Ani Shahbazian

Proline and γ-Carboxylated Glutamate Residues in Matrix Gla Protein Are Critical for Binding of Bone Morphogenetic Protein-4

Arterial calcification is ubiquitous in vascular disease and is, in part, prevented by matrix Gla protein (MGP). MGP binds calcium ions through &ggr;-carboxylated glutamates (Gla residues) and inhibits bone morphogenetic protein (BMP)-2/-4. We hypothesized that a conserved proline (Pro)64 is essential for BMP inhibition. We further hypothesized that calcium binding by the Gla residues is a prerequisite for BMP inhibition. Site-directed mutagenesis was used to modify Pro64 and the Gla residues, and the effect on BMP-4 activity, and binding of BMP-4 and calcium was tested using luciferase reporter gene assays, coimmunoprecipitation, crosslinking, and calcium quantification. The results showed that Pro64 was critical for binding and inhibition of BMP-4 but not for calcium binding. The Gla residues were also required for BMP-4 binding but flexibility existed. As long as 1 Gla residue remained on each side of Pro64, the ability to bind and inhibit BMP-4 was preserved. Chelation of calcium ions by EDTA or warfarin treatment of cells led to loss of ability of MGP to bind BMP-4. Our results also showed that phenylalanine could replace Pro64 without loss of function and that zebrafish MGP, which lacks upstream Gla residues, did not function as a BMP inhibitor. The effect of MGP mutagenesis on vascular calcification was determined in calcifying vascular cells. Only MGP proteins with preserved ability to bind and inhibit BMP-4 prevented osteogenic differentiation and calcification. Together, our results suggest that BMP and calcium binding in MGP are independent but functionally intertwined processes and that the BMP binding is essential for prevention of vascular calcification.

High-Density Lipoproteins Affect Endothelial BMP-Signaling by Modulating Expression of the Activin-Like Kinase Receptor 1 and 2

Objective—High-density lipoproteins (HDL) have antiinflammatory effects on the vascular endothelium. Because bone morphogenetic proteins (BMPs) are known to be inflammatory mediators, we examined the effect of HDL on BMP signaling. Methods and Results—Increasing concentrations of HDL progressively enhanced expression of the activin-like kinase receptor (ALK)1 and ALK2 in human aortic endothelial cells as determined by real-time polymerase chain reaction and immunoblotting. Induction of ALK1 was a result of enhanced ALK2 expression as determined by siRNA interference, and was associated with increased levels of vascular endothelial growth factor (VEGF) and matrix Gla protein (MGP). The HDL-induction of ALK2 was dependent on BMP-signaling, and affected coregulation of the ALK2 gene by the homeodomain proteins MSX2, DLX3, and DLX5, as determined by reporter gene assays, siRNA interference, and chromatin immunoprecipitation. Apolipoprotein A-I transgenic mice, known to have high HDL and inhibition of atherogenesis, exhibited similar changes in aortic gene expression as seen in endothelial cells treated with HDL in vitro. Conclusions—We conclude that HDL benefits the arterial wall by allowing for enhanced ALK1 and ALK2 signaling, resulting in an increase of VEGF and MGP, essential for endothelial cell survival and prevention of vascular calcification, respectively.

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.

Novel anti-inflammatory functions for endothelial and myeloid cyclooxygenase-2 in a new mouse model of Crohn's disease

Cyclooxygenase-2 (COX-2) is an important regulator of inflammation implicated in the development of a variety of diseases, including inflammatory bowel disease (IBD). However, the regulation of intestinal inflammation by COX-2 is poorly understood. We previously reported that COX-2(-/-) mice fed a cholate-containing high-fat (CCHF) diet had high mortality of unknown mechanisms attributable to severe intestinal inflammation in the ileo-ceco-colic junction that presented characteristics similar to Crohns disease (CD). To further characterize the role of COX-2 in intestinal inflammation, we established cell-specific conditional COX-2(-/-) mice. Endothelial cell-specific (COX-2(-E/-E)) and myeloid cell-specific (COX-2(-M/-M)) COX-2(-/-) mice, but not wild-type mice, on the CCHF diet developed localized CD-like pathology at the ileo-ceco-colic junction that was associated with cellular infiltration, increased expression of myeloperoxidase and IL-5, and decreased IL-10 expression. The CD-like pathology in COX-2(-E/-E) mice was also accompanied by increased expression of cytokines (IL-6, TNF-alpha, and INF-gamma), compared with wild-type mice and COX-2(-M/-M) mice. In contrast, the ileo-ceco-colic inflammation in COX-2(-M/-M) mice was associated with more pronounced infiltration of granulocytes and macrophages than COX-2(-E/-E) mice. COX-2(-ME/-ME) (COX-2(-M/-M) x COX-2(-E/-E)) mice on the CCHF diet developed CD-like pathology in the ileo-ceco-colic junction reminiscent of total COX-2(-/-) mice on CCHF diet and wild-type mice on CCHF diet treated with COX-2 inhibitor, celecoxib. The pathology of diet-mediated ileo-ceco-colic inflammation in COX-2(-/-) mice offers an excellent model system to elucidate the protective roles of endothelial and myeloid COX-2 and the molecular pathogenesis of CD.

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