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Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is a clinically heterogeneous disorder that is characterized by the presence of intraarticu-lar CPPD crystals. CPPD crystals deposit primarily in cartilage but can traffic within the joint space. CPPD crystals form in the normally unmineralized peri-cellular matrix of articular hyaline and fibrocartilage. The most common sites of involvement are the knee meniscus, the triangular fibrocartilage of the wrist, and the glenohumeral joint. Asymptomatic disease is common in these sites as well as in others (e.g., the symphy-sis pubis). Pathologic cartilage calcification is promoted by changes in inorganic pyrophosphate (PPi) metabolism, extracellular matrix, and chondrocyte differentiation. CPPD deposition disease has a close and complex relationship with osteoarthritis. CPPD most commonly afflicts the elderly but can occur in younger patients who have a familial variant of the condition or who are afflicted with any of several metabolic diseases that predispose to CPPD deposition. No specific evidence-based therapies for CPPD exist. However, the inflammation promoted by CPPD crystal deposition can be managed successfully in most patients through strategies typically employed for the treatment and prophylaxis of gout.

Mononuclear leukocyte–mediated inflammation is a central feature of atherosclerosis. Using measures that diminish lesion monocyte recruitment and macrophage-mediated inflammation, investigators have shown these specific events to be essential to the development and progression of atherosclerotic lesions. Lesion T lymphocytes have an activated phenotype and colocalize with lesion macrophages, particularly in early disease. However, the role of lesion T lymphocytes in atherogenesis is not as well understood as the role of macrophages. A variety of inflammatory mediators, including cytokines, growth factors, and proinflammatory lipids, are present in atherosclerotic lesions. However, even in the complex milieu of a chronic inflammatory locus, a limited number of cytokines can become dominant. This concept has most recently been illustrated by the dramatic success of inhibitors of tumor necrosis factor (TNF)-α in the treatment of rheumatoid arthritis. Thus, certain inhibitors of cytokines, or anti-inflammatory cytokines, have been proposed as potential “immunologic scalpels” to diminish the activity of several chronic mononuclear leukocyte–mediated inflammatory diseases. Interleukin (IL)-10, a prototypical anti-inflammatory cytokine, is under investigation (and in a few cases at the stage of clinical trials) for therapy of a variety of chronic diseases. These include rheumatoid arthritis, inflammatory bowel disease, psoriasis, multiple sclerosis, allergic eosinophilic inflammation, Wegener’s granulomatosis, and cardiac allograft rejection. In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology ,1 Pinderski Oslund et al present a novel chapter in an evolving story on the potential modulatory role of IL-10 in atherosclerosis. A cross-regulatory role in atherosclerosis of the activated monocyte-macrophage (and Th2 immune response) product IL-10 with the Th1 response–promoting cytokine IL-12 was first proposed by Uyemura and colleagues.2 IL-10 downregulates IL-12 production and inhibits Th1 immune responses, which would normally be associated with IL-2 and interferon-γ (IFN-γ) expression and with T-cell and macrophage activation. IL-12 was generally expressed in atherosclerotic plaques, and highly …

ObjectiveTo evaluate the reliability and validity of an instrument assessing the influence of gout (acute and chronic) on health-related quality of life (HRQOL).MethodsFocus groups were used to examine the content of an existing Gout Assessment Questionnaire (GAQ1.0). GAQ2.0 was developed, consisting of a section describing the impact of gout on HRQOL [Gout Impact (GI)] and 4 sections describing subjects’ gout overall and demographic data. The GAQ2.0 and the Medical Outcomes Study Short Form-36 Version 2 (SF-36v2) were completed by gout patients in 3 US cities. GI scales were examined using clinical judgment, review of item statistics, Rasch analysis, and confirmatory factor analysis.ResultsSubjects (n = 308) were predominantly male (90.2%), Caucasian (75.9%), with a mean age 62.2 ± 11.8 years. Half the subjects (49.7%) reported ≥ 3 attacks in the past year. Two-week test-retest reliability for each scale was good (0.77 to 0.89) for all 5 GI scales. All scales achieved high sufficient (0.86 to 0.89) or excellent (0.93 to 0.97) ratings based on 10-item adjusted alpha coefficients. Correlations and tests among known groups indicated subjects with more severe gout had higher GI scores (i.e., greater gout impact). GI scores correlated more highly with patient-reported measures of gout severity than the SF-36v2 and several traditional measures of gout severity.ConclusionThe GAQ2.0 is an instrument for measuring the impact of gout on HRQOL. The GI section exhibited acceptable reliability and validity characteristics. Future studies should assess GI responsiveness, minimally important differences, and psychometric properties in other patient populations.

gamma-Interferon (IFN-gamma) has recently been demonstrated to inhibit the ability of mononuclear phagocytes to degrade bone particles. We have further addressed the specificity, potency and mechanism of this activity using human recombinant IFN-gamma. Adherent peripheral blood mononuclear leukocytes from normal human volunteers were cultured with washed, sieved (less than or equal to 75 microns) 45Ca-labelled rat bone particles for 3 days, after which bone particle degradation (7.1 +/- 1.6%, n = 11) was calculated from the fraction of 45Ca released into the medium. As little as 5 U/ml IFN-gamma significantly suppressed bone particle degradation and 50 U/ml was associated with consistent marked suppression (74.0 +/- 3.5% inhibition, P less than 0.001, n = 11). IFN-gamma was not suppressive if added to cells 24 h or more after exposure to bone particles. Addition of indomethacin (10 microM) did not reverse the effect of IFN-gamma, suggesting that it was not prostaglandin-mediated. In addition, 1,25(OH)2D3 (10 nM) did not remove the inhibitory effect of IFN-gamma. Contact of mononuclear phagocytes with bone particles and secretion of soluble factors from these cells have both been demonstrated to play a role in their ability to degrade bone particles. IFN-gamma (50 U/ml) inhibited monocyte/macrophage interaction with another unopsonized surface, i.e., one micron fluorescent latex particles, decreasing the number of internalized particles from 12.6 +/- 2.9 per cell to 5.9 +/- 1.4 per cell (P less than 0.01, n = 15), as measured using flow cytometry. However, binding of bone particles by the cells was not diminished by IFN-gamma. Exogenous alpha-interferon and human recombinant IL-1 beta, TNF-alpha, and lymphotoxin did not alter bone particle degradation. In addition, endogenous IL-1 beta release from human monocyte/macrophages exposed to bone particles was negligible and unaffected by IFN-gamma. We conclude that IFN-gamma is a potent and specific inhibitor of monocyte/macrophage-mediated bone particle degradation, and that this activity does not appear to be due to effects on the ability of monocytes to bind bone particles or to release IL-1 in response to the particles.

Deactivating mutations in the TNSALP gene cause HPP. Akp2−/− mice model severe infantile HPP, but there is no model for the relatively mild adult form. Here we report on mice with an induced mutation in Akp2 that affects splicing. The phenotype of homozygotes mirror aspects of the adult form of HPP. Introduction: Hypophosphatasia (HPP) is a clinically varied skeletal disorder resulting from deficiency of tissue nonspecific alkaline phosphatase (TNSALP). Mice lacking Akp2 model infantile HPP characterized by skeletal hypomineralization, impaired growth, seizures, and perinatal mortality. No animal model exists to study the less severe forms of the disease that typically present in later life. Materials and Methods:N-ethyl-N-nitrosourea (ENU) mutagenesis was used to generate mouse models of human disease. A mouse with low plasma alkaline phosphatase (ALP) activity was identified by our clinical chemistry screen. Its offspring were used for inheritance studies and subjected to biochemical, histological, and radiological phenotyping. DNA was extracted for mapping and osteoblasts harvested for functional studies. Results: We showed semidominant inheritance of the low ALP phenotype and mapped the underlying point mutation to Akp2. Affected offspring bear the splice site mutation 862 + 5G>A—a hypomorphic allele named Akp2Hpp. The same mutation has been reported in a patient. Akp2Hpp/+ mice have ∼50% of normal plasma ALP but display no other biochemical or skeletal abnormalities. Unlike Akp2−/− mice, Akp2Hpp/Hpp mice have normal initial skeletal development and growth, a normal lifespan and do not have seizures. TNSALP is low but detectable in Akp2Hpp/Hpp plasma. Osteoblasts display ∼10% of normal ALP activity and reduced intracellular inorganic phosphate levels, yet are capable of normal mineralization in vitro. TNSALP substrates are significantly elevated in urine (inorganic pyrophosphate and phosphoethanolamine) and plasma (pyridoxal 5′-phosphate), whereas plasma inorganic pyrophosphate levels are normal. Akp2Hpp/Hpp mice develop late-onset skeletal disease, notably defective endochondral ossification and bone mineralization that leads to arthropathies of knees and shoulders. Conclusions:Akp2Hpp/Hpp mice mirror a number of clinical features of the human adult form of HPP. These mice provide for the first time an animal model of late onset HPP that will be valuable in future mechanistic studies and for the evaluation of therapies such as those aimed at HPP.

