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1.  C/EBP homologous protein drives pro-catabolic responses in chondrocytes 
Arthritis Research & Therapy  2013;15(6):R218.
Introduction
Excess C/EBP homologous protein (CHOP) expression is one feature of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress. Here, we focused on CHOP expression and function in chondrocytes.
Methods
We studied human knee osteoarthritis (OA) cartilage, bovine chondrocytes cultured in alginate and subjected to sub-lethal biomechanical injury, and knee chondrocytes of human autopsy donors. We performed siRNA knockdown and transfection.
Results
UPR activation was increased in human knee OA cartilage in situ, and in biomechanically injured cultured chondrocytes in vitro. In normal human chondrocytes, CHOP “gain of function” sensitized chondrocytes to IL-1β induced nitric oxide (NO) and matrix metalloproteinase (MMP)-3 release without inducing these responses by itself. Excess CHOP expression, by itself, induced superoxide production and apoptosis. Conversely, siRNA knockdown of CHOP and the UPR-specific mediator X-box binding protein (XBP1) inhibited NO release by >80% (P <0.0005) in response to IL-1β, and blunted MMP-3 release, whereas there were only minimal effects of the UPR mediator GRP78 on these responses. The anti-inflammatory metabolic “super-regulator” AMP kinase (AMPK) is known to limit UPR activation in vascular muscle cells. Here, CHOP supported the capacity of IL-1β to suppress AMPK activity in chondrocytes. We also observed that inhibition of AMPK activity promoted an increase in chondrocyte CHOP expression. Conversely, pharmacologic activation of AMPK by 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) blunted chondrocyte CHOP expression in response to biomechanical injury.
Conclusions
Biomechanical injury and IL-1 signaling stimulate UPR activation in chondrocytes. CHOP mediates chondrocyte catabolic and apoptotic responses to IL-1β, and does so partly by inhibiting AMPK activity. Conversely, development of excess CHOP activity is limited by AMPK activity in chondrocytes. Our findings suggest a mechanism for potential chondroprotection by AICAR and other AMPK activators. The work is of translational relevance for OA, since several drugs that activate AMPK are already in the clinic for arthritis (for example, allosteric AMPK activators sodium salicylate and high dose aspirin, and methotrexate, which activates AMPK by generating AICAR).
doi:10.1186/ar4415
PMCID: PMC3978428  PMID: 24351550
4.  Linked decreases in liver kinase B1 and AMP-activated protein kinase activity modulate matrix catabolic responses to biomechanical injury in chondrocytes 
Introduction
AMP-activated protein kinase (AMPK) maintains cultured chondrocyte matrix homeostasis in response to inflammatory cytokines. AMPK activity is decreased in human knee osteoarthritis (OA) chondrocytes. Liver kinase B1 (LKB1) is one of the upstream activators of AMPK. Hence, we examined the relationship between LKB1 and AMPK activity in OA and aging cartilages, and in chondrocytes subjected to inflammatory cytokine treatment and biomechanical compression injury, and performed translational studies of AMPK pharmacologic activation.
Methods
We assessed activity (phosphorylation) of LKB1 and AMPKα in mouse knee OA cartilage, in aging mouse cartilage (6 to 24 months), and in chondrocytes after mechanical injury by dynamic compression, via immunohistochemistry or western blot. We knocked down LKB1 by siRNA transfection. Nitric oxide, matrix metalloproteinase (MMP)-3, and MMP-13 release were measured by Griess reaction and ELISA, respectively.
Results
Knockdown of LKB1 attenuated chondrocyte AMPK activity, and increased nitric oxide, MMP-3 and MMP-13 release (P <0.05) in response to IL-1β and TNFα. Both LKB1 and AMPK activity were decreased in mouse knee OA and aged knee cartilage, and in bovine chondrocytes after biomechanical injury. Pretreatment of bovine chondrocytes with AMPK activators AICAR and A-769662 inhibited both AMPKα dephosphorylation and catabolic responses after biomechanical injury.
Conclusion
LKB1 is required for chondrocyte AMPK activity, thereby inhibiting matrix catabolic responses to inflammatory cytokines. Concurrent loss of LKB1 and AMPK activity in articular chondrocytes is associated with OA, aging and biomechanical injury. Conversely, pharmacologic AMPK activation attenuates catabolic responses to biomechanical injury, suggesting a potentially novel approach to inhibit OA development and progression.
doi:10.1186/ar4254
PMCID: PMC3979085  PMID: 23883619
osteoarthritis; cartilage; aging; MMP-3; nitric oxide
5.  The Growing Array of Innate Inflammatory Ignition Switches in Osteoarthritis 
Arthritis and rheumatism  2012;64(7):2055-2058.
