Abstract-Arterial calcification is ubiquitous in vascular disease and is, in part, prevented by matrix Gla protein (MGP).MGP binds calcium ions through ␥-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. 1 Patients with defined mutations in the MGP gene (Keutel syndrome) have widespread calcification in the tracheobronchial tree, and the quality and duration of life appears to be determined by the extent of pulmonary disease. 2,3 We and others have identified MGP as an inhibitor of bone morphogenetic protein (BMP). 4 -7 Specifically, we have shown that MGP binds and inhibits BMP-2 and -4, which are both detected in calcified atherosclerotic lesions. 8 -11 We have also shown that MGP-deficient cells have increased susceptibility to BMP-2-induced cell differentiation 12 and that MGP interferes with BMP-4 activity in the developing lungs of MGP transgenic mice. 13 MGP is known to be a calcium-binding protein. Calcium is bound through ␥-carboxylated glutamates, so-called "Gla residues," and induces conformational changes in the MGP protein. 4,14 Warfarin interferes with the vitamin K-dependent ␥-carboxylation and renders MGP nonfunctional, 15 presumably through loss of calcium binding. The majority of the Gla residues are located in the center of the MGP protein (amino acid residue 56 to 71 in human MGP), a region that has been implicated in the binding of BMP. 4 However, the precise amino acid residues involved in BMP binding have not been identified.In the BMP type I receptor, a phenylalanine residue (Phe85) plays a key role in binding BMP-2. 16 The aromatic side chain of Phe85, with "knob-into-hole" packing, points into a hydrophobic pocket formed at the interfaces of 2 BMP-2 monomers. Similarly, a critical proline residue (Pro35) in the BMP inhibitor Noggin occupies the same pocket, 17,18 thereby preventing ligand-receptor binding. When examining the protein sequence of human MGP, we found a conserved proline residue (Pro64) centrally located in the BMP-bin...

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 Bone morphogenetic proteins (BMPs)-2, -4, and -6 have been detected in atherosclerotic plaques 3-5 and may to contribute to their formation. In endothelial cells (ECs), BMP-2 and -4 have been identified as genes mediating the response to inflammation and oscillatory shear stress. 6,7 Noggin and matrix Gla protein (MGP), both able to inhibit BMPs, are also induced by oscillatory shear stress, 8 suggesting that the balance of BMP signaling is essential in the vascular endothelium. BMP-2 and -4 may contribute to the progression of vascular calcification because of their osteoinductive properties. 9 Indeed, BMP-2 has been shown to augment signaling through MSH homeodomain 2 (MSX2)-Wnt signaling in aortic myofibroblasts, thereby stimulating osteogenic differentiation and calcification. 10 In osteoblastic cells, MSX2 and distal-less homeodomains 3 and 5 (DLX3 and DLX5) have been shown to coregulate genes that are essential in bone development, including the RUNX2 and the osteocalcin genes. 11 The BMPs belong to the transforming growth factor (TGF)-ß superfamily of growth factors. 12 These growth factors elicit their responses via 2 types of serine/threonine receptors, termed type I and type II receptors. BMP binds to the type I receptor, and this complex then binds the type II receptor with increased affinity. 12 The receptor complex phosphorylates specific receptorregulated (R)-SMAD proteins, which associate with a common partner (Co)-SMAD4 and translocate into the nucleus, where they regulate target gene expression. 12 Activin-like kinase receptor (ALK)2, ALK3 and ALK6 are categorized as type I BMP receptors, which interact with the BMP type II receptor (BMPRII) to phosphorylate SMAD1/ 5/8. 12 ALK1 is also a type I receptor, which resembles ALK2, but was initially reported to be a receptor for TGF-ß1 and -3. However, BMP-9 and -10 were recently identified as ligands, and ALK1 was shown to interact with BMPRII similar to Original

Objective Abnormal function of high density lipoprotein (HDL) has been implicated as a potential mechanism for the increased cardiovascular disease (CVD) in patients with rheumatoid arthritis. The current work evaluated changes in HDL function and HDL-associated proteins over two years of follow-up in early RA patients receiving either methotrexate (MTX) monotherapy or combination therapies in the Treatment of Early Rheumatoid Arthritis (TEAR) trial. Methods The anti-oxidant capacity of HDL, paraoxonase 1 (PON-1) activity, HDL-associated haptoglobin (HDL-Hp), HDL-associated apolipoprotein AI (HDL-apoA-I), and myeloperoxidase (MPO) levels were measured in 550 TEAR participants at 4 time points (0 [pre-treatment], 24, 48, and 102 weeks). Repeated measures analysis was performed using mixed effect linear models with autoregressive covariate structure to model the within-subject covariance over time. Results Mixed effect models controlling for traditional CV risk factors, treatment regimen, prednisone use, and statin use demonstrated significant associations of RA disease activity measured by the disease activity score with 28 joint count (DAS28), erythrocyte sedimentation rate (ESR), or C-reactive protein (CRP) with the HDL function profile over time. Specifically, decreases in RA disease activity over time were associated with increases in PON1 activity and HDL-apoA-I levels, and decreases in the HDL inflammatory index (HII), MPO, and HDL-Hp. Conclusion Decreases in disease activity in early RA patients treated with MTX, MTX + etanercept, or triple therapy in the TEAR trial were associated with improvements in the HDL function profile. Additional work is warranted to evaluate abnormal HDL function as a potential mechanism and therapeutic target for CV risk in patients with RA.

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 geno-type (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.

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.