Background Previous studies have shown that long-term urate-lowering therapy is required for improvements in gout flare frequency and tophi reduction, and that lower serum uric acid (sUA) levels may result in greater benefit. However, optimal sUA levels to jointly achieve these outcomes within a year using single or combination oral therapies are uncertain. Objectives To assess the impact of sustained sUA lowering to determine if the magnitude of continual low sUA levels results in greater tophus size reduction and fewer patients experiencing gout flares requiring treatment (GFRT). Methods Patients, irrespective of treatment assignment, were combined from 3 Phase III clinical studies examining the efficacy of lesinurad, a selective uric acid reabsorption inhibitor (SURI), in combination with a xanthine oxidase inhibitor (allopurinol: CLEAR 1 [NCT01510158], CLEAR 2 [NCT01493531]), or febuxostat: CRYSTAL [NCT01510769]). For this analysis, GFRTs were assessed during the last quarter of study treatment (end of Month 9 to end of Month 12) and reduction in target tophus area (measured using Vernier calipers) was assessed over the duration of the study. Patients were categorized by on-study median sUA levels of ≥6, 5– Results In total, 1537 patients were eligible for analysis; 95% were male, 78% were white, and mean (SD) age was 42 (11) years. A total of 474 patients (30.8%) had ≥1 target tophi that were followed in the studies. A positive relationship between lower sUA and greater tophus area reduction and GFRT was observed. In patients with ≥1 target tophi at baseline in the 3 studies, those with lowest on-study median sUA levels achieved greatest reduction in tophus area (Fig. 1A). Patients with median sUA Similarly, those with lower sUA levels were less likely to have GFRT. During the last quarter of the study, 12.2% with median sUA Conclusions These findings demonstrate that degree of clinical benefit (reduction in GFRT or tophus area) within 12 months correlates with the magnitude of sustained sUA lowering using oral sUA-lowering therapy. The results provide additional confirmation of the validity of sUA lowering as a surrogate for clinically meaningful effects in gout patients and point to potential benchmarks. In this context, patients with median sUA Acknowledgements This study was funded by Ardea Biosciences/AstraZeneca. Editorial support was provided by PAREXEL and was funded by AstraZeneca. Disclosure of Interest R. Terkeltaub Consultant for: Ardea Biosciences, AstraZeneca, Takeda, Relburn, REVIVE, F. Perez-Ruiz Consultant for: Menarini, AstraZeneca, Pfizer, Speakers bureau: Menarini, AstraZeneca, Pfizer, C. Storgard Shareholder of: Ardea Biosciences, Inc., a member of the AstraZeneca Group., Employee of: Ardea Biosciences, Inc., a member of the AstraZeneca Group., M. Fung Employee of: Ardea Biosciences, Inc., a member of the AstraZeneca Group., J. Kopicko Employee of: Ardea Biosciences, Inc., a member of the AstraZeneca Group., N. Dalbeth Grant/research support from: AstraZeneca, Fonterra, Novartis, Speakers bureau: Savient, Menarini, Novartis, Teijin, and Advisory Boards for AstraZeneca, Fonterra, Takeda, Pfizer, Metabolex

Background Lesinurad (LESU; RDEA594) is a selective uric acid reabsorption inhibitor (SURI) being investigated for the treatment of gout in combination with a xanthine oxidase inhibitor. Objectives The CRYSTAL study is a multinational, randomized, double-blind, placebo-controlled, phase III clinical trial of LESU in combination with febuxostat (FBX) to determine efficacy and safety of combination therapy compared with FBX monotherapy in patients with tophaceous gout (NCT01510769). Methods Patients with gout, ages 18-85 yrs, with serum uric acid (sUA) ≥8 mg/dL (≥6 mg/dL on urate lowering therapy) and ≥1 tophus were given FBX 80 mg qd for 3 weeks before randomization to LESU (200 mg or 400 mg oral, qd) in combination with FBX or placebo (PBO) + FBX. Primary endpoint was proportion of patients with sUA Results Patients (N=324) were white (79.9%) and male (95.4%) with mean ± SD age of 54.1±11.0 yrs and 14.7±10.9 yrs since gout diagnosis. sUA was 8.7±1.6 mg/dL at screening and 5.3±1.6 mg/dL on FBX at randomization (28% with sUA ≥6 mg/dL). More patients achieved sUA levels Conclusions In patients with tophaceous gout, LESU (200 or 400 mg) in combination with FBX increased the proportion of patients achieving sUA Acknowledgements Research sponsored by Ardea Biosciences/AstraZeneca. Editorial support was provided by PAREXEL and funded by AstraZeneca. Disclosure of Interest N. Dalbeth Grant/research support from: AstraZeneca, Fonterra, Novartis, Speakers bureau: Savient, Menarini, Novartis, Takeda, and Advisory Boards for AstraZeneca, Fonterra, Takeda, Metabolex, G. Jones Grant/research support from: Abbvie, Ardea, Novartis, Auxilium, Consultant for: Pfizer, Roche, Hospira and Janssen, Speakers bureau: UCB, Roche, Janssen, Abbvie, Novartis, Mundipharma, Amgen, BMS, Pfizer, R. Terkeltaub Consultant for: AstraZeneca, Takeda, Revive, Relburn, UCB, D. Khanna Grant/research support from: AstraZeneca, Consultant for: AstraZeneca, Takeda, J. Kopicko Employee of: Ardea Biosciences, Inc., a member of the AstraZeneca group, N. Bhakta Employee of: Ardea Biosciences, Inc., a member of the AstraZeneca group, M. Fung Employee of: Ardea Biosciences, Inc., a member of the AstraZeneca group, C. Storgard Employee of: Ardea Biosciences, Inc., a member of the AstraZeneca group, S. Baumgartner Employee of: Ardea Biosciences, Inc., a member of the AstraZeneca group, F. Perez-Ruiz Grant/research support from: Spanish Health Ministry, Spanish Rheumatology Foundation, and Cruces Hospital Rheumatologists Association, Consultant for: AstraZeneca, Menarini, Metabolex, Novartis, Pfizer, and SOBI, Speakers bureau: AstraZeneca and Menarini

Mechanistic and therapeutic advances in gout have been moving swiftly in the past decade. Clinically, the disease is changing in character. This review discusses several of the pertinent recent developments in understanding gout and in novel therapeutics for the disease. Subjects addressed include the role of URAT1-mediated renal proximal tubule epithelial cell urate anion reabsorption in hyperuricemia. We discuss the therapeutic limitations of allopurinol and uricosurics and the potential applications of novel xanthine oxidase inhibitors and of recombinant uricase preparations. Last, we summarize understanding of the central role of the early induced innate immune response in gouty inflammation, which has suggested the potential value of new strategies for treating gouty inflammation by targeting caspase-1 or IL-1beta.

Background & Aims: Mallory body (MB) inclusions are a characteristic feature of several liver disorders and share similarities with cytoplasmic inclusions observed in neural diseases and myopathies. MBs consist primarily of keratins 8 and 18 (K8/K18), require a K8-greater-than-K18 ratio for their formation, and contain glutamine-lysine cross-links generated by transglutaminase (TG). We hypothesized that protein transamidation is essential for MB formation. Methods: Because TG2 is the most abundant hepatocyte TG, we tested our hypothesis using TG2−/− and their wild-type counterpart mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an established MB inducer. Keratin cross-linking was further examined using recombinant proteins or transgenic mice that overexpress K8 or K18. Results: TG2−/− livers have markedly reduced TG2 activity as compared with TG2+/+ livers. The DDC-fed TG2−/− mice have dramatic decreases in MB formation and liver hypertrophy response as contrasted with DDC-fed TG2+/+ mice. Despite similar hepatocellular damage, TG2−/− mice had more gallstones, jaundice, and ductal proliferation than wild-type mice. Inhibition of MB formation in TG2−/− mice was associated with marked attenuation of ubiquitination and K8-containing protein cross-linking. MB formation and resolution paralleled the generation then disappearance of cross-linked K8, respectively. K8 is a preferential TG2 substrate when compared to K18, as examined in vitro or in DDC-fed transgenic mice that overexpress K8 or K18. Conclusions: We demonstrate an essential role for TG2 in determining injury-mediated liver enlargement and the necessity of K8 and TG2 for generating cross-linked keratins and MBs. The role of TG in inclusion formation might extend to nonkeratin intermediate filament protein-related diseases.