Osteoarthritis (OA) is the most common form of arthritis and is a major cause of chronic pain and disability. We currently lack disease-modifying OA medical therapeutics that effectively slow or halt the progression to destruction and failure of articular cartilage. Importantly, OA is a disease of the whole joint, including not only meniscal fibrocartilage and hyaline articular cartilage, but also subchondral bone, periarticular musculature, tendons and ligaments, articular adipose tissue, synovium, and synovial fluid (SF). Clinically, varying degrees of synovitis and joint effusion in OA contribute to signs and symptoms of inflammation (1). Multiple lines of evidence suggest that OA progression is promoted by low-grade innate articular inflammation and by synovitis (1,2). “Conventional” inflammatory cytokines expressed in cartilage and synovium likely play a role, and interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), IL-6, IL-8, and IL-17 are among the players in synovitis (1,2). The report by Nair et al in this issue of Arthritis & Rheumatism reveals increased levels of soluble CD14 (sCD14) in SF to be a biomarker of innate inflammation in patients undergoing arthroscopic knee meniscectomy for treatment of meniscal tears (3). Investigators in this group previously characterized this population as “enriched for patients with preradiographic disease” (4), given the associated symptoms, synovitis, and evidence of articular cartilage damage detected by arthroscopy.
doi:10.1002/art.34492
PMCID: PMC3711830  PMID: 22488656
6.  Imaging joints for calcium pyrophosphate crystal deposition: a knock to the knees 
With advanced age, articular calcium pyrophosphate crystal deposition (CPPD) is common. Defining who has CPPD is of growing importance, given increases in longevity in many countries and the frequent association of chondrocalcinosis with osteoarthritis. Chondrocalcinosis detected by plain radiography serves as a major screening tool, but how many and which sites to screen have not been adequately defined in the past. The work of Abhishek and colleagues in the previous issue of Arthritis Research and Therapy sheds new light on the incomplete information from knee radiographs, and helps position us to learn more about the epidemiology, pathophysiology, diagnosis, and clinical impact of CPPD.
doi:10.1186/ar4097
PMCID: PMC3674586  PMID: 23270654
7.  RAGE signaling mediates post-injury arterial neointima formation by suppression of liver kinase B1 and AMPK activity 
Atherosclerosis  2012;222(2):417-425.
Objective
Intima formation involves smooth muscle cell (SMC) proliferation and migration that ultimately drives arterial stenosis, thrombosis, and ischemia in atherosclerosis, hypertension, and arterial revascularization. Receptor for advanced glycation endproducts (RAGE) is a transmembrane signaling receptor implicated in diabetic renal and vascular complications, and post-injury intima formation, partly via Signal transducer and activator of transcription 3 (STAT3) activation. The metabolic super-regulator Adenosine monophosphate kinase (AMPK) inhibits SMC proliferation and intima formation. AMPK activation is promoted by liver kinase B1 (LKB1), and LKB1 inhibits STAT3 activation. Here, we tested the hypothesis that RAGE promotes arterial intima formation by modulating both LKB1 and AMPK.
Methods and Results
RAGE ligands (the calgranulin S100A11, and glycated albumin) suppressed AMPK activation in conjunction with increased proliferation and migration of cultured SMCs. These effects were inhibited both by RAGE deficiency and by prior AMPK activation. In SMCs, RAGE ligands decreased LKB1 activity. Moreover, knockdown of both LKB1 and AMPK were associated with increased STAT3 phosphorylation levels. In response to murine carotid artery ligation, expression of RAGE and S100A11 increased, whereas AMPK and LKB1 activity decreased in situ. Conversely, LKB1 and AMPK activity increased in situ, and neointima formation was attenuated in Rage−/− mice.
Conclusion
The linkage of decreased LKB1 and AMPK activity with increased STAT3 in SMCs mediates the capacity of RAGE ligand-induced signaling to promote neointima formation in response to arterial injury.
doi:10.1016/j.atherosclerosis.2012.04.001
PMCID: PMC3361645  PMID: 22552116
RAGE; AMPK; LKB1; Arterial restenosis; Neointimal hyperplasia
8.  Are Either or Both Hyperuricemia and Xanthine Oxidase Directly Toxic to the Vasculature? A critical appraisal 
Arthritis and Rheumatism  2012;64(2):327-338.
