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. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: Arthritis Rheum. 2012 Jan 9;64(6):1828–1837. doi: 10.1002/art.34363

Proteomic profiling following immunoaffinity capture of HDL: Association of acute phase proteins and complement factors with pro-inflammatory HDL in Rheumatoid Arthritis

Junji Watanabe 1,*, Christina Charles-Schoeman 2,*, Yunan Miao 3,*, David Elashoff 2, Yuen Yin Lee 2, George Katselis 3,#, Terry D Lee 3, Srinivasa T Reddy 4
PMCID: PMC3330163  NIHMSID: NIHMS347793  PMID: 22231638

Abstract

Objective

To utilize proteomic analysis to identify protein biomarkers associated with pro-inflammatory HDL in patients with active rheumatoid arthritis.

Methods

Liquid chromatography-mass spectrometry (LC-MS) was used to analyze proteins associated with immunoaffinity purified HDL from plasma of two sets of RA patients carrying distinct HDL (anti- or pro-) inflammatory properties. Proteins were fractionated by Offgel electrophoresis and analyzed by LC-MS/MS equipped with a high capacity high performance liquid chromatography chip (HPLC-Chip) incorporating C18 reverse phase trapping and analytical columns. Sandwich enzyme-linked immunosorbent assays were used to validate select HDL-associated proteins in a second RA cohort.

Results

Seventy-eight proteins were identified in the HDL complexes. Twelve proteins were significantly increased in RA patients with pro-inflammatory HDL compared to RA patients with anti-inflammatory HDL. These proteins included acute phase proteins, including apolipoprotein J, fibrinogen, haptoglobin, serum amyloid A, and complement factors (B, C3, C9). Four of the proteins associated with HDL were validated in a second RA cohort.

Conclusion

Pro-inflammatory HDL in patients with RA contains a significantly altered proteome including increased amounts of acute phase proteins and proteins involved in the complement cascade. These findings suggest that HDL is significantly altered in the setting of chronic inflammation from active RA with resultant loss of its anti-inflammatory function. The characterization of the biomarkers reported here may identify novel molecular connections that contribute to the higher risk of CVD in RA patients.

Patients with RA have a significantly increased risk of cardiovascular disease (CVD) including myocardial infarction and sudden cardiac death which is not explained by traditional CVD risk factors (1). Previous work has suggested that systemic inflammation contributes to CVD in RA, and patients with active disease and high inflammatory burden are at significantly increased risk (2–4). Although some studies have suggested that RA mortality rates may be falling in response to new therapies (5), survival trends are not keeping pace with the general population and CVD remains the leading cause of death (6). The investigation of novel mechanisms for accelerated atherosclerosis in patients with RA therefore becomes important both for appropriate treatment and for aggressive primary prevention.

The inverse relationship between HDL cholesterol and the risk of CVD is well established (7, 8). However, a significant number of CVD events occur in patients with normal HDL and LDL cholesterol levels (9, 10). Previous work has suggested that the inflammatory nature of HDL may be a more sensitive marker of CVD than HDL cholesterol levels. Thus, there is a need for further investigation of biomarkers with better predictive value based on HDL function (11–13).

HDL plays numerous anti-inflammatory and athero-protective roles by promoting reverse cholesterol transport and preventing the oxidation of LDL (14, 15). However, HDL protective function is impaired and becomes pro-inflammatory during pathologic processes that accelerate CVD events (16–18). The molecular changes and mechanisms that promote the conversion of anti-inflammatory HDL to pro-inflammatory HDL are currently unknown. The knowledge of molecular profiles that distinguish pro-inflammatory and anti-inflammatory HDL may facilitate better understanding of the alterations in HDL’s protein cargo, which adversely affect its normal antioxidant and anti-inflammatory functions.

Pro-inflammatory HDL is increased in RA patients compared to healthy controls (19). We recently reported that levels of pro-inflammatory HDL are positively correlated with disease activity in RA patients (20). However, how pro-inflammatory HDL is involved in RA activity and its relationship to CVD is unknown. In this report, we evaluated HDL carrying distinct (anti-or pro-) inflammatory properties from plasma of RA patients. HDL was isolated using immunocapture columns and further subjected to our newly developed Offgel electrophoresis and LC-MS/MS method to identify protein markers that distinguish the inflammatory nature of HDL. Multiple proteins were identified in HDL including several proteins significantly associated with pro-inflammatory HDL. Four of these HDL-associated proteins were validated in a second RA cohort by HDL capturing ELISA. Since the inflammatory nature of HDL has previously been directly linked to CVD, and is also significantly correlated with disease activity in RA, characterization of the biomarkers reported here may identify novel molecular connections that contribute to the higher risk of CVD in RA patients.

PATIENTS AND METHODS

Reagents for proteomic analysis

HPLC grade and proteomic reagents were purchased from the following companies: acetonitrile from Fisher (Pittsburgh, PA); 2,2,2-trifluoroethanol (TFE) and formic acid from Fluka (Milwaukee, WI); dithiothreitol (DTT) and iodoacetamide (IAA) from Sigma (St. Louis, MO); sequencing-grade modified trypsin from Promega (Madison, WI); iTRAQ™ 8-plex reagents from Applied Biosystems (Foster City, CA); Immobiline DryStrip and IPG Buffer (pH3–10) for Offgel Fractionation from GE Healthcare (Piscataway, NJ)

Human subjects

RA patients were recruited from the UCLA rheumatology offices through flyers posted in the offices and in the UCLA Medical Center. Patients inquiring about the study from posted flyers in their rheumatologist’s office or elsewhere were referred to one of the study physicians if the treating rheumatologist was not involved in the protocol. All RA patients met the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 revised criteria for the classification of RA (21), which was verified by chart review. All subjects gave written informed consent for the study under a protocol approved by the Human Research Subject Protection Committee at UCLA.