Objective Chondrocalcinosis, which can promote joint inflammation and cartilage degeneration, is highly prevalent in elderly white subjects. Data on its prevalence are scarce in other ethnic populations. This study was undertaken to compare the prevalence of chondrocalcinosis in Chinese subjects with that in white subjects. Methods We recruited a random sample of Beijing residents ages ≥60 years. Participants underwent standard weight-bearing anteroposterior knee radiography and posteroanterior hand radiography using the protocols developed in the Framingham Osteoarthritis Study. Radiographic chondrocalcinosis was defined as present in a knee or wrist when there was evidence of definite linear cartilage calcification. We compared the prevalence of chondrocalcinosis in Chinese subjects with that in white subjects using age-standardized prevalence ratios. We used identical methods to collect samples of tap water from 2 cities and measured their levels of calcium, magnesium, and phosphate in the same laboratory. Results Chinese subjects had a much lower prevalence of knee chondrocalcinosis (1.8% in men, 2.7% in women) than did white subjects (6.2% in men, 7.7% in women), with the age-standardized prevalence ratio being 0.34 (95% confidence interval [95% CI] 0.20–0.54) and 0.43 (95% CI 0.31–0.59) in men and women, respectively. Wrist chondrocalcinosis was rare in elderly Chinese subjects (prevalence 0.3% in men and 1.0% in women), with the age-standardized prevalence ratio being 0.06 (95% CI 0.01–0.18) in Chinese men and 0.18 (95% CI 0.10–0.30) in Chinese women. Calcium levels in the tap water in Beijing were 15-fold higher than in Framingham, whereas no difference was found in magnesium and phosphate levels. Conclusion Knee chondrocalcinosis and wrist chondrocalcinosis are far less common in Chinese subjects in Beijing than in US white subjects in Framingham, Massachusetts. Given the current lack of understanding of the etiology of chondrocalcinosis, further epidemiologic studies of the impact of genetic and environmental factors on occurrence of chondrocalcinosis are indicated.

Objective Monosodium urate monohydrate (MSU) crystals have remarkable inflammatory potential. However, gouty inflammation is spontaneously self-limited, an occurrence recognized since antiquity. Gouty synovitis is driven and sustained by neutrophil influx. Importantly, macrophage phagocytosis of apoptotic (but not necrotic) neutrophils is antiinflammatory. Therefore, we tested the hypothesis that efficient clearance of apoptotic neutrophils by macrophages is one of the factors that restrains the progression of gouty inflammation. Macrophage expression of transglutaminase 2 (TG2), a multifunctional protein with reciprocally regulated transamidation and purine nucleotide–binding activities, promotes apoptotic leukocyte uptake. In this study, we tested the specific role of macrophage TG2 expression in MSU crystal–induced inflammation. Methods We studied MSU crystal–induced peritonitis in TG2−/− and congenic TG2+/+ mice. We also studied the effects of TG2 on apoptotic cell uptake by cultured macrophages. Results TG2−/− mice demonstrated more progressive neutrophilic accumulation than did TG2+/+ mice, which was associated with delayed clearance of apoptotic neutrophils during MSU crystal–induced peritonitis. We observed defective phagocytosis of apoptotic leukocytes by TG2−/− peritoneal macrophages, which was corrected by soluble extracellular TG2. Transamidation catalytic activity of TG2 was not required to mediate macrophage uptake of apoptotic leukocytes. In contrast, the TG2 nucleotide binding site residue K173 was critical for this TG2 function. TG2 bound to GDP, ADP, or ATP (but not to GTP) rescued defective apoptotic leukocyte uptake by TG2−/− macrophages. Conclusion Enhancement of apoptotic neutrophil uptake by macrophage-derived TG2 restrains gout-like neutrophilic peritoneal inflammation. Differential binding of TG2 by purine nucleotides may contribute to clinical variability in the extent and duration of gouty inflammation.

Increased levels of ePPi in mice deficient in TNALP (i.e., Akp2−/−) lead to elevated OPN concentrations. We examined the skeletal phenotype of mice lacking both OPN and TNALP and concluded that the increased OPN levels contribute to the hypophosphatasia phenotype characteristic of Akp2−/− mice. We also found that extracellular OPN regulates the PPi output by osteoblasts. Introduction:Akp2−/− display mineralization deficiencies characterized by rickets/osteomalacia. This defect has been attributed to the increased levels of extracellular inorganic pyrophosphate (ePPi), a substrate of tissue-nonspecific alkaline phosphatase (TNALP) and a potent inhibitor of mineral deposition. Because elevated levels of ePPi induce Opn gene expression, the Akp2−/− mice also display increased levels of osteopontin (OPN), another inhibitor of mineralization. Materials and Methods:Akp2−/− mice were bred into the Opn−/− line. The resulting double knockout mice were analyzed for skeletal abnormalities by histology and μCT. Calvarial osteoblasts were assayed for their ability to mineralize in vitro and were probed for changes in gene expression. Results: Mice lacking both Akp2 and Opn showed partial normalization at the histological level with regard to mineral deposition and BMD. However, high ePPi levels remained in Akp2−/− mice. We found that Opn−/− mice have themselves elevated levels of ePPi attributable to an increase in Enpp1 and Ank expression and a concomitant downregulation of Akp2 expression in Opn−/− osteoblasts, but that Opn−/− mice have more mineralized osteoid than wildtype (WT) controls despite their elevated ePPi levels. Addition of exogenous OPN to Opn−/− osteoblasts results in downregulation of Enpp1 and Ank gene expression and a reduction of the PPi output by these cells. Conclusions: Deletion of both Akp2 and Opn can partially rescue the hypomineralized phenotype of Akp2−/− mice. However, these double knockout mice do not display corrected ePPi levels, and we conclude that regulation of hydroxyapatite deposition requires the coordinated actions of both PPi and OPN and that the hypophosphatasia phenotype in Akp2−/− mice results from the combined inhibitory action of increased levels of both ePPi and OPN. Our data also suggest that the ePPi-mediated regulation of OPN and the OPN-mediated regulation of ePPi are linked counterregulatory mechanisms that control the concentrations of these two important mineralization inhibitors, OPN and ePPi.

Objective To evaluate the relationship between chondrocalcinosis and the progression of knee osteoarthritis (OA) using longitudinal magnetic resonance imaging (MRI) assessments. Methods Longitudinal knee MRIs were obtained in the Boston OA Knee Study (BOKS) and in the Health, Aging and Body Composition (Health ABC) Study. Chondrocalcinosis was determined as present or absent on baseline knee radiographs. Cartilage morphology was graded on paired longitudinal MRIs using the Whole-Organ Magnetic Resonance Imaging Score in 5 cartilage subregions of each of the medial and lateral tibiofemoral joints. Cartilage loss in a subregion was defined as an increase in the cartilage score of ≥1 (0–4 scale). The risk for change in the number of subregions with cartilage loss was assessed using Poisson regression, with generalized estimating equations to account for correlations. Analyses were adjusted for age, sex, body mass index, baseline cartilage score, and presence of damaged menisci. Results In BOKS, 23 of the 265 included knees (9%) had chondrocalcinosis. In Health ABC, 373 knees were included, of which 69 knees (18.5%) had chondrocalcinosis. In BOKS, knees with chondrocalcinosis had a lower risk of cartilage loss compared with knees without chondrocalcinosis (adjusted risk ratio [RR] 0.4, 95% confidence interval [95% CI] 0.2–0.7) (P = 0.002), and there was no difference in risk in Health ABC (adjusted RR 0.9, 95% CI 0.6–1.5) (P = 0.7). Stratification by intact versus damaged menisci produced similar results within each cohort. Conclusion In knees with OA, the presence of chondrocalcinosis was not associated with increased cartilage loss. These findings do not support the hypothesis that chondrocalcinosis worsens OA progression.

