High- density lipoprotein function is abnormal in idiopathic inflammatory myopathies.

OBJECTIVE: Damage to the vascular endothelium is strongly implicated in the pathogenesis of idiopathic inflammatory myopathies (IIM). Normally, high-density lipoprotein (HDL) protects the vascular endothelium from damage from oxidized phospholipids, which accumulate under conditions of oxidative stress. The current work evaluated the antioxidant function of HDL in IIM patients. METHODS: HDL's antioxidant function was measured in IIM patients using a cell-free assay, which assesses the ability of isolated patient HDL to inhibit oxidation of low-density lipoproteins and is reported as the HDL inflammatory index (HII). Cholesterol profiles were measured for all patients, and subgroup analysis included assessment of oxidized fatty acids in HDL and plasma MPO activity. A subgroup of IIM patients was compared with healthy controls. RESULTS: The antioxidant function of HDL was significantly worse in patients with IIM (n = 95) compared with healthy controls (n = 41) [mean (S.d.) HII 1.12 (0.61) vs 0.82 (0.13), P < 0.0001]. Higher HII associated with higher plasma MPO activity [mean (S.d.) 13.2 (9.1) vs 9.1 (4.6), P = 0.0006] and higher oxidized fatty acids in HDL. Higher 5-hydroxyeicosatetraenoic acid in HDL correlated with worse diffusion capacity in patients with interstitial lung disease (r = -0.58, P = 0.02), and HDL's antioxidant function was most impaired in patients with autoantibodies against melanoma differentiation-associated protein 5 (MDA5) or anti-synthetase antibodies. In multivariate analysis including 182 IIM patients, higher HII was associated with higher disease activity and DM diagnosis. CONCLUSION: The antioxidant function of HDL is abnormal in IIM patients and may warrant further investigation for its role in propagating microvascular inflammation and damage in this patient population.

Association of paraoxonase 1 gene polymorphism and enzyme activity with carotid plaque in rheumatoid arthritis.

OBJECTIVE: To investigate the relationship of genetic and biochemical determinants of paraoxonase 1 activity to carotid plaque as a surrogate marker of cardiovascular (CV) risk in patients with rheumatoid arthritis (RA). METHODS: The relationships between paraoxonase 1 activity, PON1 genotype (for the functional polymorphism at position 192), and carotid plaque presence were determined in 168 RA patients. After an overnight fast, blood was collected for lipoprotein analysis, and paraoxonase 1 activity was measured using paraoxon as the substrate. The PON1 Q192R genotype was determined for all patients. Lipoprotein cholesterol levels, traditional CV risk factors, medication use, and RA disease characteristics were assessed for all patients. RESULTS: Paraoxonase 1 activity values in the RA patients were highest for the RR genotype, intermediate for the QR genotype, and lowest for the QQ genotype (P < 0.0001). Compared to patients with either the QQ genotype or the QR genotype, patients with the RR genotype demonstrated decreased risk of carotid plaque on multivariate analysis, controlling for traditional CV risk factors, high-sensitivity C-reactive protein levels, prednisone use, and cholesterol-lowering medication use (P < 0.05). Additional multivariate logistic regression analysis controlling for the above factors also revealed a significant association of plasma paraoxonase 1 activity with carotid plaque in RA patients. Lower plasma paraoxonase 1 activity was associated with increased risk of carotid plaque (P < 0.05). CONCLUSION: The current findings suggest a relationship of the genetic determinants and activity of paraoxonase 1 to CV risk in RA patients, as assessed by the presence or absence of carotid plaque. Further CV outcome studies are warranted to validate the utility of paraoxonase 1 as a biomarker of CV risk in patients with RA.

Proteomic profiling following immunoaffinity capture of high-density lipoprotein: association of acute-phase proteins and complement factors with proinflammatory high-density lipoprotein in rheumatoid arthritis.

OBJECTIVE: To identify protein biomarkers associated with proinflammatory high-density lipoprotein (HDL) in patients with active rheumatoid arthritis (RA) by proteomic analysis. METHODS: Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze proteins associated with immunoaffinity-purified HDL from plasma obtained from 2 sets of RA patients, 1 with antiinflammatory HDL and 1 with proinflammatory HDL. Proteins were fractionated by Offgel electrophoresis and analyzed using an LC-MS/MS system equipped with a high-capacity high-performance liquid chromatography chip incorporating C18 reverse-phase trapping and analytical columns. Sandwich enzyme-linked immunosorbent assays were used to validate the association between select proteins and proinflammatory HDL in a second cohort of RA patients. RESULTS: Seventy-eight proteins were identified in the HDL complexes. The levels of 12 proteins were significantly increased in RA patients with proinflammatory HDL compared to RA patients with antiinflammatory HDL. These proteins included the acute-phase proteins apolipoprotein J, fibrinogen, haptoglobin, serum amyloid A, and complement factors (B, C3, and C9). The associations between proinflammatory HDL and 4 of the proteins were validated in a second RA cohort. CONCLUSION: Our findings indicate that proinflammatory 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 in active RA, with resultant loss of its antiinflammatory function. The characterization of the biomarkers described herein may identify novel molecular connections that contribute to the higher risk of cardiovascular disease in RA patients.