Clinical tests for RA patients

The Disease Activity Score using 28 joints (DAS28) (22), inflammatory markers (high-sensitive C-reactive protein [CRP] and ESR), and fasting lipid profiles were performed for individual patients as described previously (20). Disease-related disability was assessed with the disability index (DI) of the Health Assessment Questionnaire (HAQ) (23).

Assays to Determine the Inflammatory Properties of HDL

HDL anti-oxidative property was determined by cell-free assay (CFA) with 2,7,7’-dichlorofluorescein diacetate (H2DCFDA) as described previously (24). Briefly, plasma samples were isolated from fasted RA patients, cryopreserved in 10% sucrose, and freshly frozen at −80°C until use. HDL-containing supernatants were isolated by LipiDirect HDL reagent (Polymedco, Cortland Manor, NY) and assayed for cholesterol content (Thermo DMA, San Jose, CA). 50 μl of HDL (100μg HDL-cholesterol/ml) was tested for each sample. Oxidized DCF was used as an indicator of reactive oxygen species measured by fluorescence intensity at 485/530 nm. Readings with DCF and LDL were normalized to 1.0 (HDL inflammatory index). After the addition of HDL to LDL, HII < 1.0 defined HDL as anti-inflammatory, while HII ≥ 1.0 as pro-inflammatory.

Paraoxonase and arylesterase activity assays

Both assays were performed as described previously with minor modifications (14, 25). For the arylesterase assay plasma was diluted at 1:50 dilution with assay buffer and incubated with phenyl acetate. Arylesterase activity was measured by kinetic reading at A270 for 2 min. The kinetic rate at A270 was multiplied by the molecular extinction coefficient of 0.7633 and dilution factor of 2.5 to represent as Unit of arylesterase activity per ml of plasma (U/mL).

Paraoxonase-1 western blot analysis

Equal amounts of plasma protein (50μg) of all samples, (determined by measuring absorbance at 280nm using a Thermo Scientific Nanodrop 2000 Spectrometer), were loaded into 10% Mini-Protean TGX Gels (Bio-Rad) for separation and transferred to nitrocellulose membranes (Bio-Rad). After blocking with 5% skim milk in Tris-buffered saline-Tween 20 (TBS-T) for 60 minutes at room temperature, the membranes were incubated overnight at 4°C with a goat anti-human paraoxonase-1 antibody (1:2000) (R&D Systems; Minneapolis, MN, USA). After washes the membranes were further incubated with horseradish peroxidase-conjugated anti-goat IgG antibody (1:5000) (Santa Cruz Biotechnology; Santa Cruz, CA, USA), and developed with ECL Plus (GE Healthcare).

HDL isolation by anti-HDL IgY spin columns for proteomic analysis

6.5 mg of plasma protein was loaded onto anti-HDL chicken IgY immunocapture spin columns according to manufacturer’s protocol (GenWay Biotech, San Diego, CA). The protein concentrations in plasma were measured by the BCA protein assay. The first wash was combined with the initial sample solution to make the flow through sample. Eluted HDL was desalted using a YM-3 centrifugal filter unit (Millipore, Billerica, MA). The protein concentration of HDL captured by the IgY column was determined by Nanodrop spectrophotometer (Thermo, Wilmington, DE).

Separation of HDL proteins by SDS-PAGE

HDL (30 μg proteins) was loaded onto a NuPAGE® 4–12% Bis-Tris precast gel (1.5 mm thick). Proteins were visualized by staining gels overnight in SimplyBlue™ SafeStain (Invitrogen). The gel lanes were cut into 12 sections for trypsin digestion and analysis by LC-MS/MS.

Separation of HDL proteins by Offgel electrophoresis

Proteins were separated by isoelectric point (pH3–10) with Offgel electrophoresis according to the manufacturer’s protocol (Agilent, Santa Clara CA). Briefly, HDL (200 μg proteins) was loaded per well and fractionated by Agilent 3100 Offgel electrophoresis and fractionation at pH 3–10. The proteins were focused with a maximum current of 50 μA until 20 kVh was reached. Fractions were collected by first pipetting the liquid from each well. Next, 100 μL of water/methanol/formic acid (49/50/1 by volume) was added to each well and incubated for 90 minutes without voltage. The liquid was then removed, combined with the first portion removed from the well, acidified with formic acid, and concentrated by vacuum centrifugation prior to LC-MS/MS analysis. LC-MS/MS analysis was performed using 25% of each fraction.

8-Plex iTRAQ Analysis of HDL in RA Patients

iTRAQ™ 8-plex reagent kit was used to test all eight HDL samples from RA patients in a single experiment. Equivalent amounts of each isolated HDL protein were digested with trypsin and derivatized with one of the iTRAQ reagents.

Since glycine in the IgY elution buffer is incompatible with the iTRAQ reagent, eluted HDL was desalted by a reverse phase C18 HPLC column (4.6 mm i.d. × 50 mm, 5um particle size, Agilent) at a temperature of 80 °C using a trifluoroacetic acid (TFA) buffer system (solvent A – 0.1% aqueous TFA, solvent B – 0.08% TFA in acetonitrile). Samples were loaded in 10% B then eluted with a gradient from 10–70% B over two min then ramped to 100% over 1 min and held at 100% B for 2 min. The entire protein peak was collected and concentrated using a vacuum centrifuge. The protein concentration was determined using the Nanodrop spectrophotometry.