Macrophage-mediated inflammation is central to atherogenesis. We have determined previously that the CXC chemokine receptor CXCR2 is involved in advanced atherosclerosis. We sought to determine whether one of the ligands of CXCR2, KC/GRO-alpha, can also modulate atherogenesis. KC/GRO-alpha(-/-) mice were generated and mated with the atherosclerosis-prone LDLR(-/-) mice. There was a significant reduction in atherosclerosis in mice lacking KC/GRO-alpha; however, this reduction was only approximately half that seen previously in mice lacking CXCR2 in the leukocyte. To determine whether CXCR2 is involved in the early formation of atherosclerosis, leukocyte-specific CXCR2(-/-) chimeric mice on LDLR(-/-) background were generated. Early fatty streak lesion formation in these mice was not affected by leukocyte CXCR2 deficiency whereas lesions were less developed in mice lacking leukocyte CXCR2 when atherosclerosis was allowed to progress to the intermediate stage. Macrophages were relatively sparse in the lesions of leukocyte CXCR2(-/-) mice despite robust MCP-1 expression. These studies indicate that KC/GRO-alpha/CXCR2 does not play a critical role in recruitment of macrophages into early atherosclerotic lesions but both arterial KC/GRO-alpha and leukocyte-specific CXCR2 expression are central to macrophage accumulation in established fatty streak lesions.

Purpose of review Gout is the most common inflammatory arthritis in men, affecting approximately 1-2% of adult men in Western countries. United States gout prevalence has approximately doubled over the past two decades. In recent years, key prospective epidemiological and open-labeled dietary studies, coupled with recent advances in molecular biology elucidating proximal tubular urate transport, have provided novel insights into roles of diet and alcohol in hyperuricemia and gout. This review focuses on recent developments and their implications for clinical practice, including how we advise patients on appropriate diets and alcoholic beverage consumption. Recent findings Studies have observed an increased risk of gout among those who consumed the highest quintile of meat, seafood and alcohol. Although limited by confounding variables, low-fat dairy products, ascorbic acid and wine consumption appeared to be protective for the development of gout. Summary The most effective forms of dietary regimen for both hyperuricemia and gout flares remains to be unidentified. Until confirmed by a large, controlled study, it is prudent to advise patients to consume meat, seafood and alcoholic beverages in moderation, with special attention to food portion size and content of non-complex carbohydrates which are essential for weight loss and improved insulin sensitivity.

Acute gouty arthritis is a characteristically intense acute inflammatory reaction that erupts in response to articular tophaceous deposits of monosodium urate monohydrate (MSU) crystals. Full-blown acute gouty arthritis is dependent on the influx into the joint space of neutrophils, a cell type normally absent from this locus. Hence, the clinical term “gout,” classically described in the 5th century BC by Hippocrates, was derived in a remarkably prescient manner from the Latin word “gutta,” which literally means “drop.” Specifically, gout was named to reflect the notion of “evil humors” aberrantly seeping into affected joints. Recent advances have revealed the uncanny link between this ancient disease and innate immune inflammatory responses that are phylogenetically primeval. Innate immunity provides a first line of defense against infection via primitive responses that are nonspecific and broad in spectrum (1). Unlike adaptive immune responses, stereotypic innate immune “early induced” responses do not directly induce immunologic memory or lasting protective immunity, consistent in acute gout with the recurrent paroxysmal nature of the disease and the primary role in pathogenesis of “professional phagocytes.” Central to rapid innate immune inflammatory and killing responses is the remarkable capacity of all eukaryotic organisms to recognize the same constituents of microbes (termed pathogenassociated molecular patterns [PAMPs]). Seminal identification of Toll and Toll-like receptors (TLRs) that recognize PAMPs in Drosophila indicates that human innate immune mechanisms for discrimination of nonself PAMPs such as lipopolysaccharide (LPS) and peptidoglycan from self are phylogenetically primordial. The majority of encounters with microorganisms expressing PAMPs do not result in disease, but breaching of tissue barriers by pathogens can stimulate innate immune alternative complement pathway activation, the expression of chemokines and certain other inflammatory cytokines, and uptake of the pathogen by resident and recruited professional phagocytes in a rapid effort to eliminate the noxious agent.

Gouty arthritis is a characteristically intense acute inflammatory reaction that erupts in response to articular deposits of monosodium urate (MSU) crystals. Important recent molecular biologic advances in this field have given us a clear picture of the mechanistic basis of gouty inflammation. The innate immune inflammatory response is critically involved in the pathology of gout. Specifically, MSU crystals promote inflammation directly by stimulating cells via Toll-like receptor signaling and by providing a surface for cleavage of C5 and formation of complement membrane attack complex (C5b-9), culminating in secretion of cytokines, chemokines, and other inflammatory mediators with a dramatic influx of neutrophils into the joint. Despite the detailed mechanistic picture for gouty inflammation, there are no placebo-controlled, randomized clinical studies for any of the therapies commonly used, although comparative studies have demonstrated that many nonsteroidal anti-inflammatory drugs are equivalent to indomethacin with respect to controlling acute gouty attacks. In general, the first line of anti-inflammatory therapy for acute gout is nonsteroidal anti-inflammatory drugs, and the selective cyclo-oxygenase-2 inhibitor celecoxib can be used where appropriate. The second line of treatment is glucocorticosteroids, given systemically (oral, intravenous, or intramuscular) or intra-articularly. Alternatively, synthetic adrenocorticotropic hormone is effective, partly via induction of adrenal glucocorticosteroids and partly via rapid peripheral suppression of leukocyte activation by melatonin receptor 3 signaling. The third line of treatment is oral colchicine, which is highly effective when given early in an acute gouty attack, but it is poorly tolerated because of predictable gastrointestinal side effects.

Hyperuricemia treatment: is creatinine clearance a safer allopurinol dosing meter than plasma creatinine levels?

Objective In gout, incompletely defined molecular factors alter recognition of dormant articular and bursal monosodium urate monohydrate (MSU) crystal deposits, thereby inducing self-limiting bouts of characteristically severe neutrophilic inflammation. To define primary determinants of cellular recognition, uptake, and inflammatory responses to MSU crystals, we conducted a study to test the role of Toll-like receptor 2 (TLR-2), TLR-4, and the cytosolic TLR adapter protein myeloid differentiation factor 88 (MyD88), which are centrally involved in innate immune recognition of microbial pathogens. Methods We isolated bone marrow–derived macrophages (BMDMs) in TLR-2−/−, TLR-4−/−, MyD88−/−, and congenic wild-type mice, and assessed phagocytosis and cytokine expression in response to endotoxin-free MSU crystals under serum-free conditions. MSU crystals also were injected into mouse synovium-like subcutaneous air pouches. Results TLR-2−/−, TLR-4−/−, and MyD88−/− BMDMs demonstrated impaired uptake of MSU crystals in vitro. MSU crystal–induced production of interleukin-1β (IL-1β), tumor necrosis factor α, keratinocyte-derived cytokine/growth-related oncogene α, and transforming growth factor β1 also were significantly suppressed in TLR-2−/− and TLR-4−/− BMDMs and were blunted in MyD88−/− BMDMs in vitro. Neutrophil influx and local induction of IL-1β in subcutaneous air pouches were suppressed 6 hours after injection of MSU crystals in TLR-2−/− and TLR-4−/− mice and were attenuated in MyD88−/− mice. Conclusion The murine host requires TLR-2, TLR-4, and MyD88 for macrophage activation and development of full-blown neutrophilic, air pouch inflammation in response to MSU crystals. Our findings implicate innate immune cellular recognition of naked MSU crystals by specific TLRs as a major factor in determining the inflammatory potential of MSU crystal deposits and the course of gouty arthritis.

We have shown previously that the hypomineralization defects of the calvarium and vertebrae of tissue nonspecific alkaline phosphatase (TNAP)-deficient (Akp2−/−) hypophosphatasia mice are rescued by simultaneous deletion of the Enpp1 gene, which encodes nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Conversely, the hyperossification in the vertebral apophyses typical of Enpp1−/− mice is corrected in [Akp2−/−; Enpp1−/−] double-knockout mice. Here we have examined the appendicular skeletons of Akp2−/−, Enpp1−/−, and [Akp2−/−; Enpp1−/−] mice to ascertain the degree of rescue afforded at these skeletal sites. Alizarin red and Alcian blue whole mount analysis of the skeletons from wild-type, Akp2−/−, and [Akp2−/−; Enpp1−/−] mice revealed that although calvarium and vertebrae of double-knockout mice were normalized with respect to mineral deposition, the femur and tibia were not. Using several different methodologies, we found reduced mineralization not only in Akp2−/− but also in Enpp1−/− and [Akp2−/−; Enpp1−/−] femurs and tibias. Analysis of calvarial- and bone marrow-derived osteoblasts for mineralized nodule formation in vitro showed increased mineral deposition by Enpp1−/− calvarial osteoblasts but decreased mineral deposition by Enpp1−/− long bone marrow-derived osteoblasts in comparison to wild-type cells. Thus, the osteomalacia of Akp2−/− mice and the hypomineralized phenotype of the long bones of Enpp1−/− mice are not rescued by simultaneous deletion of TNAP and NPP1 functions.