Basic research and clinical studies have implicated a role for hyperuricemia and for xanthine oxidoreductase (XOR), the enzyme that generates uric acid (UA), in not only gout but also vascular diseases. At present, asymptomatic hyperuricemia (i.e., in the absence of gout, urate nephrolithiasis, or tumor lysis syndrome) is not an indication for therapy. With the rise over the past several decades in prevalence of both gout and hyperuricemia, clarifying the potential adverse effects of hyperuricemia (in patients with and without gout) is of public health importance. UA is not simply an inert end-product of purine metabolism in humans, but rather has potential antioxidant, pro-oxidant, and pro-inflammatory effects. However controversy remains as to which, if any, of these effects are of clinical relevance in development and complications of human vascular diseases in gout and asymptomatic hyperuricemia. Clearly, not all individuals with hyperuricemia develop gout, and studies to date have also been unable to clarify in which subjects hyperuricemia may have detrimental effects on the vasculature. Further, studies of urate-lowering therapy with XOR inhibition or uricosuric agents have not been able to definitively identify whether any such effects may be mediated by UA versus XO. Adequately sized, prospective randomized clinical trials of sufficient duration, and employing appropriate biomarkers, now appear critical to resolve the putative toxic roles of UA and XO in the human arterial circulation.
doi:10.1002/art.33369
PMCID: PMC3265683  PMID: 21953377
urate; uric acid; hypertension; endothelium; smooth muscle cell; nitric oxide; myeloperoxidase; xanthine oxidase; allopurinol; atherosclerosis
9.  Rilonacept in the treatment of acute gouty arthritis: a randomized, controlled clinical trial using indomethacin as the active comparator 
Introduction
In phase-3 clinical trials, the interleukin (IL-1) blocker, rilonacept (IL-1 Trap), demonstrated efficacy for gout flare prevention during initiation of urate-lowering therapy. This trial evaluated rilonacept added to a standard-of-care, indomethacin, for treatment of acute gout flares.
Methods
Adults, aged 18-70 years, with gout presenting within 48 hours of flare onset and having at least moderate pain as well as swelling and tenderness in the index joint were randomized to subcutaneous (SC) rilonacept 320 mg at baseline plus oral indomethacin 50 mg TID for 3 days followed by 25 mg TID for up to 9 days (n = 74); SC placebo at baseline plus oral indomethacin as above (n = 76); or SC rilonacept 320 mg at baseline plus oral placebo (n = 75). The primary efficacy endpoint was change in pain in the index joint (patient-reported using a Likert scale (0 = none; 4 = extreme)) from baseline to the average of values at 24, 48 and 72 hours (composite time point) for rilonacept plus indomethacin versus indomethacin alone. Comparison of rilonacept monotherapy with indomethacin monotherapy was dependent on demonstration of significance for the primary endpoint. Safety evaluation included clinical laboratory and adverse event (AE) assessments.
Results
Patient characteristics were comparable among the groups; the population was predominantly male (94.1%), white (75.7%), with mean ± SD age of 50.3 ± 10.6 years. All treatment groups reported within-group pain reductions from baseline (P < 0.0001). Although primary endpoint pain reduction was greater with rilonacept plus indomethacin (-1.55 ± 0.92) relative to indomethacin alone (-1.40 ± 0.96), the difference was not statistically significant (P = 0.33), so formal comparison between monotherapy groups was not performed. Pain reduction over the 72-hour period with rilonacept alone (-0.69 ± 0.97) was less than that in the other groups, but pain reduction was similar among groups at 72 hours. Treatment with rilonacept was well-tolerated with no reported serious AEs related to rilonacept. Across all groups, the most frequent AEs were headache and dizziness.
Conclusions
Although generally well-tolerated, rilonacept in combination with indomethacin and rilonacept alone did not provide additional pain relief over 72 hours relative to indomethacin alone in patients with acute gout flare.
Trial registration
ClinicalTrials.gov registration number NCT00855920.
doi:10.1186/ar4159
PMCID: PMC3672764  PMID: 23375025
10.  Npp1 promotes atherosclerosis in ApoE knockout mice 
Objective
Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PPi), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in humans. NPP1 and PPi have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification.
Methods and Results
Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high fat/high cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE−/− and ttw/+ ApoE−/− mice demonstrated decreased Opn expression. The 28 weeks old ttw/ttw ApoE−/− and ttw/+ ApoE−/− had significantly smaller atherosclerotic lesions compared with wild type congenic ApoE−/− mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE−/− mice without Npp1 deficiency.