The desalted HDL sample (200 μg proteins) was reduced, alkylated, and digested with trypsin using trifluoroethanol as a detergent. A 1D LC-MS/MS analysis was performed on each digest mixture to ensure the quality of the digestion reaction. A 60 μg aliquot of each sample was labeled with one of the iTRAQ reagents according to the manufacturer’s protocol (Applied Biosystems). Briefly, one vial of iTRAQ reagent was used for each digested HDL sample. The iTRAQ reagent was solubilized in 100% isopropanol and added to the peptide sample at the final concentration of 60% (v/v). The solution pH was adjusted to 7.5–8.5 to optimize the labeling efficiency (>99%). After labeling for 2 hours at room temperature the samples were combined and concentrated by vacuum centrifugation. The labeled samples (200 μg proteins) were separated by Offgel electrophoresis. Each fraction was analyzed in triplicate by LC-MS/MS.

Mass Spectrometry

All mass spectrometry analyses were performed on an Agilent 6520 Q-TOF mass spectrometer equipped with an Agilent 1200 series liquid chromatograph and an Agilent Chip Cube LC-MS interface. LC separations used a high capacity HPLC chip consisting of a 160 nL enrichment column and a 75 μm × 150 mm analytical column both packed with Zorbax 300 SB-C18, 5 μm reverse phase support. Peptides were loaded onto the enrichment column with 97% solvent A (water with 0.1% formic acid) and 3% solvent B (90% acetonitrile, 10% water with 0.1% formic acid) at a flow rate of 4 μL/min and then eluted with a linear gradient 8% to 30% B in 45 min then 30% to 80% B in 1 min at a flow rate of 0.3 μL/min. Positive ion electrospray mass spectra were acquired using a capillary voltage set at 1850 V, the ion fragmentor set at 175 V, and the drying gas set at 300 °C and 4 L/min. MS spectra were collected in centroid mode over the mass range of 375 to 2500 m/z at a scan rate of 4 spectra per second. MS/MS spectra were collected in centroid mode over the range of 50–3000 m/z and an isolation width of 4 amu. The collision energy was ramped at a slope of 2.5 and an offset of 3.7. The top six most intense precursor ions for each MS scan were selected for tandem MS with active exclusion for 0.33 min.

Peptide and Protein Identification

The iTRAQ labels are designed such that the same peptide from each sample has the same molecular weight and chromatographic retention time, but yields a different fragment or reporter ion when the MS/MS spectrum is acquired. The relative intensity of the reporter ion signals in an individual MS/MS spectrum is a direct measure of the relative levels of that peptide in each of the original samples.

The spectral data were converted to m/z Data format using Agilent MassHunter Qualitative Analysis Software (B.03.01) and processed through the Computational Proteomics Integrated Environment (COPINE) hosted at the City of Hope National Medical Center. COPINE is a collection of comprehensive proteomics data analysis tools, which comprise the GPM from The Global Proteome Machine Organization. The X!Tandem Tornado (2009.04.01.1) was used as the database search engine. Spectra were searched against the Uniprot Human database together with the reverse Uniprot Human database and a custom database of common contaminants. Search parameters included a fragment mass error of 50 ppm, a parent mass error of 50 ppm, trypsin cleavage specificity, and carbamidomethyl as a fixed modification of cysteine. Deamidation of asparagine and glutamine, oxidation of methionine to methionine sulfoxide and sulfone, and acetylation of the n-terminus were specified as variable modifications. For the X!Tandem search of the iTRAQ derivatized samples, the iTRAQ™ 8 plex was specified for peptide n-terminus and lysine residues. A merged, non-redundant output file was generated for protein identifications with log(e) values less than -1. A concatenated decoy database consisting of the reversed sequences in the Uniprot database was used for all searches.

Results of the database search were analyzed by Scaffold (v 3.00.01) running on COPINE. Peptide identifications were accepted if they could be established at greater than 90% probability as specified by the Peptide Prophet algorithm (26). Protein identifications were accepted if they could be established at greater than 99% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (27). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

Reporter ion intensity data were collected from the iTRAQ LC-MS/MS runs using the Agilent Spectrum Mill database search program. Peptides less than seven amino acid residues were excluded because of the high probability that they would match more than one protein. Only proteins with two or more unique iTRAQ peptides in each of the triplicate runs were included in the results. Values reported are for the sum of the reporter ion intensities for all the peptides assigned to a protein. Reporter ion values for each of the replicated runs were averaged to yield a total reporter ion intensity for each sample. The average coefficient of variation for the set of proteins in the anti-inflammatory data set was 0.37 and for the pro-inflammatory data set the value was 0.34.

Analysis of HDL-associated proteins by ELISA

Individual plasma samples from randomly selected RA patients containing anti-inflammatory or pro-inflammatory HDL were assayed by HDL-capturing sandwich enzyme-linked immunosorbent assays (ELISA) as described previously (16). The following antibodies were used in the ELISA experiments reported in this paper: chicken antibody for human HDL (GenWay Biotech); mouse antibodies for human apolipoprotein J (apoJ), serum amyloid A (SAA), HRP-conjugated apolipoprotein A-I (apoA-I), fibrinogen (Fg), and haptoglobin (Hp) (Abcam, Cambridge, MA); HRP-conjugated secondary antibodies for anti-mouse antibody (GE Healthcare).

