Basic calcium phosphate crystals induce the expression of extracellular matrix remodelling enzymes in tenocytes.

OBJECTIVES: Basic calcium phosphate (BCP) crystals contribute to several syndromes associated with tendon disease, including acute calcific tendinitis and Milwaukee shoulder syndrome. Interactions between BCP crystals and tenocytes (tendon cells) may contribute to these clinical syndromes. This study aimed to determine the direct effects of BCP crystals on tenocyte function and viability. METHODS: In vitro assays were used to assess changes in human tenocytes cultured with BCP crystals. Real-time PCR was used to determine changes in the expression of tendon-related genes and extracellular matrix remodelling enzymes (MMPs; a disintegrin and metalloproteases, ADAMTS; and tissue inhibitor of metalloproteinases, TIMPs). ELISA was used to measure protein concentrations in tenocyte supernatants. MTT and alamarBlue™ assays were used to determine changes in cell viability. RESULTS: BCP crystals upregulated tenocyte gene expression of MMP-1, MMP-3, ADAMTS-4 and TIMP-1 after 24 h. Time-course experiments showed expression peaked at 8 h for TIMP-1 and 48 h for MMP-1 and ADAMTS-4. Cyclooxygenase (COX)-1 gene expression was upregulated after 48 h. Tenocytes did not alter expression of scleraxis and tendon collagens, and expression of pro-inflammatory cytokines was not induced with BCP crystals. BCP crystals increased tenocyte release of prostaglandin E2 (PGE2) and MMP-1 protein after 24 h. However, neither COX-1 inhibition nor COX-2 inhibition led to consistent change in BCP crystal-induced tenocyte gene expression of extracellular matrix remodelling enzymes. BCP crystals had no effect on tenocyte viability. CONCLUSION: BCP crystals induce extracellular matrix remodelling enzymes, but not inflammatory cytokines, in tenocytes.

Factors secreted by monosodium urate crystal-stimulated macrophages promote a proinflammatory state in osteoblasts: a potential indirect mechanism of bone erosion in gout.

BACKGROUND: Tophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts. METHODS: Conditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay. RESULTS: In MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs. CONCLUSIONS: Factors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

Limitations of dual-energy CT in the detection of monosodium urate deposition in dense liquid tophi and calcified tophi.

OBJECTIVE: Dual-energy CT (DECT) detection of monosodium urate (MSU) crystal deposition has demonstrated good sensitivity and specificity in patients with established gout. However, limitations have been reported with early disease and with low urate burden. We aimed to study the performance of DECT in the detection and quantification of MSU deposition in solid and liquid tophi. MATERIALS AND METHODS: Patient-derived solid and liquid tophi, suspensions of commercial synthetic, and in-house synthetic MSU crystals were prepared at varying concentrations. DECT was performed at 80 kVp and 150 kVp, and post-processed using Syngo Via gout software (Siemens) that color-coded urate and cortical bone as green and purple, respectively. DECT findings were correlated with ultrasound and microscopic findings. The protocol was reviewed by IRB and considered a non-human subject research. RESULTS: DECT did not detect urate deposition in either patient-derived liquid tophi or in-house synthetic crystals at any concentration. Lowering the post-processing minimum threshold increased the detection of in-house synthetic crystals but did not change the detection of patient-derived liquid tophi. Areas of calcium-rich purple color-coded regions, masking detection of urate, within the solid tophi and surrounding liquid tophi were noted on DECT. Histology showed co-presence of calcium along with MSU deposition in these. CONCLUSION: This study illustrates important limitations of DECT for liquid tophi due to subthreshold CT attenuation and for calcified tophi due to the obscuration of urate by calcium. Urate may be either undetectable or underestimated by DECT when these conditions are present.

Human Cartilage Homogenates Influence the Crystallization of Monosodium Urate and Inflammatory Response to Monosodium Urate Crystals: A Potential Link Between Osteoarthritis and Gout.