Conclusion
We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.
doi:10.1111/j.1582-4934.2011.01327.x
PMCID: PMC3154990  PMID: 21477221
atherosclerosis; plaque calcification; osteopontin; inorganic pyrophosphate
11.  Chondrocyte AMP-activated Protein Kinase Activity Suppresses Matrix Degradation Responses to Inflammatory Cytokines IL-1β and TNFα 
Arthritis and rheumatism  2011;63(7):1928-1937.
Objective
IL-1β and TNFα stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in OA. AMPK, which regulates energy homeostasis and cellular metabolism, also exerts anti-inflammatory effects in multiple tissues. Here, we tested the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL-1β and TNFα.
Methods
Expression of AMPK subunits was examined, and AMPKα activity was ascertained by phosphorylation status of AMPKα Thr172 in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Pro-catabolic responses to IL-1β and TNFα such as release of GAG, NO, MMP-3 and MMP-13 were determined by DMMB assay, Griess reaction and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by siRNA approach.
Results
Normal human knee articular chondrocytes express AMPKα1, α2, β1, β2 and γ1 subunits. AMPK activity is constitutively present in normal, but is decreased in OA articular chondrocytes and cartilage, and in normal chondrocytes treated with IL-1β and TNFα. Knockdown of AMPKα results in enhanced catabolic responses to IL-1β and TNFα in chondrocytes. Moreover, AMPK activators suppress cartilage/chondrocyte pro-catabolic responses to IL-1β and TNFα and the capacity of TNFα and CXCL8 (IL-8) to induce type X collagen expression.
Conclusions
AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL-1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and pro-catabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation-induced degradation.
doi:10.1002/art.30333
PMCID: PMC3128233  PMID: 21400477
12.  Minimally important differences of the gout impact scale in a randomized controlled trial 
Rheumatology (Oxford, England)  2011;50(7):1331-1336.
Objective. The Gout Impact Scale (GIS) is a gout-specific quality of life instrument that assesses impact of gout during an attack and impact of overall gout. The GIS has five scales and each is scored from 0 to 100 (worse health). Our objective was to assess minimally important differences (MIDs) for the GIS administered in a randomized controlled trial (RCT) assessing rilonacept vs placebo for prevention of gout flares during initiation of allopurinol therapy.
Methods. Trial subjects ( n = 83) included those with two or more gout flares (self-reported) in the past year. Of these, 73 had data for Weeks 8 vs 4 and formed the MID analysis group and were analysed irrespective of the treatment assignment. Subjects completed the GIS and seven patient-reported anchors. Subjects with a one-step change (e.g. from very poor to poor) were considered as the MID group for each anchor. The mean change in GIS scores and effect size (ES) was calculated for each anchor’s MID group. The average of these created the overall summary MID statistics for each GIS. An ES of 0.2–0.5 was considered to represent MID estimates.
Results. Trial subjects (n = 73) were males (96.0%), White (90.4%), with mean age of 50.5 years and serum uric acid of 9.0 mg/dl. The mean change score for the MID improvement group for scales ranged from −5.24 to −7.61 (0–100 scale). The ES for the MID improvement group for the four scales ranged from 0.22 to 0.38.
Conclusion. The MID estimates for GIS scales are between 5 and 8 points (0–100 scale). This information can aid in interpreting the GIS results in future gout RCTs.
Trial Registration. Clinicaltrials.gov, www.clinicaltrials.gov, NCT00610363.
doi:10.1093/rheumatology/ker023
PMCID: PMC3307519  PMID: 21372003
Gout assessment questionnaire; Gout impact scale; Minimally important difference; Minimal clinically important differences; Rilonacept; Clinical trial design; Health-related quality of life; Health status
13.  Vanin-1 Pantetheinase Drives Smooth Muscle Cell Activation in Post-Arterial Injury Neointimal Hyperplasia 
PLoS ONE  2012;7(6):e39106.
The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn1−/− SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARγ expression. Vnn1−/− mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARγ, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization.
doi:10.1371/journal.pone.0039106
PMCID: PMC3374784  PMID: 22720042
14.  Arterial Calcification Is Driven by RAGE in Enpp1–/– Mice 
Journal of Vascular Research  2010;48(3):227-235.
Background/Aims
Ectopic osteochondral differentiation, driven by ENPP1-catalyzed generation of the chondrogenesis and calcification inhibitor inorganic pyrophosphate (PPi), promotes generalized arterial calcification of infancy. The multiligand receptor for advanced glycation end-products (RAGE), which promotes atherosclerosis and diabetic cardiovascular and renal complications, also mediates chondrocyte differentiation in response to RAGE ligand calgranulins such as S100A11. Here, we tested RAGE involvement in ENPP1 deficiency-associated arterial calcification.