Briefly, 96-well PVC microtiter plates were pre-coated with 2 μg/ml of chicken anti-human HDL antibodies overnight at 4°C. Following incubation of the pre-coated plates with individual plasma samples diluted at 1:10 with 1X PBS, the plates were washed thoroughly, blocked with 5% non-fat milk in PBS, and incubated with corresponding primary antibodies to apoJ, SAA or horseradish peroxidase (HRP) conjugated antibodies to apoA-I, Fg, or Hp at 1:2500 dilution. The non-conjugated primary antibodies were detected by HRP-conjugated secondary antibodies for mouse immunoglobulin at 1:2500 dilution. Following incubation with TMB solution, HRP activity was measured at OD 450 nm (OD450). The HRP-conjugated antibody of each target protein was coated in empty wells at a series of different concentrations as a standard to convertthe OD450 of each sample to the concentration of the HRP-conjugated antibody of each target protein. The value for each protein in a given sample was represented as a relative concentration to the average of that protein in plasma samples of RA patients containing anti-inflammatory HDL, which was set at 1.

Statistical analysis

The distributions of all data were examined carefully to determine the appropriate parametric or nonparametric test for analysis. For the proteomics data, a t-test for a two-sample with unequal variance (with Satterwaite adjustment) was used. P-values were not corrected for multiple corrections given the exploratory nature of the proteomics analyses. For the clinical measures and HDL- associated protein ELISAs, (Tables 1 and 2), patient groups were compared using t-test for continuous variables, and the chi-square test of association for categorical variables, along with Fisher’s exact test in cases where there were small sample sizes in individual categories. When needed, nonparametric Wilcoxon rank-sum tests were used to compare groups for skewed continuous variables. DAS28 is known from the literature to be normally distributed and was treated as such even with a small sample (Table 1). The significance level for all comparisons was prespecified using a two tailed test with p<0.05.

Table 1.

Clinical data and HDL function data in RA patients with pro-inflammatory HDL compared with those with anti-inflammatory HDL.

HDL (A) N=4 HDL (P) N=4 p
Age (years) 53.2 (9.7) 55.3 (9.2) 0.76
Sex (% Female) 100 100 1.0
DAS28 (Disease Activity Score) 2.12 (0.84) 7.84 (0.71) < 0.0001
HAQ DI scores (Disability) 0.0 (0.2) 2.5 (1.7) 0.027
High-sensitivity CRP (mg/L) 0.3 (0.6) 39.9 (64.9) 0.029
ESR (mm/hr) 7.25 (7.09) 84.3 (30.1) 0.012
Total cholesterol (mg/dL) 185.3 (7.3) 184.8 (31.4) 0.98
LDL cholesterol (mg/dL) 91.5 (16.3) 100.0 (20.4) 0.54
HDL cholesterol (mg/dL) 68 (40) 52 (10) 0.06
Triglycerides (mg/dL) 90 (90) 173 (170) 0.31
CFA(HII) 0.22 (0.11) 1.34 (0.29) 0.002
PON (U/mL) (Paraoxon) 243 (233) 59 (18) 0.19
PON (U/mL) (Arylesterase) 163(144) 238(127) 0.47
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Each number represents the average (± one standard deviation of 4 RA patients per group) for normally distributed variables, and the median (interquartile range) for non-normally distributed variables as described in the methods section. DAS28 = disease activity score in 28 joints; HAQ DI = health assessment questionnaire disability index; CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; LDL = low-density lipoprotein; HDL = high-density lipoprotein; CFA = cell free assay; HII = HDL inflammatory index; PON = paraoxonase.

Table 2.

Clinical data and HDL function data in RA patients with pro-inflammatory HDL compared with those with anti-inflammatory HDL.

HDL (A) N=16 HDL (P) N=15 P
Age (years) 56.3 (10.7) 58.3 (13.3) 0.66
Sex (% Female) 94 93 1.0
Ethnicity (% Caucasian) 69 67 1.0
ESR (mm/hr) 10(8) 45(35) 0.0001
High-sensitivity CRP (mg/L) 1.3(1.9) 4.7(16.2) 0.005
DAS28 (Disease Activity Score) 3.1 (0.9) 6.0(1.4) <0.0001
HAQ DI scores(Disability) 0.13 (0.84) 1.31(1.46) 0.002
Total cholesterol (mg/dL) 184.2 (38.2) 208.7 (47.2) 0.12
LDL cholesterol (mg/dL) 101.3 (31.8) 114.9 (29.7) 0.23
HDL cholesterol (mg/dL) 59 (23) 71 (42) 0.28
Triglycerides (mg/dL) 107 (75) 102 (64) 0.84
CFA(HII) 0.32 (0.09) 1.38 (0.46) <0.0001
PON (U/mL) (Paraoxon) 201(233) 133 (124) 0.12
PON (U/mL) (Arylesterase) 221 (180) 195 (87) 0.38
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Each number represents the average (± one standard deviation of 15 or 16 RA patients per group) for normally distributed variables, and the median (interquartile range) for non-normally distributed variables as described in the methods section. DAS28 = disease activity score in 28 joints; HAQ DI = health assessment questionnaire disability index; CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; LDL = low-density lipoprotein; HDL = high-density lipoprotein; CFA = cell free assay; HII = HDL inflammatory index; PON = paraoxonase.

RESULTS

HDL isolation by immunocapture spin columns

HDL complexes were isolated using immunocapture spin columns with IgY antibody for human HDL. For these particular patient samples the protein assays of the amount of protein obtained from the IgY capture were significantly higher for the pro-inflammatory group. This suggests that there is either a significant difference in the amount of ApoA-I or ApoA-II present in the samples, or that the ratio of the amount of ApoA-I or ApoA-II relative to the total amount of other proteins is different. When corrected for the fraction of the sample used, the MS analysis indicated that there was no significant difference in the amounts of ApoA-I and ApoA-II, but that the levels of many other proteins were significantly higher in the pro-inflammatory patients (see below). The ability of this method to enrich for HDL associated proteins is evident in the SDS-PAGE analysis of the starting plasma (input), the fraction that does not bind to the column (flow through), and the fraction that comes off with the elution buffer (bound) (Figure 1).