The clinical implication of articular deposits of calcium-containing crystals (specifically of calcium pyrophosphate dihydrate and hydroxyapatite) in osteoarthritis is unknown. Recent longitudinal studies have suggested that in some instances calcium crystals are direct participants in cartilage damage, while in other situations they are merely markers of joint damage. Better understanding of the mechanisms of crystal formation, especially in relation to inorganic pyrophosphate regulation, has lead to potential avenues for therapeutic intervention. The current treatment of osteoarthritis associated with calcium-containing crystals should involve nonsteroidal anti-inflammatory drugs, intra-articular steroids, and in resistant cases, joint irrigation can be considered. While preliminary studies suggest the possibility of favorable benefits from colchicine and hydroxycholorquine in this osteoarthritis disease subset, more rigorous studies need to be conducted to establish their roles.

Objective. Certain mutations in ANKH, which encodes a multiple-pass transmembrane protein that regulates inorganic pyrophosphate (PPi) transport, are linked to autosomal-dominant familial chondrocalcinosis. This study investigated the potential for ANKH sequence variants to promote sporadic chondrocalcinosis. Methods. ANKH variants identified by genomic sequencing were screened for association with chondrocalcinosis in 128 patients with severe sporadic chondrocalcinosis or pseudogout and in ethnically matched healthy controls. The effects of specific variants on expression of common markers were evaluated by in vitro transcription/translation. The function of these variants was studied in transfected human immortalized CH-8 articular chondrocytes. Results. Sporadic chondrocalcinosis was associated with a G-to-A transition in the ANKH 5'-untranslated region (5'-UTR) at 4 bp upstream of the start codon (in homozygotes of the minor allele, genotype relative risk 6.0, P = 0.0006; overall genotype association P = 0.02). This -4-bp transition, as well as 2 mutations previously linked with familial and sporadic chondrocalcinosis (+ 14 bp C-to-T and C-terminal GAG deletion, respectively), but not the French familial chondrocalcinosis kindred 143-bp T-to-C mutation, increased reticulocyte ANKH transcription/ANKH translation in vitro. Transfection of complementary DNA for both the wild-type ANKH and the -4-bp ANKH protein variant promoted increased extracellular PPi in CH-8 cells, but unexpectedly, these ANKH mutants had divergent effects on the expression of extracellular PPi and the chondrocyte hypertrophy marker, type X collagen. Conclusion. A subset of sporadic chondrocalcinosis appears to be heritable via a -4-bp G-to-A ANKH 5'-UTR transition that up-regulates expression of ANKH and extracellular PPi in chondrocyte cells. Distinct ANKH mutations associated with heritable chondrocalcinosis may promote disease by divergent effects on extracellular PPi and chondrocyte hypertrophy, which is likely to mediate differences in the clinical phenotypes and severity of the disease.

Microcrystals of calcium pyrophosphate dihydrate (CPPD) and monosodium urate (MSU) deposited in synovium and articular cartilage initiate joint inflammation and cartilage degradation in large part by binding and directly activating resident cells. TLRs trigger innate host defense responses to infectious pathogens, and the expression of certain TLRs by synovial fibroblasts has revealed the potential for innate immune responses to be triggered by mesenchymally derived resident cells in the joint. In this study we tested the hypothesis that chondrocytes also express TLRs and that one or more TLRs centrally mediate chondrocyte responsiveness to CPPD and MSU crystals in vitro. We detected TLR2 expression in normal articular chondrocytes and up-regulation of TLR2 in osteoarthritic cartilage chondrocytes in situ. We demonstrated that transient transfection of TLR2 signaling-negative regulator Toll-interacting protein or treatment with TLR2-blocking Ab suppressed CPPD and MSU crystal-induced chondrocyte release of NO, an inflammatory mediator that promotes cartilage degeneration. Conversely, gain-of-function of TLR2 in normal chondrocytes via transfection was associated with increased CPPD and MSU crystal-induced NO release. Canonical TLR signaling by parallel pathways involving MyD88, IL-1R-associated kinase 1, TNF receptor-associated factor 6, and IκB kinase and Rac1, PI3K, and Akt critically mediated NO release in chondrocytes stimulated by both CPPD and MSU crystals. We conclude that CPPD and MSU crystals critically use TLR2-mediated signaling in chondrocytes to trigger NO generation. Our results indicate the potential for innate immunity at the level of the articular chondrocyte to directly contribute to inflammatory and degenerative tissue reactions associated with both gout and pseudogout.

Objective— We recently linked human arterial media calcification of infancy to heritable PC-1/nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) deficiency. NPP1 hydrolyzes ATP to generate PP i , a physicochemical inhibitor of hydroxyapatite crystal growth. But pathologic calcification in NPP1 deficiency states is tissue-restricted and in perispinal ligaments is endochondral differentiation–mediated rather than simply a dystrophic process. Because ectopic chondro-osseous differentiation promotes artery calcification in atherosclerosis and other disorders, we tested the hypothesis that NPP1 and PP i deficiencies regulate cell phenotype plasticity to promote artery calcification. Methods and Results— Using cultured multipotential NPP1−/− mouse bone marrow stromal cells, we demonstrated spontaneous chondrogenesis inhibitable by treatment with exogenous PP i . We also demonstrated cartilage-specific gene expression, upregulated alkaline phosphatase, decreased expression of the physiological calcification inhibitor osteopontin, and increased calcification in NPP1−/− aortic smooth muscle cells (SMCs). Similar changes were demonstrated in aortic SMCs from ank/ank mice, which are extracellular PP i –depleted because of defective ANK transmembrane PP i transport activity. Moreover, NPP1−/− and ank/ank mice demonstrated aortic media calcification by von Kossa staining, and intra-aortic cartilage-specific collagen gene expression was demonstrated in situ in NPP1−/− mice. Conclusions— NPP1 and PP i deficiencies modulate phenotype plasticity in artery SMCs and chondrogenesis in mesenchymal precursors, thereby stimulating artery calcification by modulating cell differentiation.

Generalized arterial calcification of infancy (GACI), is characterized by calcification of the internal elastic lamina of large and medium-sized arteries and stenosis due to myointimal proliferation. Although survival to adulthood has been reported, most patients die within the first six months of life. Recently, we found mutations of ENPP1 coding for ecto-nucleotide pyrophosphatase/phosphodiesterase 1 to be associated with GACI in 8 of 11 families. In this study, we analyzed ENPP1 in affected individuals of another 12 unrelated families. We identified 11 novel homozygous or compound heterozygous mutations in 10 of the 12 new families. The mutations (1 nonsense, 7 missense, 1 single amino acid deletion, and 2 frame shift mutations) were scattered over the whole coding region with a slightly more condensed distribution within the catalytic and nuclease-like domain as compared to the first survey. In this study, three mutations were found repeatedly in apparently unrelated patients, 7 x c.913CA (p.Pro305Thr) and c.2662C [corrected]T (p.Arg888Trp) as well as c.2320CT (p.Arg774Cys) each twice. However, haplotype analysis suggested a founder effect of British extraction for mutation c.913CA (p.Pro305Thr). The fact that the two other mutations c.2662C [corrected]T (p.Arg888Trp) and c.2320CT (p.Arg774Cys) occurred twice within a single allele also suggests a single founder. This study confirms the role of ENPP1 mutations as the main cause of GACI and adds considerably to the mutational spectrum of ENPP1.