Methods
Because ectopic artery calcification in Enpp1–/– mice is Pi-dependent and mediated by PPi deficiency, in vitro studies on effects of S100A11 and RAGE on mouse aortic explants were conducted using exogenous Pi, as well as alkaline phosphatase to hydrolyze ambient PPi.
Results
S100A11 induced cartilage-specific collagen IX/XI expression and calcification dependent on RAGE in mouse aortic explants that was inhibited by the endogenous RAGE signaling inhibitor soluble RAGE (sRAGE). Enpp1–/– aortic explants demonstrated decreased Pi-stimulated release of sRAGE, and increased calcification and type IX/XI collagen expression that were suppressed by exogenous sRAGE and by Rage knockout. Last, Rage knockout suppressed spontaneous aortic calcification in situ in Enpp1–/– mice.
Conclusion
Cultured Enpp1–/– aortic explants have decreased Pi-stimulated release of sRAGE, and RAGE promotes ectopic chondrogenic differentiation and arterial calcification in Enpp1–/– mice.
doi:10.1159/000318805
PMCID: PMC2997448  PMID: 21099228
Artery calcification; Receptor for advanced glycation end-products; Calgranulin; ENPP1; Pit-1
15.  Transglutaminase 2 is a Marker of Chondrocyte Hypertrophy and Osteoarthritis Severity in the Hartley Guinea Pig Model of Knee OA 
Objective
The transglutaminase (TG) isoenzyme TG2, which catalyzes protein cross-linking via transamidation, influences healing phenotype in multiple forms of tissue injury. Moreover, TG2 knockout suppresses cartilage destruction but promotes osteophyte formation in instability-induced mouse knee OA. TG2 is marker of growth plate chondrocyte hypertrophy. Moreover, TG2 secreted by chondrocytes acts in part by promoting chondrocyte maturation to hypertrophy, a differentiation state linked with MMP-13 expression and disease progression in OA. Moreover, glucosamine, which is currently under investigation as an OA therapy, binds and inhibits TG2. Here, we examined TG2 as a potential marker of cartilage hypertrophy in the spontaneous guinea pig model of OA.
Methods
Synovial fluid ELISA and cartilage Immunohistochemistry and quantitative RT-PCR, were used to examine TG2 expression and TG transamidation-catalyzed isopeptide bonds.
Results
TG isopeptide bonds and TG2 were most abundant in articular cartilage in early knee OA. TG2 expression was robust at sites of early but not established osteophytes. Synovial fluid TG2 correlated with knee OA total histological score (r=0.47, p=0.01), as did medial tibial plateau cartilage TG2 mRNA (r=1.0, p=0.003). At 12 months of age, medial tibial plateau cartilage TG2 mRNA expression rose markedly in association with elevated type X collagen, as well as ADAMTS-5, and MMP-13 expression, changes not shared in age-matched Strain 13 guinea pigs that are less susceptible to knee OA.
Conclusion
Hartley guinea pig knee TG2 expression associates with enhanced articular chondrocyte hypertrophy and is a biomarker of OA severity.
doi:10.1016/j.joca.2009.02.015
PMCID: PMC3249463  PMID: 19328881
Osteoarthritis Biomarkers; Chondrocyte Hypertrophy; MMP-13; ADAMTS-5; Strain 13
16.  Genetics in Arterial Calcification 
Circulation research  2011;109(5):578-592.
Artery calcification reflects an admixture of factors such as ectopic osteochondral differentiation with primary host pathological conditions. We review how genetic factors, as identified by human genome-wide association studies, and incomplete correlations with various mouse studies, including knockout and strain analyses, fit into “pieces of the puzzle” in intimal calcification in human atherosclerosis, and artery tunica media calcification in aging, diabetes mellitus, and chronic kidney disease. We also describe in sharp contrast how ENPP1, CD73, and ABCC6 serve as “cogs in a wheel” of arterial calcification. Specifically, each is a minor component in the function of a much larger network of factors that exert balanced effects to promote and suppress arterial calcification. For the network to normally suppress spontaneous arterial calcification, the “cogs” ENPP1, CD73, and ABCC6 must be present and in working order. Monogenic ENPP1, CD73, and ABCC6 deficiencies each drive a molecular pathophysiology of closely related but phenotypically different diseases (generalized arterial calcification of infancy (GACI), pseudoxan-thoma elasticum (PXE) and arterial calcification caused by CD73 deficiency (ACDC)), in which premature onset arterial calcification is a prominent but not the sole feature.
doi:10.1161/CIRCRESAHA.111.247965
PMCID: PMC3248761  PMID: 21852556
ENPP1; CD73; ABCC6; generalized arterial calcification of infancy; pseudoxanthoma elasticum
17.  CARTILAGE CELL CLUSTERS 
Arthritis and rheumatism  2010;62(8):2206-2218.