Cholesterol efflux by high density lipoproteins is impaired in patients with active rheumatoid arthritis.

OBJECTIVES: Reverse cholesterol transport (RCT) is a major antiatherogenic function of high density lipoprotein (HDL). In the current work, the authors evaluated whether the RCT capacity of HDL from rheumatoid arthritis (RA) patients is impaired when compared to healthy controls. METHODS: HDL was isolated from 40 patients with RA and 40 age and sex matched healthy controls. Assays of cholesterol efflux, HDL's antioxidant function and paraoxanase-1 (PON-1) activity were performed as described previously. Plasma myeloperoxidase (MPO) activity was assessed by a commercially available assay. RESULTS: Mean cholesterol efflux capacity of HDL was not significantly different between RA patients (40.2% ± 11.1%) and controls (39.5% ± 8.9%); p=0.75. However, HDL from RA patients with high disease activity measured by a disease activity score using 28 joint count (DAS28>5.1), had significantly decreased ability to promote cholesterol efflux compared to HDL from patients with very low disease activity/clinical remission (DAS28<2.6). Significant correlations were noted between cholesterol efflux and the DAS28 (r=-0.39, p=0.01) and erythrocyte sedimentation rate, (r=-0.41, p=0.0009). Higher plasma MPO activity was associated with worse HDL function (r=0.41/p=0.009 (antioxidant capacity); r=0.35, p=0.03 (efflux)). HDL's ability to promote cholesterol efflux was modestly but significantly correlated with its antioxidant function (r=-0.34, p=0.03). CONCLUSIONS: The cholesterol efflux capacity of HDL is impaired in RA patients with high disease activity and is correlated with systemic inflammation and HDL's antioxidant capacity. Attenuation of HDL function, independent of HDL cholesterol levels, may suggest a mechanism by which active RA contributes to increased cardiovascular (CV) risk.

Suppression of inflammatory arthritis in human serum paraoxonase 1 transgenic mice.

Paraoxonase 1(PON1) is an HDL-associated protein, which metabolizes inflammatory, oxidized lipids associated with atherosclerotic plaque development. Because oxidized lipid mediators have also been implicated in the pathogenesis of rheumatoid arthritis (RA), we evaluated the role of PON1 in murine inflammatory arthritis. K/BxN serum transfer (STIA) or collagen antibody transfer (CAIA) was used for arthritis induction in B6 mice homozygous for the PON1 human transgene [PON1Tg], PON1 knock-out mice [PON1KO], and wild type littermate control mice [WT]. Experiments were also performed in K/BxN mice with chronic arthritis, and in RA patients and healthy controls. Arthritis activity in K/BxN mice was associated with a marked dyslipidemia, lower PON1 activity and higher bioactive lipid mediators (BLM), as well as a dysregulated hepatic lipid gene expression profile. Higher serum PON1 activity correlated with lower BLM and lower arthritis activity in both K/BxN mice and RA patients. Overexpression of the human PON1 transgene was associated with reduced inflammatory arthritis, which correlated strongly with higher circulating PON1 activity, upregulation of the hepatic glutathione pathway, and reduction of circulating BLM. These results implicate PON1 as a potential novel therapeutic target for joint disease in RA with potential for vascular benefit, which warrants further investigation.

High levels of oxidized fatty acids in HDL are associated with impaired HDL function in patients with active rheumatoid arthritis.

The objective of this study was to evaluate oxidation products of arachidonic acid and linoleic acid in lipoproteins and synovial fluid (SF) from patients with active rheumatoid arthritis (RA) compared to non-RA controls. High-density lipoproteins (HDL) and low-density lipoproteins (LDL) were isolated from plasma using fast protein liquid chromatography and HDL was isolated from SF using dextran sulfate precipitation. 5-Hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, 9 hydroxyoctadecadienoic (HODE), and 13-HODE levels were measured in HDL, LDL, and SF by liquid chromatography-tandem mass spectrometry. HDL's anti-inflammatory function, cholesterol levels, myeloperoxidase (MPO) and paraoxonase 1 (PON1) activities were determined as previously. 5-HETE, 15-HETE, 9-HODE, and 13-HODE levels were significantly increased in HDL and LDL from patients with active RA (n = 10) compared to healthy controls (n = 8) and correlated significantly with measures of systemic inflammation, particularly in HDL (r = 0.65-0.80, p values < 0.004). Higher HETES and HODES in HDL were also significantly correlated with impaired HDL function as measured by the HDL inflammatory index (HII) (r = 0.54-0.58; p values < 0.03). 15-HETE levels and MPO activity were higher in RA SF (n = 10) compared to osteoarthritis (OA) SF(n = 11), and HDL from RA SF had worse function compared to OA SF HDL (HII = 2.1 ± 1.9 and 0.5 ± 0.1), respectively (p < 0.05). Oxidation products of arachidonic acid and linoleic acid are increased in HDL and LDL from patients with active RA compared to healthy controls, and are associated with worse anti-oxidant function of HDL. These results suggest a potential mechanism by which oxidative stress from active RA increases oxidized fatty acids in HDL, promoting HDL dysfunction, and thereby increasing atherosclerotic risk.