Objective Monosodium urate monohydrate (MSU) crystals promote gouty inflammation that is critically mediated by neutrophil recruitment and activation. Interleukin-8 (IL-8) and closely related chemokines are major neutrophil chemotaxins in experimental gout. But MSU crystals also activate the classical and alternative pathways of complement, and MSU crystals directly cleave C5 on the crystal surface. Unlike IL-8, the roles in acute gout of individual complement-derived peptides and of the terminal C5b–9 complement components that comprise the membrane attack complex (MAC) are unclear. Hence, we studied rabbits deficient in the MAC component C6 to determine if MAC mediated urate crystal–induced arthritis. Methods We injected C6-deficient and C6-sufficient rabbit knee joints with 10 mg of pyrogen-free urate crystals and analyzed IL-8 levels, leukocyte influx, and joint inflammation 24 hours later. Results There was a significant decrease (>60%) in swelling in MSU crystal–injected knees of C6-deficient animals as compared with C6-sufficient animals (P < 0.05). An attenuated rise in MSU crystal–induced joint effusion levels of IL-8 also was observed, which was concordant with diminished numbers of neutrophils (P < 0.05) but not monocytes in MSU crystal–induced knee synovial fluid from C6-deficient animals. Synovial tissue analysis confirmed mononuclear leukocyte infiltration in response to MSU crystal injection in all animals, but substantial neutrophil infiltration only in C6-sufficient animals. Conclusion MAC activation appears to play a major role in intraarticular IL-8 generation and in neutrophil recruitment in experimental acute gouty arthritis of the rabbit knee. C6 and MAC activation may represent novel therapeutic targets for suppression of neutrophil-mediated joint inflammation in gout.

Summary Objective Growth plate chondrocytes up-regulate calcium-sensing receptor (CaR) expression as they mature to hypertrophy. In cells other than chondrocytes, extracellular calcium-sensing via the CaR functions partly to promote expression of parathyroid hormone-related protein (PTHrP), a critical regulator of endochondral development. Moreover, PTHrP is up-regulated in human osteoarthritis (OA) and surgically induced rabbit OA cartilages and may promote both chondrocyte proliferation and osteophyte formation therein. Hence, we examined chondrocyte CaR-mediated calcium-sensing in OA pathogenesis. Methods We studied spontaneous knee OA in male Hartley guinea pigs. We also evaluated cultured bovine knee chondrocytes and immortalized human articular chondrocytes (CH-8 cells), employing the CaR calcimimetic agonist NPS R-467 or altering physiologic extracellular calcium (1.8mM). Results Immunohistochemistry revealed that CaR expression became up-regulated in the superficial zone at 4 months of age in the guinea pig medial tibial plateau cartilage as early OA developed. CaR expression later became up-regulated in the middle zone. PTHrP content, measured by immunoassay, was significantly increased in the medial tibial plateau cartilage as OA developed and progressed. In cultured chondrocytic cells, CaR-mediated extracellular calcium-sensing, stimulated by the calcimimetic NPS R-467, induced PTHrP and matrix metalloproteinase (MMP)-13 expression and suppressed expression of tissue inhibitor of metalloproteinase (TIMP)-3 dose-dependently, effects shared by elevated extracellular calcium (3mM). Extracellular calcium-sensing appeared essential for PTHrP and interleukin (IL)-1 to induce MMP-13 and for PTHrP 1-34 to suppress TIMP-3 expression. Conclusions Chondrocyte CaR expression becomes up-regulated early in the course of spontaneous guinea pig knee OA. Chondrocyte CaR-mediated extracellular calcium-sensing promotes PTHrP expression, modulates the effects of PTHrP and IL-1, and promotes MMP-13 expression and TIMP-3 depletion. Our results implicate up-regulated extracellular calcium-sensing via the CaR as a novel mediator of OA progression.

The ectonucleoside pyrophosphatase phosphodiesterase 1 (NPP1/PC-1) is a member of the NPP enzyme family that is critical in regulating mineralization. In certain mineralizing sites of bone and cartilage, membrane-limited vesicles [matrix vesicles (MVs)] provide a sheltered internal environment for nucleation of calcium-containing crystals, including hydroxyapatite. MV formation occurs by budding of vesicles from the plasma membrane of mineralizing cells. The MVs are enriched in proteins that promote mineralization. Paradoxically, NPP1, the type II transmembrane protein that generates the potent hydroxyapatite crystal growth inhibitor inorganic pyrophosphate (PPi), is also enriched in MVs. Although osteoblasts express NPP1, NPP2, and NPP3, only NPP1 is enriched in MVs. Therefore, this study uses mineralizing human osteoblastic SaOS-2 cells, a panel of NPP1 mutants, and NPP1 chimeras with NPP3, which does not concentrate in MVs, to investigate how NPP1 preferentially targets to MVs. We demonstrated that a cytosolic dileucine motif (amino acids 49–50) was critical in localizing NPP1 to regions of the plasma membrane that budded off into MVs. Moreover, transposition of the NPP1 cytoplasmic dileucine motif and flanking region (AAASLLAP) to NPP3 conferred to NPP3 the ability to target to the plasma membrane and, subsequently, concentrate in MVs. Functionally, the cytosolic tail dileucine motif NPP1 mutants lost the ability to support MV PPiconcentrations and to suppress calcification. The results identify a specific targeting motif in the NPP1 cytosolic tail that delivers PPi-generating NPP activity to osteoblast MVs for control of calcification.

Objective Because articular chondrocytes reside in a hypoxic milieu, anaerobic glycolysis is central in generating ATP to support chondrocyte matrix synthesis and viability, with mitochondrial oxidative phosphorylation possibly providing physiologic reserve ATP generation. Nitric oxide (NO) potently suppresses mitochondrial oxidative phosphorylation. Because enhanced cartilage NO generation occurs in osteoarthritis (OA), we systematically tested for mitochondrial dysfunction in the pathogenesis of OA. Methods We assessed chondrocytes for ATP depletion and for in situ changes in mitochondrial ultrastructure prior to and during the evolution of spontaneous knee OA in male Hartley guinea pigs, a model in which chondrocalcinosis also supervenes. Results Spontaneous NO release from knee cartilage samples in organ culture doubled between ages 2 months and 8 months as knee OA developed. Concomitantly, chondrocyte intracellular ATP levels declined by ∼50%, despite a lack of mitochondrial ultrastructure abnormalities in knee chondrocytes. As ATP depletion progressed with aging in knee chondrocytes, an increased ratio of lactate to pyruvate was observed, consistent with an adaptive augmentation of glycolysis to mitochondrial dysfunction. Furthermore, we observed progressive elevation of chondrocyte ATP-scavenging nucleotide pyrophosphatase/phosphodiesterase (NPP) activity and extracellular levels of the NPP enzymatic end product inorganic pyrophosphate (PPi), which stimulate chondrocalcinosis. Conclusion Profound chondrocyte ATP depletion develops in association with heightened NO generation in guinea pig knee OA. Increased NPP activity and concordant increases in extracellular PPi, which are strongly associated with human aging-associated degenerative arthropathy and directly stimulate chondrocalcinosis, may be primarily driven by chondrocyte ATP depletion. Our findings implicate a decreased mitochondrial bioenergetic reserve as a pathogenic factor in both degenerative arthropathy and chondrocalcinosis in aging.

Commentary on: Zhang Y, Neogi T, Chen C, et al. Cherry consumption and decreased risk of recurrent gout attacks. Arthritis Rheum 2012;64:4004–11.[OpenUrl][1][CrossRef][2][PubMed][3][Web of Science][4] The prevalence of gout has risen in the USA and many other countries, owing to a ‘perfect storm’ of factors, including increased prevalence of comorbid conditions, and diet and alcohol consumption megatrends. Resultant increase in clinical complexity of gouty arthritis and hyperuricaemia management is compounded by relatively poor patient adherence compared to many other medical conditions. This is partly because of gaps in patient and physician education. Notwithstanding, there is substantial interest by patients and clinicians in inexpensive and health-promoting non-pharmacological modalities to potentially improve gout management. Cherries and cherry products, buttressed by an initial uncontrolled study,1 are a popular folk remedy for gout, as evidenced by more than 1 600 000 ‘hits’ (as of January 2013) in an internet search for … [1]: {openurl}?query=rft.jtitle%253DArthritis%2Band%2Brheumatism%26rft.stitle%253DArthritis%2BRheum%26rft.aulast%253DZhang%26rft.auinit1%253DY.%26rft.volume%253D64%26rft.issue%253D12%26rft.spage%253D4004%26rft.epage%253D4011%26rft.atitle%253DCherry%2Bconsumption%2Band%2Bdecreased%2Brisk%2Bof%2Brecurrent%2Bgout%2Battacks.%26rft_id%253Dinfo%253Adoi%252F10.1002%252Fart.34677%26rft_id%253Dinfo%253Apmid%252F23023818%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [2]: /lookup/external-ref?access_num=10.1002/art.34677&link_type=DOI [3]: /lookup/external-ref?access_num=23023818&link_type=MED&atom=%2Febmed%2F18%2F6%2F230.atom [4]: /lookup/external-ref?access_num=000311706300021&link_type=ISI