The formation of new cell clusters is a histological hallmark of arthritic cartilage but the biology of clusters and their role in disease are poorly understood. This is the first comprehensive review of clinical and experimental conditions associated with cluster formation. Genes and proteins that are expressed in cluster cells, the cellular origin of the clusters, mechanisms that lead to cluster formation and the role of cluster cells in pathogenesis are discussed.
doi:10.1002/art.27528
PMCID: PMC2921934  PMID: 20506158
18.  Chondrocyte Innate Immune MyD88-dependent Signaling Drives Pro-Catabolic Effects of the Endogenous TLR2/TLR4 Ligands LMW-HA and HMGB1 
Arthritis and rheumatism  2010;62(7):2004-2012.
Objective
TLR2/TLR4-mediated innate immunity serves in frontline antimicrobial host defense, but also modulates tissue remodeling and repair responses to endogenous ligands released in low-grade inflammatory diseases. Here, we assessed if the endogenous TLR2/TLR4 ligands low molecular weight hyaluronan (LMW-HA) and high mobility group box protein 1 (HMGB1), which are increased in osteoarthritic (OA) joints, drive pro-catabolic chondrocyte responses dependent on TLR2 and TLR4 signaling through the cytosolic adaptor MyD88.
Methods
We studied mature femoral head cap cartilage explants and immature primary knee articular chondrocytes from TLR2/TLR4 double knockout (TLR2/TLR4−/−), MyD88 knockout (MyD88−/−) and congenic wild type mice.
Results
IL-1β and TLR2 and TLR4 ligands induced both HMGB1 release from chondrocytes and extracellular LMW-HA generation in normal chondrocytes. TLR2/TLR4−/− and MyD88−/− mouse cartilage explants and chondrocytes lost the capacity to mount pro-catabolic responses to both LMW-HA and HMGB1, demonstrated by >95% suppression of nitric oxide (NO) production (p<0.01), and attenuated induction of MMP-3 and MMP-13. Combined deficiency of TLR2/4, or of MyD88 alone, also attenuated release of NO and blunted induction of MMP-3 and MMP-13 release. Last, MyD88 was required for HMGB1 and hyaluronidase 2 (hyal2) (which generates LMW-HA) to induce chondrocyte hypertrophy, which is implicated in OA progression.
Conclusions
MyD88-dependent TLR2/TLR4 signaling is essential for pro-catabolic responses to LMW-HA and HMGB1, and MyD88 drives chondrocyte hypertrophy. Therefore, LMW-HA and HMGB1 act as innate immune cytokine-like signals with the potential to modulate chondrocyte differentiation and function in OA progression.
doi:10.1002/art.27475
PMCID: PMC2902571  PMID: 20506365
19.  Serum Urate Is Not Associated with Coronary Artery Calcification: The NHLBI Family Heart Study 
The Journal of rheumatology  2010;38(1):111-117.
Objective
Urate may have effects on vascular remodeling and atherosclerosis. We had shown an association between serum uric acid (SUA) and carotid atherosclerotic plaques. Inflammation and vascular remodeling in atherosclerosis promote coronary artery calcification (CAC), a preclinical marker for atherosclerosis. Here, we examined whether SUA is associated with CAC, using the same study sample and methods as for our previous carotid atherosclerosis study.
Methods
The National Heart, Lung, and Blood Institute Family Heart Study is a multicenter study designed to assess risk factors for heart disease. Participants were recruited from population-based cohorts in the US states of Massachusetts, North Carolina, Minnesota, Utah, and Alabama. CAC was assessed with helical computed tomography (CT). We conducted sex-specific and family-cluster analyses, as well as additional analyses among persons without risk factors related to both cardiovascular disease and hyperuricemia, adjusting for potential confounders as we had in the previous study of carotid atherosclerosis.