Figure 1. Proteins in HDL isolated by IgY immuno-affinity spin columns.

Figure 1

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10 or 30 μg of total proteins in plasma (Input), proteins that do not bind (Flow Through) or captured by HDL IgY antibody (Bound) were loaded on SDS-PAGE. The lane for the 30 μg Bound was cut into 12 sections as indicated and the extracted proteins were analyzed by LC-MS/MS. The major proteins identified in each section are indicated on the right side of the image.

Proteomic analysis on pro-inflammatory HDL in RA patients

We recently reported that the pro-inflammatory properties of HDL are positively correlated with systemic inflammation in CVD (28) and RA patients (20). For the present study, we chose plasma samples from RA patients that contained either pro-inflammatory HDL (HII>1.0) or anti-inflammatory HDL (HII<0.5). Plasma samples showed no difference in age, sex, or levels of cholesterol and triglycerides (Table 1). As reported, disease activity in RA patients is significantly higher in the pro-inflammatory HDL group (Table 1). Proteins associated with HDL complexes were labeled with iTRAQ reagents as described previously (29) and subjected to LC-MS/MS following Offgel electrophoresis. A total of 78 proteins were identified in HDL (Figure 2), including lipid metabolism factors and transporters, protease inhibitors, acute phase proteins, complement proteins, and coagulation factors. Twelve of these proteins were significantly increased in pro-inflammatory HDL compared to anti-inflammatory HDL in RA patients: Fg, complement factors, α-1-antitrypsin, Hp, apoJ, immunoglobin heavy chain, serpin D1 (heparin cofactor II) and SAA (Figure 2). The total reporter ion intensity assigned to each protein provided a rough measure of the relative amount of each protein in the sample. The value of the total reporter ion intensity for the two patient groups was within 5% (p=0.71), providing confirmation that the protein amounts analyzed were well matched (data not shown).

Figure 2. Proteins associated with HDL.

Figure 2

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HDL was isolated by IgY immunoaffinity columns from plasma in RA patients containing anti-inflammatory (A) or pro-inflammatory (P) HDL. Proteins were separated by off-gel electrophoresis and analyzed by LC-MS/MS as described on materials and methods. Each bar represents the average + one standard deviation of relative reporter ion intensity of identified protein in anti-inflammatory HDL (n=4 per group). X-axis shows relative reporter ion intensity (RII) of iTRAQ™ reporter ion in log2 scale. Each number in () is the average of percentage change of individual pro-inflammatory HDL samples based on the average of anti-inflammatory HDL. * = p<0.05, # = p<0.07.

Pro-inflammatory mediators associated with pro-inflammatory HDL in RA patients

To validate the discovery of protein biomarkers of pro-inflammatory HDL in RA patients (Fig 2), we tested additional plasma samples from RA patients containing anti-inflammatory HDL or pro-inflammatory HDL. Sandwich ELISA was performed to capture HDL and validate four markers involved in the acute phase inflammation: apoJ, Fg, Hp and SAA. We chose these four markers because i) they were highly significant, ii) they are increased during vascular inflammation and iii) they have been linked specifically to rheumatoid arthritis, a disease associated with increased CV risk secondary to systemic inflammation. All four markers were significantly associated with pro-inflammatory HDL (Figure 3). No significant differences in HDL-associated apoA-1 levels were noted between the two groups (Figure 3).

Figure 3. Association of inflammatory mediators with pro-inflammatory HDL.

Figure 3

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HDL associated apoA-I, apoJ, Fg, Hp and SAA contents in plasma from RA patients containing anti-inflammatory (A, n=16) or pro-inflammatory (P, n=15) HDL-associated protein concentrations were determined by ELISA as described in materials and methods. Each bar represents the average + one standard deviation of relative concentration to plasma containing anti-inflammatory HDL. *=p<0.05. Rel [] = relative concentration of the targeted protein.

A trend was noted for decreased PON-1 protein levels in the initial proteomic analysis (p <0.07; Table 1). Follow-up assessment of PON-1 protein levels by western blotting followed by densitometry in the validation cohort (n=15 per group) revealed a significant decrease (21% ± 16.4%) in PON-1 protein in pro-inflammatory HDL compared to anti-inflammatory HDL. PON-1 activity was also assessed by means of the arylesterase and paraoxonase assays for both cohorts of patients. Anon-significant trend for higher PON-1 activity (paraoxonase assay), in anti-inflammatory HDL compared to pro-inflammatory HDL was noted in the proteomics samples (p= 0.19; Table 1). A similar trend was noted in the larger validation cohort (Table 2). This data is consistent with previous work suggesting a link between PON-1 activity and inflammation in patients with rheumatoid arthritis (30).

DISCUSSION

Oxidation of LDL is one of the major factors in the development of human atherosclerosis (31, 32). Entrapment and oxidation of LDL in the sub-endothelial space and the subsequent interactions between endothelial cells and monocytes is a key process in the initiation of atherosclerotic lesion development (33, 34). The inverse relationship between HDL cholesterol and the risk of atherosclerosis is well established (8). Although there does not appear to be a single explanation for the anti-atherogenic role of HDL, it has become clear that the functional status of HDL, which is largely dependent on its protein components, is probably an important determinant of CVD (18).

Over a decade ago, it was reported that the anti-inflammatory properties of HDL are impaired in rabbits (14), mice (15), and humans (13) during inflammatory processes. This impaired HDL is pro-inflammatory in nature, as characterized by (i) decreased levels and activity of anti-inflammatory, antioxidant factors (17); (ii) gain of pro-inflammatory proteins (14); (iii) increased lipid hydroperoxide (LOOH) content (18); (iv) reduced potential to efflux cholesterol (35); and (v) diminished ability to prevent LDL oxidation (36). Previous work has also suggested that HDL inflammatory properties may be a more sensitive marker of CVD than HDL cholesterol levels (13). However, the molecular changes and mechanisms that promote anti-inflammatory HDL conversion to pro-inflammatory HDL are currently not well understood.

