, Billerica, MA). Because the C18 Zip Tip was the most effective method of sample purification after the trypsin digestion of CRP, each trypsin-digested sample was cleaned by using a C18 Zip Tip. All trypsin-digested samples were subsequently analyzed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and LC-MS/MS. SDS-PAGE Analysis of Trypsin-Digested CRP. After trypsin digestion, CRP standard was subjected to SDS-PAGE (5.12%) analysis, which was performed according to standard procedures (see Supporting Information for details). LC-MS/MS Analysis of CRP. For the characterization of CRP, CRP peptide or trypsin-digested CRP were analyzed by LC-MS/MS. A 20-μL solution containing CRP was introduced
into a Waters high-performance LC (HPLC) system (Waters Corporation, Milford, MA). The HPLC instrument was equipped with a photodiode array detector (Waters 966). The column was a Waters Sunfire C18 column (particle size, 3.5 μm; internal diameter, 4.6 mm; length, 15 cm). The mobile phases tested were (a)
acetonitrile.water (HPLC grade acetonitrile; Echo Chemical, Miaoli, Taiwan) containing 0.1% formic acid (30:70, v/v), (b) acetonitrile.water containing 0.1% formic acid (50:50, v/v), (c) acetonitrile.water containing 0.1% acetic acid (50:50, v/v), (d) acetonitrile.water containing 0.1% formic acid (75:25, v/v), and (e) methanol.water containing 0.1% formic acid (75:25, v/v). The HPLC mobile phase flow rates tested were 0.1 and 0.2 mL/min. Column temperature was maintained at
room temperature, and column pressure was between 850 and 900 psi. Chromatograms were analyzed by Millennium32 Chromatography Manager software (version 3.2; Waters Corporation). The HPLC system was coupled to an LCQ iontrap mass spectrometer (Finnigan Corporation, San Jose, CA). Samples were analyzed in positive ion mode. The ion spray voltage used was 5 kV.
Nitrogen was used as sheath gas, and the pressures tested were 35, 50, 65, and 80 psi. The capillary voltage was maintained at 43 V. The capillary temperatures tested were 200, 225, 250, 275, and 300 °C. The tube lens offset voltages tested were .5 and 20 V. The collision energies tested were 25%, 35%, and 45%. Nitrogen was used as auxiliary gas, and the pressures tested were 0, 10, and 30 psi. The
isolation widths of parent ion m/z values tested were 1, 3, and 10. The microscan was set at 3. The maximum injection time used was 200 ms. Data were analyzed by Xcalibur software (version 1.2; Finnigan Corporation).
■ RESULTS AND DISCUSSION
Characterization of Ox-LDL. LDL from one healthy donor was oxidized in vitro with 5 μMCu2+, and CE was performed to compare the results with those from native LDL.41 The average effective mobilities for the major peaks of native and oxidized LDL were .18.45 ± 0.14 and .25.55 ± 0.15 × 10.5 cm2 V.1 s.1, respectively, suggesting that LDL particles become more negatively charged upon oxidation. The electropherograms were dramatically different between the two types of LDL (Figure S-1), as previously shown.41 Ox-LDL induces dysfunction of HAECs.43.47 We confirmed the effect of ox-LDL on the morphologic features of cultured HAECs by using light microscopy. HAECs were treated with PBS, EGM-2 medium, native LDL, or oxidized LDL for 12 or 24 h, respectively (Figure S-2). Incubating HAECs with ox-LDL for 12 or 24 h increased the number of shrinking cells and cells detached from the culture dish. However, the control experiments showed that PBS, EGM-2 medium, and native LDL did not impair the morphologic features of HAECs. Purification of CRP by DEAE Chromatography. DEAE chromatography was used to purify CRP by using procedures modified from those previously reported by Tsujimoto and 1.0, or 2.0 M NaCl were compared. As shown in Figure 1a, fractions 2.5 had higher absorbance at 280 nm than the other fractions; fraction 2 eluted with buffer containing 2.0 M NaCl showed the highest absorbance. The absorbance for fractions eluted with buffers containing 0.5, 1.0, or 2.0 M NaCl were similar, whereas fractions eluted with buffer containing 0.0 M NaCl had absorbance close to 0, suggesting that buffer containing no salt was not able to elute protein. The higher the salt concentration, the more the interference exists between analyte and the ion-exchange resin; therefore, 2.0MNaCl was chosen as the optimal salt concentration in the elution buffer. The pH conditions in the elution buffer were also compared. We analyzed the elution of intact CRP standard, as well as BSA and EGM-2 medium, in elution buffer containing 0.05Msodium phosphate (pH 6.5) with 2.0MNaCl. As shown in Figure 1b, the absorbance at 280 nm was higher for fractions 2.5 in all three groups; in addition, the absorbance was higher for intact CRP standard than for BSA and EGM-2 medium. However, the highest absorbance of intact CRP standard detected at pH 6.5 was lower than that detected at pH 4.5 (Figure 1a). We also examined the elution of intact CRP standard, BSA, and EGM-2 medium with elution buffer