Results
For the CAC study, 2412 subjects had both SUA and helical CT results available (55% women, age 58 ± 13 yrs, body mass index 27.6 ± 5.3). We found no association of SUA with CAC in men or women [OR in men: 1.0, 1.11, 0.86, 0.90; women: 1.0, 0.83, 1.00, 0.87 for increasing categories of SUA: < 5 (referent group), 5 to < 6, 6 to < 6.8, ≥ 6.8 mg/dl, respectively], nor in subgroup analyses.
Conclusion
Replicating the methods used to demonstrate an association of SUA with carotid atherosclerosis did not reveal any association between SUA and CAC, suggesting that SUA likely does not contribute to atherosclerosis through effects on arterial calcification. The possibility that urate has divergent pathophysiologic effects on atherosclerosis and artery calcification merits further study.
doi:10.3899/jrheum.100639
PMCID: PMC3119360  PMID: 20889594
URIC ACID; CORONARY ARTERY CALCIFICATION; ATHEROSCLEROSIS
20.  Gout disease-specific quality of life and the association with gout characteristics 
Purpose
Assess the association of gout characteristics with health-related quality of life (HRQoL) using a new gout-specific HRQoL instrument, the Gout Impact Scale (GIS).
Patients and methods
Gout patients completed the GIS (five scales [0–100 score each] representing impact of gout overall [three scales] and during an attack [two scales]) and other questions describing recent gout attacks, treatment, gout history, comorbidities, and demographics. Physicians confirmed gout diagnosis, presence of tophi, and most recent serum uric acid (sUA) level. Relationships between gout characteristics and GIS scores were examined using analysis of variance and correlation analyses.
Results
The majority of patients were male (90.2%) with a mean age of 62.2 (±11.8) years. Approximately one-half (49.7%) reported ≥3 gout attacks in the past year and the majority (57.9%) reported experiencing gout-related pain between attacks. Patients had appreciable concern about their gout (“gout concern overall” scale, 63.1 ± 28.0) but believed their treatment was adequate (“unmet gout treatment need” scale (38.2 ± 21.4) below scale mid-point). Significantly worse GIS scores were associated with increasing attack frequency and greater amount of time with pain between attacks (most scales, P < 0.001). Common objective measures such as sUA level, presence of tophi and the number of joints involved in a typical attack did not appear to be good indicators of the impact of gout on the patients' HRQoL.
Conclusion
Attack frequency and gout pain between attacks were important correlates of patients' ratings of gout impact on their HRQoL. Further studies are needed to determine the minimal important difference for each GIS scale and interpret our results relative to other patient populations with gout.
doi:10.2147/PROM.S8310
PMCID: PMC3113652  PMID: 21686040
Gout impact scale; GIS; patient-reported outcomes
21.  Shifting HIFs in osteoarthritis 
Nature medicine  2010;16(6):641-644.
There is no cure for osteoarthritis—the most common disease of the joints. By piecing together the molecular events that drive the progression of this debilitating disease, recent studies published in Nature Medicine put hypoxia-inducible factor-2α (HIF-2α) in the driver's seat, opening up new avenues for early detection and treatment (pages 678–686 and 687–693).
doi:10.1038/nm0610-641
PMCID: PMC2917906  PMID: 20526316
22.  Perceptions of disease and health-related quality of life among patients with gout 
Rheumatology (Oxford, England)  2009;48(5):582-586.
Objective. To assess the impact of gout on health-related quality of life (HRQoL) among patients in three large US cities.
Methods. Gout patients completed the Short Form-36 (SF-36) and a series of questions regarding their gout, comorbidities and demographics. Their physicians confirmed the gout diagnosis and evaluated the severity of patient's gout. The differences in mean norm-based SF-36 scores between the US norms and gout patients and between subgroups of gout patients were calculated. The relative weight and significance of gout-related characteristics associated with patients’ HRQoL were also calculated.
Results. The majority of the patients were males with a mean age of 62.2 years and median disease duration of 13.8 years. Most were overweight/obese with several comorbidities. Half of the patients experienced three or more gout attacks per year with a typical gout attack involving five joints and lasting for at least 4 days. The Physical Component Summary (PCS) and Mental Component Summary (MCS) was significantly lower for gout patients (P < 0.002 and P < 0.001, respectively). Among gout patients, the mean PCS and MCS were lower for those with more frequent gout attacks and greater number of affected joints (P < 0.005 and P < 0.001, respectively). After adjusting for age, gender and comorbidities, the number of joints involved during a typical and the worst gout attack had the greatest impact on patient's PCS and MCS.