Association of Triple Therapy With Improvement in Cholesterol Profiles Over Two-Year Followup in the Treatment of Early Aggressive Rheumatoid Arthritis Trial.

OBJECTIVE: To evaluate long-term changes in cholesterol levels in patients with early rheumatoid arthritis (RA) who were randomized to begin treatment with methotrexate (MTX) monotherapy, MTX plus etanercept, or triple therapy (MTX plus sulfasalazine plus hydroxychloroquine) in the Treatment of Early Aggressive Rheumatoid Arthritis (TEAR) trial. METHODS: Levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol were analyzed in 416 patients participating in the TEAR trial, during 102 weeks of followup. Associations of cholesterol changes with disease activity and drug treatment were evaluated using repeated-measures analysis with mixed-effect linear models to model within-subject covariance over time. RESULTS: Mixed-effect models controlling for traditional cardiovascular (CV) risk factors, TEAR treatment, and baseline prednisone and statin use demonstrated significant inverse associations of RA disease activity with changes in cholesterol over time. Decreases in the 28-joint Disease Activity Score, the C-reactive protein level, or the erythrocyte sedimentation rate were associated with increases in levels of HDL cholesterol, LDL cholesterol, and total cholesterol in all treatment groups (P < 0.001-0.035). Triple therapy was strongly associated with higher levels of HDL cholesterol, lower levels of LDL cholesterol, and higher ratios of total cholesterol:HDL cholesterol (P < 0.001 for all) compared to MTX monotherapy or MTX plus etanercept therapy over the 2-year followup. CONCLUSION: Decreases in RA disease activity over long-term followup were associated with increases in cholesterol levels in patients with early RA treated with either biologic or nonbiologic therapies. The use of triple therapy during 2 years of followup was associated with higher HDL cholesterol levels, lower LDL cholesterol levels, and lower total cholesterol:HDL cholesterol ratios compared to those observed in patients who received MTX monotherapy or MTX plus etanercept combination therapy. Additional studies are needed to assess the effects of these cholesterol changes on CV events in patients with RA.

Abnormal function of high-density lipoprotein is associated with poor disease control and an altered protein cargo in rheumatoid arthritis.

OBJECTIVE: To characterize the antiinflammatory function of high-density lipoprotein (HDL) in patients with rheumatoid arthritis (RA) and to identify specific differences in HDL-associated proteins and enzymes that distinguish proinflammatory HDL from normal, antiinflammatory HDL. METHODS: We studied 132 RA patients. The antiinflammatory function of HDL was assessed by a cell-free assay, and proinflammatory HDL was defined by an HDL inflammatory index > or =1. Plasma and HDL-associated protein levels of apolipoprotein A-I (Apo A-I), haptoglobin, hemopexin, hemoglobin, and myeloperoxidase (MPO) were measured by direct and sandwich enzyme-linked immunosorbent assays, respectively. Lecithin:cholesterol acyltransferase (LCAT) activity was measured by a commercially available assay. RESULTS: Age, disease activity, the presence of erosive disease, non-Caucasian race, and smoking were significantly associated with proinflammatory HDL on multivariate analysis. Patients with proinflammatory HDL had higher measures of systemic inflammation, and a significant correlation was observed between RA disease activity (using the Disease Activity Score in 28 joints) and the HDL inflammatory index (r = 0.54, P < 0.0001). Compared with patients with antiinflammatory HDL, patients with proinflammatory HDL had significantly higher levels of haptoglobin, hemoglobin, Apo A-I, and MPO associated with HDL (P < 0.05 for all comparisons except MPO, which was P = 0.05). LCAT activity was lowest in patients with proinflammatory HDL, but was also significantly reduced in RA patients with antiinflammatory HDL as compared with healthy controls (P = 0.001). CONCLUSION: Proinflammatory HDL in this RA patient cohort was associated with active disease and an altered protein cargo as compared with antiinflammatory HDL in RA patients and in healthy controls. The antiinflammatory function of HDL was inversely correlated with systemic inflammation in RA patients and may warrant further investigation as a mechanism by which active RA increases cardiovascular morbidity and mortality.