Rheumatoid arthritis is a systemic inflammatory disease associated with high cardiovascular risk (1). Previous work by our group has shown that HDL from patients with RA has abnormal anti-inflammatory properties compared to healthy controls, and that the anti-inflammatory function of HDL is significantly correlated with disease activity and systemic inflammation; higher disease activity was associated with worsened ability of HDL to inhibit LDL oxidation (20). Previous studies also suggested alterations in the levels of select proteins associated with HDL (16, 28).

In the current work, we developed an Offgel electrophoresis and LC-MS/MS method to identify proteins that distinguish the inflammatory nature of HDL. Among 78 proteins identified in HDL, 12 proteins were significantly associated with pro-inflammatory HDL (Figure 2): Fg (α, β, and γ chains), complement factors (C3, C9 and B), α-1-antitrypsin, Hp, apoJ, immunoglobin heavy chain, serpin D1 (heparin cofactor II), and SAA. These results suggest that alternative proteins, particularly acute phase proteins, associate with HDL that might exert pro-inflammatory and pro-atherogenic functions.

Interestingly, eleven out of the twelve proteins were also identified in HDL from CVD patients (37), except Fg, a plasma glycoprotein that is converted to fibrin by thrombin during blood coagulation. Fg is considered as a thrombotic marker that contributes to the high CVD mortality in RA patients (38, 39). How HDL-associated Fg is involved in thrombosis needs to be further investigated in order to understand the pathological mechanisms involved in CVD events in RA patients.

We previously identified hemoglobin (Hb) and its accessory proteins, Hp and hemopexin (Hx) as biomarkers of pro-inflammatory HDL in CVD patients (16, 28). Hp was also significantly associated with pro-inflammatory HDL in RA patients in the current work (Figure 2 and 3). In contrast, Hb and Hx were associated with HDL, but were found to be independent of HDL inflammatory properties in this study (Figure 2), which may be attributable to a small sample size. Heme regulatory factors associated with pro-inflammatory HDL regulate the reductive-oxidative (redox) reaction and vascular tone in the circulation (16). The oxidative properties of heme produce reactive oxygen species causing oxidative stress (40). Thus, based on our previous findings and this study, Hp appears to be the most predictive biomarker of pro-inflammatory HDL to evaluate the protective role on oxidative status.

Pro-inflammatory HDL can be converted to anti-inflammatory HDL by diet (37) or therapeutic interventions such as apoA-I mimetic peptides (41). Previous studies showed that HDL function in RA patients may be modestly improved with high dose statin therapy (42), methotrexate (MTX) (20), and infliximab (30). The current study identifies significant alterations in the proteome of pro-inflammatory HDL in RA patients, which may aid in the development of additional therapeutics to improve HDL anti-inflammatory properties. Large, prospective controlled studies are necessary to confirm the direct cause and effect relationship between HDL function and disease activity in RA and its relationship to CVD.

It should be noted, however, that the protein changes reported in our studies are limited to the fact that we are analyzing the entire HDL population through the immunoaffinity method. Given the fact that HDL is highly heterogeneous in particle composition, the current approach cannot identify the specific particles of HDL with which the protein cargo is associated. The method can be further improved by employing a size exclusion step following the immunoaffinity capture of HDL to further identify specific changes in protein cargo that are associated with specific HDL particles in RA patients. Furthermore, it should also be noted that for most of the high abundant proteins including Fg, we suspect that HDL-associated component is quite small compared to the corresponding serum concentrations.

In summary, the current work is the first to use immunoaffinity capture of HDL with subsequent proteomic analysis to describe the protein cargo of HDL in patients with RA, a chronic inflammatory disease associated with significantly increased cardiovascular morbidity and mortality. The work implicates HDL as an active participant in the inflammatory response, carrying multiple complement proteins, serine protease inhibitors, and other proteins involved in cell signaling and the coagulation cascade. In addition, marked differences were seen in the HDL cargo of patients with active disease and pro-inflammatory HDL, compared to patients with low disease activity and anti-inflammatory HDL. These findings fuel a known link between inflammation and accelerated CVD in RA, and suggest that HDL-associated biomarkers may warrant further investigation including network and pathway analyses. Future work on the proteins identified in these studies will determine whether any single or combination of these markers is i) predictive of CV events in RA patients, and ii) reversed with anti-inflammatory therapies.

Supplementary Material

Supp Table S1

Acknowledgments

This work was supported by National Institutes of Health Grants, NHLBI, 5R01HL082823 (STR) and 5K23HL094834 (CCS), and Arthritis Foundation--Southern California Chapter Grant.