OBJECTIVE: Monosodium urate (MSU) crystal deposition and gout flares frequently affect osteoarthritic joints. This study was undertaken to examine the effects of human cartilage homogenates on MSU crystallization and MSU crystal-induced inflammation. METHODS: Human cartilage homogenates were prepared from macroscopically healthy and macroscopically diseased knee joint samples. Crystallization assays were used to test the effects of cartilage homogenates or individual cartilage factors on MSU crystallization. Changes in urate solubility, crystal nucleation, crystal growth, and total crystal mass were determined. THP-1 cell assays were used to assess cytokine release following culture with MSU crystals grown in the presence or absence of cartilage homogenates or individual proteins. RESULTS: Addition of either 5% or 10% healthy cartilage homogenate increased the total mass of MSU crystals formed and resulted in formation of shorter MSU crystals compared to controls without cartilage homogenate. MSU crystal bows were observed in both the presence and absence of cartilage homogenate; however, bows formed in the presence of cartilage homogenates were significantly shorter than bows formed in their absence. There were no effect differences between macroscopically healthy and macroscopically diseased cartilage homogenates in all assessments. Addition of either type II collagen or albumin also led to the formation of shorter MSU crystals. In THP-1 cell assays, MSU crystals grown with healthy cartilage homogenate increased the release of interleukin-8, whereas MSU crystals grown with type II collagen or albumin had no effect on inflammatory cytokine release. CONCLUSION: In the presence of elevated urate levels, human cartilage homogenates increase MSU crystal formation and promote the formation of smaller crystals, which have greater inflammatory potential. These processes may contribute to the predilection of osteoarthritic joints to develop gout.

Liposome-Mediated Drug Delivery in Larval Zebrafish to Manipulate Macrophage Function.

Chemical interventions are regularly used to examine and manipulate macrophage function in larval zebrafish. Given chemicals are typically administered by simple immersion or injection, it is not possible to resolve whether their impact on macrophage function is direct or indirect. Liposomes provide an attractive strategy to target drugs to specific cellular compartments, including macrophages. As an example, injecting liposomal clodronate into animal models, including zebrafish, is routinely used to deliver toxic levels of clodronate specifically to macrophages for targeted cell ablation. Here we show that liposomes can also target the delivery of drugs to zebrafish macrophages to selectively manipulate their function. We utilized the drugs etomoxir (a fatty acid oxidation inhibitor) and MitoTEMPO (a scavenger of mitochondrial reactive oxygen species [mROS]), that we have previously shown, through free drug delivery, suppress monosodium urate (MSU) crystal-driven macrophage activation. We generated poloxamer 188 modified liposomes that were readily phagocytosed by macrophages, but not by neutrophils. Loading these liposomes with etomoxir or MitoTEMPO and injecting into larvae suppressed macrophage activation in response to MSU crystals, as evidenced by proinflammatory cytokine expression and macrophage-driven neutrophil recruitment. This work reveals the utility of packaging drugs into liposomes as a strategy to selectively manipulate macrophage function.

Basic Calcium Phosphate Crystals Induce Osteoarthritis-Associated Changes in Phenotype Markers in Primary Human Chondrocytes by a Calcium/Calmodulin Kinase 2-Dependent Mechanism.

Chondrocytes in osteoarthritis undergo a phenotype shift leading to increased production of cartilage-degrading enzymes. There are similarities between the phenotype of osteoarthritic chondrocytes and those of growth plate chondrocytes. Hydroxyapatite can promote chondrocyte differentiation in the growth plate. Basic calcium phosphate (BCP) crystals (which consist of hydroxyapatite, octacalcium apatite and tricalcium phosphate) are frequently found in osteoarthritic joints. The objective of this study was to determine whether BCP crystals induce disease-associated changes in phenotypic marker expression in chondrocytes. Primary human chondrocytes isolated from macroscopically normal cartilage were treated with BCP for up to 48 h. Expression of indian hedgehog (IHH), matrix metalloproteinase 13 (MMP13), interleukin-6 (IL-6) and type X collagen (COLX) were higher, and expression of sry-box 9 (SOX9) lower, in BCP-treated chondrocytes (50 µg/mL) compared to untreated controls. COLX protein was also present in BCP-treated chondrocytes. Intracellular calcium and levels of phosphorylated and total calcium/calmodulin kinase 2 (CaMK2) were elevated following BCP treatment due to BCP-induced release of calcium from intracellular stores. CaMK2 inhibition or knockdown ameliorated the BCP-induced changes in SOX9, IHH, COLX, IL-6 and MMP13 expression. BCP crystals induce osteoarthritis-associated changes in phenotypic marker expression in chondrocytes by calcium-mediated activation of CaMK2. The presence of BCP crystals in osteoarthritic joints may contribute to disease progression.