Foci of chondrocyte hypertrophy that commonly develop in osteoarthritic (OA) cartilage can promote dysregulated matrix repair and pathologic calcification in OA. The closely related chemokines IL-8/CXCL8 and growth-related oncogene α (GROα)/CXCL1 and their receptors are up-regulated in OA cartilage chondrocytes. Because these chemokines regulate leukocyte activation through p38 mitogen-activated protein kinase signaling, a pathway implicated in chondrocyte hypertrophic differentiation, we tested whether IL-8 and GROα promote chondrocyte hypertrophy. We observed that normal human and bovine primary articular chondrocytes expressed both IL-8Rs (CXCR1, CXCR2). IL-8 and the selective CXCR2 ligand GROα (10 ng/ml) induced tissue inhibitor of metalloproteinase-3 expression, markers of hypertrophy (type X collagen and MMP-13 expression, alkaline phosphatase activity), as well as matrix calcification. IL-8 and the selective CXCR2 ligand GROα also induced increased transamidation activity of chondrocyte transglutaminases (TGs), enzymes up-regulated in chondrocyte hypertrophy that have the potential to modulate differentiation and calcification. Under these conditions, p38 mitogen-activated protein kinase pathway signaling mediated induction of both type X collagen and TG activity. Studies using mouse knee chondrocytes lacking one of the two known articular chondrocyte-expressed TG isoenzymes (TG2) demonstrated that TG2 was essential for murine GROα homologue KC-induced TG activity and critically mediated induction by KC of type X collagen, matrix metalloproteinase-13, alkaline phosphatase, and calcification. In conclusion, IL-8 and GROα induce articular chondrocyte hypertrophy and calcification through p38 and TG2. Our results suggest a novel linkage between inflammation and altered differentiation of articular chondrocytes. Furthermore, CXCR2 and TG2 may be sites for intervention in the pathogenesis of OA.

Osteopontin and PP(i) both suppress hydroxyapatite deposition. Extracellular PP(i) deficiency causes spontaneous hypercalcification, yet unchallenged osteopontin knockout mice have only subtle mineralization abnormalities. We report that extracellular PP(i) deficiency promotes osteopontin deficiency and correction of osteopontin deficiency prevents hypercalcification, suggesting synergistic inhibition of hydroxyapatite deposition. Nucleotide pyrophosphatase phosphodiesterase (NPP) isozymes including PC-1 (NPP1) function partly to generate PP(i), a physiologic calcification inhibitor. PP(i) transport is modulated by the membrane channel protein ANK. Spontaneous articular cartilage calcification, increased vertebral cortical bone formation, and peripheral joint and intervertebral ossific ankylosis are associated with both PC-1 deficiency and expression of truncated ANK in ank/ank mice. To assess how PC-1, ANK, and PP(i) regulate both calcification and cell differentiation, we studied cultured PC-1 -/- and ank/ank mouse calvarial osteoblasts. PC-1 -/- osteoblasts demonstrated approximately 50% depressed NPP activity and markedly lowered extracellular PP(i) associated with hypercalcification. These abnormalities were rescued by transfection of PC-1 but not of the NPP isozyme B10/NPP3. PC-1 -/- and ank/ank cultured osteoblasts demonstrated not only comparable extracellular PP(i) depression and hypercalcification but also marked reduction in expression of osteopontin (OPN), another direct calcification inhibitor. Soluble PC-1 (which corrected extracellular PP(i) and OPN), and OPN itself (> or = 15 pg/ml), corrected hypercalcification by PC-1 -/- and ank/ank osteoblasts. Thus, linked regulatory effects on extracellular PP(i) and OPN expression mediate the ability of PC-1 and ANK to regulate calcification.

Objective Aging and osteoarthritic (OA) cartilage commonly demonstrate enhanced expression of the large, transforming growth factor β (TGFβ)–inducible glycoprotein cartilage intermediate-layer protein (CILP) as well as enhanced extracellular inorganic pyrophosphate (PPi) that promotes the deposition of calcium pyrophosphate dihydrate crystals. In normal chondrocytes, TGFβ induces elevated chondrocyte extracellular PPi. Insulin-like growth factor 1 (IGF-1) normally blocks this response and reduces extracellular PPi. However, chondrocyte resistance to IGF-1 is observed in OA and aging. Because CILP was reported to chromatographically fractionate with PPi-generating nucleotide pyrophosphatase phosphodiesterase (NPP) activity, it has been broadly assumed that CILP itself has NPP activity. Our objective was to directly define CILP functions and their relationship to IGF-1 in chondrocytes. Methods Using primary cultures of articular chondrocytes from the knee, we defined the function of the previously described CILP (CILP-1) and of a recently described 50.6% identical protein that we designated the CILP-2 isoform. Results Both CILP isoforms were constitutively expressed by primary cultured articular chondrocytes, but only CILP-1 expression was detectable in cultured knee meniscal cartilage cells. Neither CILP isoform had intrinsic NPP activity. But CILP-1 blocked the ability of IGF-1 to decrease extracellular PPi, an activity specific for the CILP-1 N-terminal domain. The CILP-1 N-terminal domain also suppressed IGF-1–induced (but not TGFβ-induced) proliferation and sulfated proteoglycan synthesis, and it inhibited ligand-induced IGF-1 receptor autophosphorylation. Conclusion Two CILP isoforms are differentially expressed by chondrocytes. Neither CILP isoform exhibits PPi-generating NPP activity. But, increased expression of CILP-1, via N-terminal domain–mediated inhibitory effects of CILP-1 on chondrocyte IGF-1 responsiveness, could impair chondrocyte growth and matrix repair and indirectly promote PPi supersaturation in aging and OA cartilage.

The major types of crystals containing calcium, which causes arthropathy and periarticular disease, are calcium pyrophosphate dihydrate and basic calcium phosphates, including hydroxyapatite. Exciting advances include the identification of mutations in the gene ANKH associated with disordered inorganic pyrophosphate (PPi) transport in some kindred with familial chondrocalcinosis linked to chromosome 5p. In addition, central basic mechanisms governing cartilage calcification and their relationship to aging and osteoarthritis have now been elucidated. These include the role of plasma cell glycoprotein-1, the PPi-generating ecto-enzyme, in chondrocalcinosis and the linkage of low- grade inflammation to expression and activation of two cartilage-expressed transglutaminase isoenzymes with direct calcification-stimulating activity. This review discusses clinically pertinent new information on pathogenesis. The authors also address, in detail, current diagnostic and therapeutic issues pertaining to calcium pyrophosphate dihydrate and hydroxyapatite crystal deposition in the joint, as well as possible therapeutic directions for the future.

Osteoblasts mineralize bone matrix by promoting hydroxyapatite crystal formation and growth in the interior of membrane-limited matrix vesicles (MVs) and by propagating the crystals onto the collagenous extracellular matrix. Two osteoblast proteins, tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein- 1 (PC-1) are involved in this process. Mutations in the TNAP gene result in the inborn error of metabolism known as hypophosphatasia, characterized by poorly mineralized bones, spontaneous fractures, and elevated extracellular concentrations of inorganic pyrophosphate (PP i ). PP i suppresses the formation and growth of hydroxyapatite crystals. PP i is produced by the nucleoside triphosphate pyrophosphohydrolase activity of a family of isozymes, with PC-1 being the only member present in MVs. Mice with spontaneous mutations in the PC-1 gene have hypermineralization abnormalities that include osteoarthritis and ossification of the posterior longitudinal ligament of the spine. Here, we show the respective correction of bone mineralization abnormalities in knockout mice null for both the TNAP ( Akp2 ) and PC-1 ( Enpp1 ) genes. Each allele of Akp2 and Enpp1 has a measurable influence on mineralization status in vivo. Ex vivo experiments using cultured double-knockout osteoblasts and their MVs demonstrate normalization of PP i content and mineral deposition. Our data provide evidence that TNAP and PC-1 are key regulators of the extracellular PP i concentrations required for controlled bone mineralization. Our results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia. Conversely, interfering with TNAP activity may correct pathological hyperossification because of PP i insufficiency.