References

  • 1.Sattar N, McCarey DW, Capell H, McInnes IB. Explaining how “high-grade” systemic inflammation accelerates vascular risk in rheumatoid arthritis. Circulation. 2003;108(24):2957–63. doi: 10.1161/01.CIR.0000099844.31524.05. [DOI] [PubMed] [Google Scholar]
  • 2.Del Rincon I, Williams K, Stern MP, Freeman GL, O'Leary DH, Escalante A. Association between carotid atherosclerosis and markers of inflammation in rheumatoid arthritis patients and healthy subjects. Arthritis Rheum. 2003;48(7):1833–40. doi: 10.1002/art.11078. [DOI] [PubMed] [Google Scholar]
  • 3.Maradit-Kremers H, Crowson CS, Nicola PJ, Ballman KV, Roger VL, Jacobsen SJ, et al. Increased unrecognized coronary heart disease and sudden deaths in rheumatoid arthritis: a population-based cohort study. Arthritis Rheum. 2005;52(2):402–11. doi: 10.1002/art.20853. [DOI] [PubMed] [Google Scholar]
  • 4.Maradit-Kremers H, Nicola PJ, Crowson CS, Ballman KV, Gabriel SE. Cardiovascular death in rheumatoid arthritis: a population-based study. Arthritis Rheum. 2005;52(3):722–32. doi: 10.1002/art.20878. [DOI] [PubMed] [Google Scholar]
  • 5.Bjornadal L, Baecklund E, Yin L, Granath F, Klareskog L, Ekbom A. Decreasing mortality in patients with rheumatoid arthritis: results from a large population based cohort in Sweden, 1964–95. J Rheumatol. 2002;29(5):906–12. [PubMed] [Google Scholar]
  • 6.Gonzalez A, Maradit Kremers H, Crowson CS, Nicola PJ, Davis JM, 3rd, Therneau TM, et al. The widening mortality gap between rheumatoid arthritis patients and the general population. Arthritis Rheum. 2007;56(11):3583–7. doi: 10.1002/art.22979. [DOI] [PubMed] [Google Scholar]
  • 7.Miller GJ, Miller NE. Plasma-high-density-lipoprotein concentration and development of ischaemic heart-disease. Lancet. 1975;1(7897):16–9. doi: 10.1016/s0140-6736(75)92376-4. [DOI] [PubMed] [Google Scholar]
  • 8.Di Angelantonio E, Sarwar N, Perry P, Kaptoge S, Ray KK, Thompson A, et al. Major lipids, apolipoproteins, and risk of vascular disease. Jama. 2009;302(18):1993–2000. doi: 10.1001/jama.2009.1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR. High density lipoprotein as a protective factor against coronary heart disease. The Framingham Study. Am J Med. 1977;62(5):707–14. doi: 10.1016/0002-9343(77)90874-9. [DOI] [PubMed] [Google Scholar]
  • 10.Castelli WP, Garrison RJ, Wilson PW, Abbott RD, Kalousdian S, Kannel WB. Incidence of coronary heart disease and lipoprotein cholesterol levels. The Framingham Study. Jama. 1986;256(20):2835–8. [PubMed] [Google Scholar]
  • 11.Navab M, Hama SY, Anantharamaiah GM, Hassan K, Hough GP, Watson AD, et al. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3. J Lipid Res. 2000;41(9):1495–508. [PubMed] [Google Scholar]
  • 12.Navab M, Hama SY, Cooke CJ, Anantharamaiah GM, Chaddha M, Jin L, et al. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1. J Lipid Res. 2000;41(9):1481–94. [PubMed] [Google Scholar]
  • 13.Ansell BJ, Navab M, Hama S, Kamranpour N, Fonarow G, Hough G, et al. Inflammatory/antiinflammatory properties of high-density lipoprotein distinguish patients from control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin treatment. Circulation. 2003;108(22):2751–6. doi: 10.1161/01.CIR.0000103624.14436.4B. [DOI] [PubMed] [Google Scholar]
  • 14.Van Lenten BJ, Hama SY, de Beer FC, Stafforini DM, McIntyre TM, Prescott SM, et al. Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures. J Clin Invest. 1995;96(6):2758–67. doi: 10.1172/JCI118345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Van Lenten BJ, Wagner AC, Nayak DP, Hama S, Navab M, Fogelman AM. High-density lipoprotein loses its anti-inflammatory properties during acute influenza a infection. Circulation. 2001;103(18):2283–8. doi: 10.1161/01.cir.103.18.2283. [DOI] [PubMed] [Google Scholar]
  • 16.Watanabe J, Chou KJ, Liao JC, Miao Y, Meng HH, Ge H, et al. Differential association of hemoglobin with proinflammatory high density lipoproteins in atherogenic/hyperlipidemic mice. A novel biomarker of atherosclerosis. J Biol Chem. 2007;282(32):23698–707. doi: 10.1074/jbc.M702163200. [DOI] [PubMed] [Google Scholar]
  • 17.Mackness MI, Durrington PN, Mackness B. The role of paraoxonase 1 activity in cardiovascular disease: potential for therapeutic intervention. Am J Cardiovasc Drugs. 2004;4(4):211–7. doi: 10.2165/00129784-200404040-00002. [DOI] [PubMed] [Google Scholar]
  • 18.Navab M, Berliner JA, Subbanagounder G, Hama S, Lusis AJ, Castellani LW, et al. HDL and the inflammatory response induced by LDL-derived oxidized phospholipids. Arterioscler Thromb Vasc Biol. 2001;21(4):481–8. doi: 10.1161/01.atv.21.4.481. [DOI] [PubMed] [Google Scholar]
  • 19.McMahon M, Grossman J, FitzGerald J, Dahlin-Lee E, Wallace DJ, Thong BY, et al. Proinflammatory high-density lipoprotein as a biomarker for atherosclerosis in patients with systemic lupus erythematosus and rheumatoid arthritis. Arthritis Rheum. 2006;54(8):2541–9. doi: 10.1002/art.21976. [DOI] [PubMed] [Google Scholar]
  • 20.Charles-Schoeman C, Watanabe J, Lee YY, Furst DE, Amjadi S, Elashoff D, et al. Abnormal function of high-density lipoprotein is associated with poor disease control and an altered protein cargo in rheumatoid arthritis. Arthritis Rheum. 2009;60(10):2870–9. doi: 10.1002/art.24802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 1988;31(3):315–24. doi: 10.1002/art.1780310302. [DOI] [PubMed] [Google Scholar]