Greater insulin response to acute fructose ingestion among Maori and Pacific people compared to European people living in Aotearoa New Zealand.

BACKGROUND: Fructose consumption has been linked with insulin resistance, obesity and diabetes, which are more prevalent in those of Maori or Pacific ethnicity compared to New Zealand European. AIM: To determine whether the acute effects of fructose consumption on serum glucose, insulin, lipids and C-reactive protein differs according to body mass index (BMI) and/or ethnicity. METHODS: Participants of Maori (n = 25), Pacific (n = 26) or New Zealand European (n = 25) ethnicity consumed a 64 g fructose/16 g glucose solution. Changes in lipids, glucose, insulin and C-reactive protein were analysed using mixed models for repeated measures. RESULTS: After adjustment for age and gender, those with higher BMI had a higher glucose (P = 0.0064) and insulin (P = 0.0007) response than those with lower BMI. Those of Maori or Pacific ethnicity had similar glucose levels (P = 0.077) to those of New Zealand European ethnicity but higher insulin responses (P = 0.0005), which remained after additional adjustment for BMI (P = 0.001). Reported sugar-sweetened beverages (SSB) intake was higher among Maori and Pacific than New Zealand European (median 1.0 vs 0.0 SSB/day P = 0.002). CONCLUSION: Even after adjustment for BMI, those of Maori and Pacific ethnicity have a significantly higher insulin response to fructose than New Zealand Europeans. Higher habitual SSB intake may be a contributing factor.

Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state.

BACKGROUND: Bone erosion is a frequent complication of gout and is strongly associated with tophi, which are lesions comprising inflammatory cells surrounding collections of monosodium urate (MSU) crystals. Osteocytes are important cellular mediators of bone remodeling. The aim of this study was to investigate the direct effects of MSU crystals and indirect effects of MSU crystal-induced inflammation on osteocytes. METHODS: For direct assays, MSU crystals were added to MLO-Y4 osteocyte cell line cultures or primary mouse osteocyte cultures. For indirect assays, the RAW264.7 macrophage cell line was cultured with or without MSU crystals, and conditioned medium from these cultures was added to MLO-Y4 cells. MLO-Y4 cell viability was assessed using alamarBlue® and LIVE/DEAD® assays, and MLO-Y4 cell gene expression and protein expression were assessed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Histological analysis was used to examine the relationship between MSU crystals, inflammatory cells, and osteocytes in human joints affected by tophaceous gout. RESULTS: In direct assays, MSU crystals reduced MLO-Y4 cell and primary mouse osteocyte viability but did not alter MLO-Y4 cell gene expression. In contrast, conditioned medium from MSU crystal-stimulated RAW264.7 macrophages did not affect MLO-Y4 cell viability but significantly increased MLO-Y4 cell expression of osteocyte-related factors including E11, connexin 43, and RANKL, and inflammatory mediators such as interleukin (IL)-6, IL-11, tumor necrosis factor (TNF)-α and cyclooxygenase-2 (COX-2). Inhibition of COX-2 in MLO-Y4 cells significantly reduced the indirect effects of MSU crystals. In histological analysis, CD68+ macrophages and MSU crystals were identified in close proximity to osteocytes within bone. COX-2 expression was also observed in tophaceous joint samples. CONCLUSIONS: MSU crystals directly inhibit osteocyte viability and, through interactions with macrophages, indirectly promote a shift in osteocyte function that favors bone resorption and inflammation. These interactions may contribute to disordered bone remodeling in gout.

Blocking fatty acid-fueled mROS production within macrophages alleviates acute gouty inflammation.