Objectives Using a national registry of gout patients cared for by rheumatologists, we sought to characterize the differences between women and men with gout, in terms of predisposing factors, comorbid conditions, and treatments, in order to tailor and improve gout care to both sexes. Methods Rheumatologists participating in the Consortium of Rheumatology Researchers of North America (CORRONA) registry agreed to enroll their gout patients regardless of gender, disease severity, disease activity or medication use. All patients enrolled between 11/1/12 and 12/11/13 were included in the current study. Data gathered from patients and their rheumatologists at study enrollment included demographics, predisposing factors (comorbid conditions, medications, diet), gout disease characteristics, current treatments, and physical exam findings. Results Fifty-four rheumatologists enrolled 239 women and 928 men with gout. Women were older (71 vs. 61 years, p Conclusions Women with gout more frequently were receiving predisposing medications, and had gout-associated comorbidities and/or contraindications to NSAIDs and colchicine, while men more commonly had dietary risk factors. Physicians should be aware of these differences, in order to tailor their treatment recommendations, to ensure all patients achieve optimal disease control. Acknowledgements This study is sponsored by CORRONA. Initial funding for the CORRONA Gout registry was provided by Savient Pharmaceuticals, Inc. In the last two years, AbbVie, Amgen, AstraZeneca, Genentech, Horizon Pharma, Lilly, Novartis, Pfizer, Savient, Vertex, and UCB have supported CORRONA through contracted subscriptions. Disclosure of Interest L. Harrold Grant/research support: Takeda and AstraZeneca, Consultant for: CORRONA, Inc., C. Etzel Employee of: CORRONA, Inc., A. Gibofsky Shareholder of: AbbVie, Amgen, BMS, GlaxoSmithKline, Johnson&Johnson, Pfizer, Roche (Genentech), Consultant for: AbbVie, Amgen, AstraZeneca, Celgene, Horizon, Iroko, Pfizer, Roche (Genentech), UCB, Takeda, Antares, Speakers bureau: Abbvie, Amgen, Pfizer, Roche (Genentech), UCB, J. Kremer Shareholder of: CORRONA, Inc., Employee of: CORRONA, Inc., M. Pillinger Grant/research support: Takeda, Savient, K. Saag Grant/research support: Takeda, Ardea, Consultant for: Takeda, Ardea, N. Schlesinger Grant/research support: Novartis, Consultant for: Novartis, Sobi, Speakers bureau: Novartis, Takeda, Savient, R. Terkeltaub Grant/research support: VA Research Service, Consultant for: Takeda, Savient, ARDEA, BioCryst, Novartis, Regeneron, Sobi, Pfizer, AbbVie, V. Cox Employee of: CORRONA, Inc., J. Greenberg Shareholder of: CORRONA, Inc., Consultant for: AstraZeneca and Pfizer, Employee of: CORRONA, Inc. DOI 10.1136/annrheumdis-2014-eular.2486

Neutrophil-dependent inflammation dependent on monosodium urate (MSU)crystal-induced IL-8 expression occurs in gout. MSU crystals activatephagocyte Src family tyrosine kinases and the serine/threonine kinasep70s6k. Thus, using monocytic THP-1 cells, we assessed the potentialfor Src family kinases and p70s6k to mediate MSU-induced IL-8expression. MSU crystals induced phosphorylation of p70s6k and the Srckinases c-Src, Lyn, Hck, and Fyn. IL-8 expression was attenuated moreby the Src kinase inhibitor PP1 than by the p70s6k inhibitor rapamycin.PP1 inhibited crystal-induced phosphorylation of ERK1/2 and IκBαand suppressed IκB kinase (IKK) activation and NF-κB binding to theIL-8 promoter, signals that mediate MSU-induced IL-8 expression.Transfection of the native Src inhibitor, C-terminal Src kinase (Csk),also suppressed crystal-induced c-Src, ERK1/2, and IκBαphosphorylation and IL-8 expression. We conclude that Src familytyrosine kinase signaling plays a significant role in MSUcrystal-induced IL-8 expression via stimulation of ERK1/2 pathway andNF-κB activation.

Two transglutaminases (TGases), Factor XIIIa and tissue TGase (tTGase), are expressed in temporal-spatial association with matrix calcification in growth plates. Meniscal and articular cartilage matrix calcification are prevalent in osteoarthritis (OA) and aging. Here, we demonstrated up-regulation of tTGase and Factor XIIIa in superficial and deep zones of knee OA articular cartilage and the central (chondrocytic) zone of OA menisci. Transforming growth factor-beta and interleukin (IL)-1beta induced Factor XIIIa and tTGase expression in cartilage and meniscal organ cultures. Thus, we studied TGase activity. Donor age-dependent, OA severity-related, and IL-1-induced increases in TGase activity were demonstrated in both knee menisci and cultured meniscal cells. Meniscal cell TGase activity was stimulated by nitric oxide donors and tumor necrosis factor-alpha, but transforming growth factor-beta did not stimulate TGase activity. The iNOS inhibitor N-monomethylarginine (NMMA) and an inhibitor of tumor necrosis factor receptor-associated factor (TRAF)2 and TRAF6 signaling (the zinc finger protein A20) suppressed IL-1 induction of TGase activity. Increased Factor XIIIa and tTGase activities, achieved via direct transfection of chondrocytic TC28 and meniscal cells, both induced matrix apatite deposition. Thus, Factor XIIIa and tTGase activities were increased in aging, degenerative cartilages and induced by IL-1. Because TGase activity promoted apatite deposition, our findings potentially implicate inflammation in the pathogenesis of cartilage matrix calcification.

In a variety of tissues, cumulative oxidative stress, disrupted mitochondrial respiration, and mitochondrial damage promote aging, cell death, and ultimately, functional failure and degeneration. Because articular cartilage chondroyctes are highly glycolytic, mitochondrially mediated pathogenesis has not been previously applied in models for pathogenesis of osteoarthritis (OA), a cartilage degenerative disease that increases markedly in aging. However, chondrocyte mitochondria respire in vitro and they demonstrate swelling and changes in number in situ in the course of OA. Normal chondrocyte mitochondrial function is hypothesized to critically support adenosine triphosphate (ATP) reserves in functional stressed chondrocytes during OA evolution. In this model, disruption of chondrocyte respiration by nitric oxide, a mediator markedly up-regulated in OA cartilage, is centrally involved in chondrocyte functional compromise. Furthermore, mitochondrial dysfunction can mediate several specific pathogenic pathways implicated in OA. These include oxidative stress, inadequacy of chondrocyte biosynthetic and growth responses, up-regulated chondrocyte cytokine-induced inflammation and matrix catabolism, increased chondrocyte apoptosis, and pathologic cartilage matrix calcification. In addition, the direct, sublethal impairment of chondrocyte mitochondrial ATP synthesis in vitro decreases matrix synthesis and increases matrix calcification (‘disease in a dish’). The weight of evidence reviewed herein strongly supports chondrocyte mitochondrial impairment as a mediator of the establishment and progression of OA.

Inorganic pyrophosphate (PPi) regulates certain intracellular functions and extracellular crystal deposition. PPiis produced, degraded, and transported by specialized mechanisms. Moreover, dysregulated cellular PPiproduction, degradation, and transport all have been associated with disease, and PPiappears to directly mediate specific disease manifestations. In addition, natural and synthetic analogs of PPiare in use or currently under evaluation as prophylactic agents or therapies for disease. This review summarizes recent developments in the understanding of how PPiis made and disposed of by cells and assesses the body of evidence for potentially significant physiological functions of intracellular PPiin higher organisms. Major topics addressed are recent lines of molecular evidence that directly link decreased and increased extracellular PPilevels with diseases in which connective tissue matrix calcification is disordered. To illustrate in depth the effects of disordered PPimetabolism, this review weighs the roles in matrix calcification of the transmembrane protein ANK, which regulates intracellular to extracellular movement of PPi, and the PPi-generating phosphodiesterase nucleotide pyrophosphatase family isoenzyme plasma cell membrane glycoprotein-1 (PC-1).

Articular cartilage, unlike growth plate cartilage, is specialized to not undergo matrix calcification. However, articular cartilage mineralization, in the form of CPPD (chondrocalcinosis) and hydroxyapatite crystals, frequently accompanies and complicates osteoarthritis and aging. Recent work has demonstrated that certain features of growth cartilage development and mineralization are shared in degenerative cartilage. These include chondrocyte proliferation, hypertrophy and increased apoptosis. Moreover, parathyroid hormone related protein (PTHrP), one of the central mediators of endochondral development, is abundant in osteoarthritic cartilage. Cartilage PPi elaboration and cytosolic transglutaminase activity are markedly increased with aging. Only recently have the molecular identities been defined for the chondrocyte inorganic pyrophosphate (PPi)-generating isozymes of the phosphodiesterase nucleotide pyrophosphatase (PDNP) family (including PC-1 and B10), and for transglutaminase in articular cartilage. This review focuses on the evolving understanding of the potential roles, in articular cartilage calcification, of PTHrP, PDNP family enzymes, PPi metabolism, and transglutaminase activity.