Conclusion. Gout patients had clinically significant lower HRQoL than their age-matched US norm. Comorbidities and several additional gout-related factors significantly impacted the overall HRQoL.
doi:10.1093/rheumatology/kep047
PMCID: PMC2722803  PMID: 19307257
Gout; Health-related quality of life; SF-36; Outcomes
23.  Gout disease-specific quality of life and the association with gout characteristics 
Purpose:
Assess the association of gout characteristics with health-related quality of life (HRQoL) using a new gout-specific HRQoL instrument, the Gout Impact Scale (GIS).
Patients and methods:
Gout patients completed the GIS (five scales [0–100 score each] representing impact of gout overall [three scales] and during an attack [two scales]) and other questions describing recent gout attacks, treatment, gout history, comorbidities, and demographics. Physicians confirmed gout diagnosis, presence of tophi, and most recent serum uric acid (sUA) level. Relationships between gout characteristics and GIS scores were examined using analysis of variance and correlation analyses.
Results:
The majority of patients were male (90.2%) with a mean age of 62.2 (±11.8) years. Approximately one-half (49.7%) reported ≥3 gout attacks in the past year and the majority (57.9%) reported experiencing gout-related pain between attacks. Patients had appreciable concern about their gout (“gout concern overall” scale, 63.1 ± 28.0) but believed their treatment was adequate (“unmet gout treatment need” scale (38.2 ± 21.4) below scale mid-point). Significantly worse GIS scores were associated with increasing attack frequency and greater amount of time with pain between attacks (most scales, P < 0.001). Common objective measures such as sUA level, presence of tophi and the number of joints involved in a typical attack did not appear to be good indicators of the impact of gout on the patients’ HRQoL.
Conclusion:
Attack frequency and gout pain between attacks were important correlates of patients’ ratings of gout impact on their HRQoL. Further studies are needed to determine the minimal important difference for each GIS scale and interpret our results relative to other patient populations with gout.
PMCID: PMC3113652  PMID: 21686040
Gout impact scale; GIS; patient-reported outcomes
24.  Inorganic pyrophosphatase induces type I collagen in osteoblasts 
Bone  2009;46(1):81.
Introduction
The physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PPi) and generates phosphate (Pi). Humans and mice deficient in the PPi-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPPiase), which generates Pi via PPi hydrolysis with neutral pH optimum, remains unknown. We assessed iPPiase in Enpp1−/− and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPPiase regulates collagen I expression.
Methods
We treated mouse calvarial osteoblasts with ascorbate and β-glycerol phosphate to promote calcification, and we assessed cytosolic Pi and PPi levels, sodium-dependent Pi uptake, Pit-1 Pi co-transporter expression, and iPPiase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts.
Results
Inorganic pyrophosphatase but not TNAP was elevated in Enpp1−/− calvariae in situ. Cultured primary Enpp1−/− calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PPi in early culture, Enpp1−/− osteoblasts maintained cytosolic Pi levels comparable to WT osteoblasts, in association with increased iPPiase, enhanced sodium-dependent Pi uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1−/− osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic Pi and decreased cytosolic but not extracellular PPi, and induced both collagen I synthesis and calcification.
Conclusions
Increased iPPiase is associated with “Pi hunger” and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPPiase induces collagen I at the levels of mRNA expression and synthesis and, unlike TNAP, stimulates calcification by osteoblasts without reducing the extracellular concentration of the hydroxyapatite crystal inhibitor PPi.
doi:10.1016/j.bone.2009.08.055
PMCID: PMC2818162  PMID: 19733704
PPi; Pi; Tissue-Nonspecific Alkaline Phosphatase; Calcification; Enpp1
25.  Gout. Novel therapies for treatment of gout and hyperuricemia 
In the past few decades, gout has increased not only in prevalence, but also in clinical complexity, the latter accentuated in part by a dearth of novel advances in treatments for hyperuricemia and gouty arthritis. Fortunately, recent research reviewed here, much of it founded on elegant translational studies of the past decade, highlights how gout can be better managed with cost-effective, well-established therapies. In addition, the advent of both new urate-lowering and anti-inflammatory drugs, also reviewed here, promises for improved management of refractory gout, including in subjects with co-morbidities such as chronic kidney disease. Effectively delivering improved management of hyperuricemia and gout will require a frame shift in practice patterns, including increased recognition of the implications of refractory disease and frequent noncompliance of patients with gout, and understanding the evidence basis for therapeutic targets in serum urate-lowering and gouty inflammation.
doi:10.1186/ar2738
PMCID: PMC2745774  PMID: 19664185

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