  • 22.Prevoo ML, van 't Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum. 1995;38(1):44–8. doi: 10.1002/art.1780380107. [DOI] [PubMed] [Google Scholar]
  • 23.Fries JF, Spitz P, Kraines RG, Holman HR. Measurement of patient outcome in arthritis. Arthritis Rheum. 1980;23(2):137–45. doi: 10.1002/art.1780230202. [DOI] [PubMed] [Google Scholar]
  • 24.Navab M, Hama SY, Hough GP, Subbanagounder G, Reddy ST, Fogelman AM. A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids. J Lipid Res. 2001;42(8):1308–17. [PubMed] [Google Scholar]
  • 25.Furlong CE, Richter RJ, Seidel SL, Motulsky AG. Role of genetic polymorphism of human plasma paraoxonase/arylesterase in hydrolysis of the insecticide metabolites chlorpyrifos oxon and paraoxon. Am J Hum Genet. 1988;43(3):230–8. [PMC free article] [PubMed] [Google Scholar]
  • 26.Keller A, Nesvizhskii AI, Kolker E, Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem. 2002;74(20):5383–92. doi: 10.1021/ac025747h. [DOI] [PubMed] [Google Scholar]
  • 27.Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem. 2003;75(17):4646–58. doi: 10.1021/ac0341261. [DOI] [PubMed] [Google Scholar]
  • 28.Watanabe J, Grijalva V, Hama S, Barbour K, Berger FG, Navab M, et al. Hemoglobin and its scavenger protein haptoglobin associate with apoA-1-containing particles and influence the inflammatory properties and function of high density lipoprotein. J Biol Chem. 2009;284(27):18292–301. doi: 10.1074/jbc.M109.017202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics. 2004;3(12):1154–69. doi: 10.1074/mcp.M400129-MCP200. [DOI] [PubMed] [Google Scholar]
  • 30.Popa C, van Tits LJ, Barrera P, Lemmers HL, van den Hoogen FH, van Riel PL, et al. Anti-inflammatory therapy with tumour necrosis factor alpha inhibitors improves high-density lipoprotein cholesterol antioxidative capacity in rheumatoid arthritis patients. Ann Rheum Dis. 2009;68(6):868–72. doi: 10.1136/ard.2008.092171. [DOI] [PubMed] [Google Scholar]
  • 31.Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88(6):1785–92. doi: 10.1172/JCI115499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Witztum JL, Steinberg D. The oxidative modification hypothesis of atherosclerosis: does it hold for humans? Trends Cardiovasc Med. 2001;11(3–4):93–102. doi: 10.1016/s1050-1738(01)00111-6. [DOI] [PubMed] [Google Scholar]
  • 33.Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA, et al. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation. 1995;91(9):2488–96. doi: 10.1161/01.cir.91.9.2488. [DOI] [PubMed] [Google Scholar]
  • 34.Navab M, Berliner JA, Watson AD, Hama SY, Territo MC, Lusis AJ, et al. The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture. Arterioscler Thromb Vasc Biol. 1996;16(7):831–42. doi: 10.1161/01.atv.16.7.831. [DOI] [PubMed] [Google Scholar]
  • 35.Hayek T, Oiknine J, Brook JG, Aviram M. Role of HDL apolipoprotein E in cellular cholesterol efflux: studies in apo E knockout transgenic mice. Biochem Biophys Res Commun. 1994;205(2):1072–8. doi: 10.1006/bbrc.1994.2775. [DOI] [PubMed] [Google Scholar]
  • 36.Navab M, Ananthramaiah GM, Reddy ST, Van Lenten BJ, Ansell BJ, Hama S, et al. The double jeopardy of HDL. Ann Med. 2005;37(3):173–8. doi: 10.1080/07853890510007322. [DOI] [PubMed] [Google Scholar]
  • 37.Heinecke JW. The HDL proteome: a marker--and perhaps mediator--of coronary artery disease. J Lipid Res. 2009;50 (Suppl):S167–71. doi: 10.1194/jlr.R800097-JLR200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.McEntegart A, Capell HA, Creran D, Rumley A, Woodward M, Lowe GD. Cardiovascular risk factors, including thrombotic variables, in a population with rheumatoid arthritis. Rheumatology (Oxford) 2001;40(6):640–4. doi: 10.1093/rheumatology/40.6.640. [DOI] [PubMed] [Google Scholar]
  • 39.Hurt-Camejo E, Paredes S, Masana L, Camejo G, Sartipy P, Rosengren B, et al. Elevated levels of small, low-density lipoprotein with high affinity for arterial matrix components in patients with rheumatoid arthritis: possible contribution of phospholipase A2 to this atherogenic profile. Arthritis Rheum. 2001;44(12):2761–7. doi: 10.1002/1529-0131(200112)44:12<2761::aid-art463>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  • 40.Puppo A, Halliwell B. Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is haemoglobin a biological Fenton reagent? Biochem J. 1988;249(1):185–90. doi: 10.1042/bj2490185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Bloedon LT, Dunbar R, Duffy D, Pinell-Salles P, Norris R, DeGroot BJ, et al. Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients. J Lipid Res. 2008;49(6):1344–52. doi: 10.1194/jlr.P800003-JLR200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Charles-Schoeman C, Khanna D, Furst DE, McMahon M, Reddy ST, Fogelman AM, et al. Effects of high-dose atorvastatin on antiinflammatory properties of high density lipoprotein in patients with rheumatoid arthritis: a pilot study. J Rheumatol. 2007;34(7):1459–64. [PubMed] [Google Scholar]

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Supplementary Materials

Supp Table S1
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