Yuen Yin Lee

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, HDLs 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)). HDLs 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 HDLs 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.

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.

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.

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.

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

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.

Improvement of High-Density Lipoprotein Function in Patients With Early Rheumatoid Arthritis Treated With Methotrexate Monotherapy or Combination Therapies in a Randomized Controlled Trial.

Abnormal function of high‐density lipoprotein (HDL) has been implicated as a potential mechanism for the increased incidence of cardiovascular (CV) disease in patients with rheumatoid arthritis (RA). This study was undertaken to evaluate changes in HDL function and HDL‐associated proteins over 2 years of follow‐up in patients with early RA receiving either methotrexate (MTX) monotherapy, MTX + etanercept (ETN) combination therapy, or MTX + sulfasalazine (SSZ) + hydroxychloroquine (HCQ) triple therapy in the Treatment of Early Aggressive Rheumatoid Arthritis (TEAR) trial.

Improvement in HDL Function in Early Rheumatoid Arthritis Patients Treated with Methotrexate Monotherapy or Combination Therapy in the TEAR Trial.

Abnormal function of high‐density lipoprotein (HDL) has been implicated as a potential mechanism for the increased incidence of cardiovascular (CV) disease in patients with rheumatoid arthritis (RA). This study was undertaken to evaluate changes in HDL function and HDL‐associated proteins over 2 years of follow‐up in patients with early RA receiving either methotrexate (MTX) monotherapy, MTX + etanercept (ETN) combination therapy, or MTX + sulfasalazine (SSZ) + hydroxychloroquine (HCQ) triple therapy in the Treatment of Early Aggressive Rheumatoid Arthritis (TEAR) trial.

Association of Triple Therapy with Improvement in Cholesterol Profiles over Two Year Follow-up in the TEAR Trial

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.

Association of Triple Therapy With Improvement in Cholesterol Profiles Over Two-Year Followup in the Treatment of Early Aggressive Rheumatoid Arthritis Trial: TRIPLE THERAPY AND CHOLESTEROL PROFILES IN EARLY RA

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.

FRI0065 Proteomic Profiling Following Immunoaffinity Capture of High-Density Lipoprotein (HDL) Identifies Changes in Multiple HDL-Associated Proteins Following Treatment with Abatacept or Adalimumab in the Ample Study of Patients with Rheumatoid Arthritis

Objectives To evaluate changes in high-density lipoprotein (HDL) function and HDL-associated proteins and their association with RA disease activity measures in a subset of patients with active RA initiating therapy with abatacept or adalimumab in the Abatacept Versus Adalimumab Comparison in Biologic-Naïve RA Subjects with Background Methotrexate (AMPLE) study.1 Methods Ultra high-pressure liquid chromatography (UHPLC) coupled with ion mobility mass spectrometry (LC-IM-MS) was used to analyse proteins associated with immunoaffinity-captured HDL from plasma of 30 patients with RA randomized to either abatacept (n=15) or adalimumab (n=15) therapy. Proteins were reduced, alkylated and digested with trypsin, and the resulting peptides were identified and quantitated by a Waters NanoAcquity UHPLC system coupled to a Synapt G2 HDMS mass spectrometer fitted with an Ionkey nanoelectrospray source. Standard cholesterol profiles, HDL anti-oxidant function and paraoxonase 1 (PON1) activity were measured by previously published assays.2 Repeated-measures analyses were performed using mixed-effect linear models with autoregressive covariate structure to model the within-subject covariance over time using available time points from AMPLE. Results In mixed-effect models controlling for age, sex and treatment group, improvement in inflammation measured by decrease in CRP was associated with increase in total cholesterol levels (p=0.01) irrespective of treatment. Improvement in inflammation (CRP) was also associated with improvement in HDL anti-oxidant function (i.e. decrease in HDL Inflammatory Index [HII]; p=0.01), which was not different between treatment groups. Decreases in CRP were significantly associated with decreases in several HDL-associated proteins including lipopolysaccharide-binding protein, serum amyloid A-I (SAA-I) and inter-alpha-trypsin inhibitor heavy chain H4 (all 3 p values <0.05). Strong trends for decreases in other HDL-associated proteins including fibrinogen (gamma chain) and galectin 3 binding protein associated with decreases in CRP were also noted (both p values=0.10). Adalimumab was associated with a greater increase in PON1 activity (p=0.01) and a trend for greater decrease in HDL-associated SAA-I compared with abatacept (p=0.06). Conclusions This pilot study showed that decreases in inflammation associated with treatment of active RA, using either abatacept or adalimumab in the AMPLE study, were associated with significant alterations in the HDL proteome, including proteins involved in the immune response, and overall improvement in HDL anti-oxidant function. As the function of HDL has previously been directly linked to CV disease, further characterization of the biomarkers reported here may identify novel molecular connections that contribute to the higher risk of CV disease in patients with active RA, which are improved with effective therapy. References Schiff M, et al. Ann Rheum Dis 2014;73:86–94. Charles-Schoeman C, et al. Arthritis Rheum 2013;65:2765–72 Disclosure of Interest C. Charles-Schoeman Grant/research support from: Bristol-Myers Squibb, G. B. Gugiu: None declared, H. Ge: None declared, A. Shahbazian: None declared, Y. Y. Lee: None declared, X. Wang: None declared, D. E. Furst Grant/research support from: AbbVie, Actelion, Amgen, BMS, Gilead, GSK, NIH, Novartis, Pfizer, Roche/Genentech, UCB, Consultant for: AbbVie, Actelion, Amgen, BMS, Cytori, Janssen, Gilead, GSK, NIH, Novartis, Pfizer, Roche/Genentech, UCB, Speakers bureau: AbbVie, Actelion, UCB (all CME only), V. K. Ranganath Grant/research support from: BMS, Genentech, M. Maldonado Shareholder of: Bristol-Myers Squibb, Employee of: Bristol-Myers Squibb, S. T. Reddy: None declared