1c, the absorbance at 280 nm was higher for fractions 2.5 in all three groups, and the absorbance was higher for intact CRP standard than for BSA or EGM-2
medium. The highest absorbance of CRP standard detected at pH 8.1 was also lower than that detected at pH 4.5 (Figure 1a). CRP has two isoelectric points (pI) of 5.3 and 7.4;42 our results indicated that, at a lower pH, more positive charges form on CRP, and thus more interference exists between CRP and the anion-exchange resin. As a result, we concluded that elution buffer containing 0.05 M sodium phosphate with 2.0 M NaCl (pH 4.5) is the optimal buffer for the elution of CRP by DEAE chromatography. For cell culture analysis, HAECs were incubated with PBS, EGM-2 medium, native LDL, or ox-LDL for 24 h, and the cell culture supernatants were concentrated and purified by DEAE chromatography. The absorbance at 280 nm was higher for fractions 2.5 in all treated groups (Figure S-3). Overall, the absorbance was higher for groups treated with native LDL and ox-LDL than for those treated with PBS or EGM-2 medium, suggesting that both native LDL and ox-LDL stimulate the release of proteins from HAECs. However, because absorbance detected at 280 nm represents total protein, we used additional methods to identify CRP. ELISA Analysis of CRP in Purified Cell
Supernatant. After the chromatographic separation of cell culture supernatants, ELISA was used to determine the concentration of CRP in each fraction. As shown in Figure 2, in the supernatant of ox-LDL-treated cells, CRP concentrations were higher in fractions 2.5, with fraction 2 showing the highest concentration.
However, in the supernatant of native LDL-treated cells, CRP was undetectable in all fractions, suggesting that CRP was released only from HAECs incubated with ox-LDL.
Real-Time PCR Analysis of CRP mRNA Levels in HAECs Treated with ox-LDL. Taqman real-time PCR of CRP mRNA levels in HAECs treated with ox-LDL (0, 25, 50, 100, 150, or 200 μg/mL protein) revealed that ox-LDL increased CRP mRNA levels in a dose-dependent manner (Figure S-4). We observed maximum levels of CRP mRNA levels in HAECs treated with 50 μg/mL ox-LDL. In HAECs treated with 100 μg/mL ox-LDL, massive cell death was induced, and CRP mRNA levels in these cells were similar to those of cells treated with 50 μg/mL ox-LDL (Figure S-4). These results indicate that CRP mRNA levels are upregulated in HAECs treated with ox-LDL. LC-MS/MS Analysis of CRP. To further characterize CRP released from ox-LDL-treated cells, optimization of LC-MS/MS
conditions (5 HPLC mobile phase compositions, 2 HPLC flow rates, and 6 MS parameters) was performed on peptides derived from the trypsin digestion of intact CRP standard. SDS-PAGE analysis confirmed the successful digestion of
intact CRP standard. Characterization of CRP peptides was based on their extracted ion chromatograms (EICs) and MRM spectra. First, five mobile phases were compared for HPLC separation: (a) acetonitrile. water containing 0.1% formic acid (30:70, v/v); (b) acetonitrile. water containing 0.1% formic acid (50:50, v/v); (c) acetonitrile. water containing 0.1% acetic acid (50:50, v/v); (d) acetonitrile. water containing 0.1% formic acid (75:25, v/v); and (e) methanol.water containing 0.1% formic acid (75:25, v/v). Our results suggested that solvent b resulted in the best resolution and reproducibility of EIC (data not shown). We found that 2+ peptide precursor ions (M + 2H)2+ were much more abundant than 1+ peptide precursor ions (M + H)1+. As a result, three 2+ peptide precursor ions (m/z = 434.3, 565.1, and 569.1) were selected for study. The relative ion intensities of the selected transition ions for the three precursor ions were used as unique MRM spectra for each peptide. Previously, Kuhn and colleagues36 used these peptide precursor ions and their MRM transitions to investigate CRP in the serum of rheumatoid arthritis patients. Other researchers have also studied CRP by LC-MS/MS analysis.34,35,37−40,48 To optimize the LC-MS/MS conditions for identifying CRP, we examined the effect of sheath gas pressures (35, 50, 65, and 80 psi), capillary temperatures (200, 225, 250, 275, and 300 °C), collision energies (25%, 35%, and 45%), tube lens offsets (.5 and 20 V), auxiliary gas pressures (0, 10, and 30 psi), and isolation width of parent ion m/z values (1, 3, and 10) on the relative abundance of three CRP peptide precursor ions in LCMS/MS analysis. The relative abundance of the three peptide precursor ions was highest for a gas pressure of 80 psi, capillary temperature of 275 °C, collision energy of 25%, tube lens offset of .5 V, auxiliary gas pressure of 0 psi, and isolation width of parent ion m/z value of 3 (Figure 3 or data not shown). Under these optimized conditions, MRM transitions of the three 2+ peptide precursor ions were monitored. For peptide-1