Gout is the most common inflammatory arthritis affecting men. Acute gouty inflammation is triggered by monosodium urate (MSU) crystal deposition in and around joints that activates macrophages into a proinflammatory state, resulting in neutrophil recruitment. A complete understanding of how MSU crystals activate macrophages in vivo has been difficult because of limitations of live imaging this process in traditional animal models. By live imaging the macrophage and neutrophil response to MSU crystals within an intact host (larval zebrafish), we reveal that macrophage activation requires mitochondrial ROS (mROS) generated through fatty acid oxidation. This mitochondrial source of ROS contributes to NF-κB-driven production of IL-1ß and TNF-α, which promote neutrophil recruitment. We demonstrate the therapeutic utility of this discovery by showing that this mechanism is conserved in human macrophages and, via pharmacologic blockade, that it contributes to neutrophil recruitment in a mouse model of acute gouty inflammation. To our knowledge, this study is the first to uncover an immunometabolic mechanism of macrophage activation that operates during acute gouty inflammation. Targeting this pathway holds promise in the management of gout and, potentially, other macrophage-driven diseases.

Lack of Evidence that Soluble Urate Directly Influences Bone Remodelling: A Laboratory and Clinical Study.

INTRODUCTION: Numerous observational studies have reported that serum urate concentration positively correlates with bone density and reduced risk of fractures. The aim of this study was to examine whether soluble urate directly influences bone remodelling. METHODS: In laboratory studies, the in vitro effects of soluble urate were examined in osteoclast, osteoblast and osteocyte assays at a range of urate concentrations consistent with those typically observed in humans (up to 0.70 mmol/L). The clinical relevance of the in vitro assay findings was assessed using serial procollagen-1 N-terminal propeptide (P1NP) and Month 12 bone density data from a randomised controlled trial of allopurinol dose escalation in people with gout. RESULTS: Addition of urate in the RAW264.7 cell osteoclastogenesis assay led to small increases in osteoclast formation (ANOVA p = 0.018), but no significant difference in bone resorption. No significant effects on osteoclast number or activity were observed in primary cell osteoclastogenesis or resorption assays. Addition of urate did not alter viability or function in MC3T3-E1 pre-osteoblast, primary human osteoblast, or MLO-Y4 osteocyte assays. In the clinical trial analysis, reducing serum urate over a 12 month period by allopurinol dose escalation did not lead to significant changes in P1NP or differences in bone mineral density. CONCLUSION: Addition of soluble urate at physiological concentrations does not influence bone remodelling in vitro. These data, together with clinical trial data showing no effect of urate-lowering on P1NP or bone density, do not support a direct role for urate in influencing bone remodelling.

Lactoferrin and parathyroid hormone are not harmful to primary tenocytes in vitro, but PDGF may be.

INTRODUCTION: Recently, bone-active factors such as parathyroid hormone and lactoferrin, have been used in pre-clinical models to promote tendon healing. How-ever, there is limited understanding of how these boneactive factors may affect the cells of the ten-don themselves. Here, we present an in vitro study assessing the effects of parathyroid hor-mone and lactoferrin on primary tendon cells (tenocytes), and compare their responses to the tenogenic factors, PDGF, IGF-1 and TGF-ß. MATERIALS AND METHODS: Tenocyte proliferation and collagen production were assessed by alamarBlue® and Sirius red as-says, respectively. To assess tenocyte trans-differentiation, changes in the expression of genes important in tenocyte, chondrocyte and osteoblast biology were determined using real-time PCR. RESULTS: Parathyroid hormone and lactoferrin had no effect on tenocyte growth or collagen production, with minimal changes in gene expression and no detrimental effects observed to suggest trans-differentiation away from tendon cell behaviour. Tenogenic factors PDGF, IGF-1 and TGF all increasetenocyte collagen production, however, the gene expression data suggests that PDGF promotes severe de-differentiation of the tenocytes. DISCUSSION: Our findings suggest that using parathyroid hormone or lactoferrin as a singular factor to promote tendon healing may not be of benefit, but for use in tendon-bone healing there would be no detrimental effect on the tendon itself.

Role of miR-146a in regulation of the acute inflammatory response to monosodium urate crystals.