(42APLTKPLK49), four transitions were monitored: 434.3 →169.0, 357.2, 586.4, or 699.5. For peptide-2 (32ESDTSYVSLK41), five transitions were monitored: 565.1 → 347.2, 446.3, 609.4, 696.4, or 912.6. For peptide-3 (66GYSIFSYATK75), five
transitions were monitored: 569.1 → 220.9, 248.1, 716.4, 829.5, or 916.4. The EIC and MRM spectra of peptide-2 and peptide-3 obtained from digested CRP standard (prepared as 1000 ppm of intact CRP standard) are shown in Figures 4 and 5. The EIC and MRMspectrum of peptide-1 are shown in Figure S-5. For further
characterization, a synthetic CRP peptide-2 standard (ESDTSYVSLK; Biomer Technology, Pleasanton, CA) prepared at three concentrations (25, 50, and 100 ppb) was also analyzed (Figure S-6). In addition to the intact CRP protein standard (1000 ppm) shown in Figures 4 and 5, we also analyzed four other low
them, three concentrations (10, 1, and 0.1 ppm) were prepared in Tris buffer, and one concentration (2.5 ppm) was prepared in cell culture media. For peptide-1, the EIC and MRM spectrum were difficult to measure, even at a concentration of 10 ppm. However, for peptide-2 and peptide-3, the EICs andMRMspectra could be measured at a concentration as low as 0.1 ppm, suggesting that peptide-1 is more difficult to
ionize than are peptide-2 and peptide-3. To identify CRP released from ox-LDL-stimulated HAECs, cell culture supernatant was collected, concentrated, purified (by DEAE chromatography), and trypsin digested. Subsequently, CRP was
analyzed by LC-MS/MS by using our optimized conditions. Figures 6 and 7 show the EICs and selected MRM spectra of CRP peptide-2 (32ESDTSYVSLK41) and peptide-3 (66GYSIFSYATK75) obtained from the digested sample. The retention time of EIC for CRP peptide-2 (Figure 6) was similarto that of peptide-2 from the intact CRP standard (Figure 4). Furthermore, the relative intensities of the five selected transition ions were similar between CRP peptide-2 and peptide-2 from intact CRP standard. Likewise, the retention time of EIC for CRP peptide-3 (Figure 7) was similar to that of peptide-3 from intact CRP standard (Figure 5), and the relative intensities of the five selected transition ions were also similar between the two (Figures 5 and 7). These results suggest that ox-LDL-stimulated HAECs do indeed synthesize CRP, but the concentration is very
low. We found that the EIC andMRMspectrum of CRP peptide-1 were difficult to measure, possibly because of the very low concentration of CRP released in cell culture and the difficulty of ionization of peptide-1 relative to peptide-2 and peptide-3. Although only CRP peptide-2 and peptide-3 were detectable, this is sufficient for the identification of CRP, according to the guidelines established by the European Commission Decision No. 2002/657/EC, which requires that the confirmation of a molecule is achieved by using one precursor ion and two daughter ions (identification points = 4). From our cell culture sample, two precursor ions each with five daughter ions were detected, which meets the minimum performance criteria of this regulation.
■ CONCLUSIONS
We have optimized analytical methods that can be used to identify CRP generated in an in vitro cell culture model of atherogenic stress. The results of our study suggest that ox-LDL induces the synthesis of CRP by HAECs. To our knowledge, this is the first report to characterize CRP released from HAECs stimulated with ox-LDL in vitro. Furthermore, we have demonstrated that MRM MS is a powerful tool for identifying proteins in a complex matrix.
■
ASSOCIATED CONTENT*
S Supporting InformationAdditional information as noted in text. This information is available free of charge via the Internet at http://pubs.acs.org
■ AUTHOR INFORMATION Corresponding Author
*E-mail: [email protected] (M.-Y.L), [email protected] (C.-H.C.). Phone: 886-4-7232105 ext 3530 (M.-Y.L). Fax: 886-4-7211190 (M.-Y.L).
Author Contributions
#These authors contributed equally to this study. Notes
The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS
This study was funded in part by grants from the National
Science Council of Taiwan (NSC-98-2113-M-018-005-MY2 and NSC-100-2113-M-018-001-MY3), and grant DOH101-TD-BFigure 111-004 from the Taiwan
Department of Health Clinical Trial and Research Center of Excellence. The
authors thank Nicole Stancel, Ph.D., ELS, of the Texas Heart Institute at St. Luke’s Episcopal Hospital in Houston, Texas, for editorial assistance.
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