OBJECTIVES: MicroRNAs (miRNA) are small non-coding RNAs that function as post-transcriptional repressors of gene expression. We hypothesised that miRNA regulate gene expression of proinflammatory cytokines in response to monosodium urate (MSU) crystals. METHODS: We stimulated human monocytic THP-1 cells with MSU crystals and examined miRNA and proinflammatory cytokine gene expression. The effects of miR-146a overexpression were examined by transfecting THP-1 cells with miR-146a precursor. miR-146a expression was examined in the urate peritonitis model, in peripheral blood mononuclear cells from people with gout and control participants, and in gouty tophus samples. RESULTS: MSU crystals increased miR-146a expression in THP-1 cells, but not other miRNA implicated in interleukin (IL)-1ß regulation. Overexpression of miR-146a expression reduced MSU crystal-induced IL-1ß, tumour necrosis factor-α (TNFα), monocyte chemoattractant protein-1 (MCP-1) and IL-8 gene expression. In the urate peritonitis model, reduced miR-146a expression was observed during the acute inflammatory response to MSU crystal injection. In people with intercritical gout, peripheral blood mononuclear cells expressed significantly higher levels of miR-146a, compared with normouricaemic and hyperuricaemic control participants and those with acute gout flares. Expression of miR-146a was also observed in all tophus samples. CONCLUSIONS: Collectively, these data suggest that miR-146a is a transcriptional brake that is lost during the acute inflammatory response to MSU crystals.

The effect of diet-induced obesity on the inflammatory phenotype of non-adipose-resident macrophages in an in vivo model of gout.

OBJECTIVE: Gout is strongly associated with obesity. The aim of this study was to determine if obesity altered the inflammatory phenotype of non-adipose tissue-resident macrophages in response to the gout-causing agent monosodium urate (MSU) crystals. METHODS: C57BL/6J mice were fed a high-fat diet for 12 weeks. Resident peritoneal macrophages were stimulated ex vivo with MSU crystals (200 µg/ml for 18 h) and the supernatants were collected. Mice were challenged with MSU crystals in vivo (3 mg, intraperitonal) and the peritoneal lavage fluid was collected (8 and 16 h). Cytokine and chemokine levels were analysed by multiplex bead array and peritoneal cell populations were analysed by flow cytometry. RESULTS: Peritoneal macrophages from obese mice produced elevated background levels of IL-6, monocyte chemoattractant protein 1 (MCP-1) and keratinocyte-derived cytokine (KC) that decreased following MSU crystal stimulation ex vivo. MSU-induced IL-1ß production was higher for macrophages from obese mice compared with controls. High background levels of IL-6, MCP-1, KC and GM-CSF, but not IL-1ß, were measured in the peritoneal fluid of unchallenged obese mice. MSU crystal challenge in vivo raised IL-1ß levels equally in both control and obese mice, whereas elevated background levels of IL-6, MCP-1, KC and GM-CSF levels dropped in obese mice. There was a consistent trend towards lower numbers of naive peritoneal resident macrophages and MSU-recruited monocytes and neutrophils in obese mice. CONCLUSION: Obesity induces a background pro-inflammatory environment orchestrated by non-adipose tissue-resident macrophages. However, this may not automatically translate into exacerbation of MSU crystal-induced inflammation in gout.

Interactions between tenocytes and monosodium urate monohydrate crystals: implications for tendon involvement in gout.

OBJECTIVES: Advanced imaging studies have demonstrated that urate deposition in periarticular structures, such as tendons, is common in gout. The aim of this study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on tenocyte viability and function. METHODS: The histological appearance of tendons in joints affected by advanced gout was examined using light microscopy. In vitro, colorimetric assays and flow cytometry were used to assess cell viability in primary rat and primary human tenocytes cultured with MSU crystals. Real-time PCR was used to determine changes in the relative mRNA expression levels of tendon-related genes, and Sirius red staining was used to measure changes in collagen deposition in primary rat tenocytes. RESULTS: In joint samples from patients with gout, MSU crystals were identified within the tendon, adjacent to and invading into tendon, and at the enthesis. MSU crystals reduced tenocyte viability in a dose-dependent manner. MSU crystals decreased the mRNA expression of tendon collagens, matrix proteins and degradative enzymes and reduced collagen protein deposition by tenocytes. CONCLUSIONS: These data indicate that MSU crystals directly interact with tenocytes to reduce cell viability and function. These interactions may contribute to tendon damage in people with advanced gout.

Myeloid-related proteins 8 and 14 contribute to monosodium urate monohydrate crystal-induced inflammation in gout.

OBJECTIVE: Monosodium urate monohydrate (MSU) crystal-induced interleukin-1ß (IL-1ß) secretion is a critical factor in the pathogenesis of gout. However, without costimulation by a proIL-1ß-inducing factor, MSU crystals alone are insufficient to induce IL-1ß secretion. The responsible costimulatory factors that act as a priming endogenous signal in vivo are not yet known. We undertook this study to analyze the costimulatory properties of myeloid-related protein 8 (MRP-8) and MRP-14 (endogenous Toll-like receptor 4 [TLR-4] agonists) in MSU crystal-induced IL-1ß secretion and their relevance in gout. METHODS: MRP-8/MRP-14 was measured in paired serum and synovial fluid samples by enzyme-linked immunosorbent assay (ELISA) and localized in synovial tissue from gout patients by immunohistochemistry. Serum levels were correlated with disease activity, and MSU crystal-induced release of MRPs from human phagocytes was measured. Costimulatory effects of MRP-8 and MRP-14 on MSU crystal-induced IL-1ß secretion from phagocytes were analyzed in vitro by ELISA, Western blotting, and polymerase chain reaction. The impact of MRP was tested in vivo in a murine MSU crystal-induced peritonitis model. RESULTS: MRP-8/MRP-14 levels were elevated in the synovium, tophi, and serum of patients with gout and correlated with disease activity. MRP-8/MRP-14 was released by MSU crystal-activated phagocytes and increased MSU crystal-induced IL-1ß secretion in a TLR-4-dependent manner. Targeted deletion of MRP-14 in mice led to a moderately reduced response of MSU crystal-induced inflammation in vivo. CONCLUSION: MRP-8 and MRP-14, which are highly expressed in gout, are enhancers of MSU crystal-induced IL-1ß secretion in vitro and in vivo. These endogenous TLR-4 ligands released by activated phagocytes contribute to the maintenance of inflammation in gout.

Influence of the ABCG2 gout risk 141 K allele on urate metabolism during a fructose challenge.

INTRODUCTION: Both genetic variation in ATP-binding cassette sub-family G member 2 (ABCG2) and intake of fructose-containing beverages are major risk factors for hyperuricemia and gout. This study aimed to test the hypothesis that the ABCG2 gout risk allele 141 K promotes the hyperuricaemic response to fructose loading. METHODS: Healthy volunteers (n = 74) provided serum and urine samples immediately before and 30, 60, 120 and 180 minutes after ingesting a 64 g fructose solution. Data were analyzed based on the presence or absence of the ABCG2 141 K gout risk allele. RESULTS: The 141 K risk allele was present in 23 participants (31%). Overall, serum urate (SU) concentrations during the fructose load were similar in those with and without the 141 K allele (PSNP = 0.15). However, the 141 K allele was associated with a smaller increase in SU following fructose intake (PSNP <0.0001). Those with the 141 K allele also had a smaller increase in serum glucose following the fructose load (PSNP = 0.002). Higher fractional excretion of uric acid (FEUA) at baseline and throughout the fructose load was observed in those with the 141 K risk allele (PSNP <0.0001). However, the change in FEUA in response to fructose was not different in those with and without the 141 K risk allele (PSNP = 0.39). The 141 K allele effects on serum urate and glucose were more pronounced in Polynesian participants and in those with a body mass index ≥25 kg/m². CONCLUSIONS: In contrast to the predicted responses for a hyperuricemia/gout risk allele, the 141 K allele is associated with smaller increases in SU and higher FEUA following a fructose load. The results suggest that ABCG2 interacts with extra-renal metabolic pathways in a complex manner to regulate SU and gout risk. CLINICAL TRIALS REGISTRATION: The study was registered by the Australian Clinical Trials Registry (ACTRN12610001036000).

The effects of monosodium urate monohydrate crystals on chondrocyte viability and function: implications for development of cartilage damage in gout.

OBJECTIVE: Cartilage damage is frequently observed in advanced destructive gout. The aim of our study was to investigate the effects of monosodium urate monohydrate (MSU) crystals on chondrocyte viability and function. METHODS: The alamarBlue assay and flow cytometry were used to assess the viability of primary human chondrocytes and cartilage explants following culture with MSU crystals. The number of dead chondrocytes in cartilage explants cultured with MSU crystals was quantified. Real-time PCR was used to determine changes in the relative mRNA expression levels of chondrocytic genes. The histological appearance of cartilage in joints affected by gout was also examined. RESULTS: MSU crystals rapidly reduced primary human chondrocyte and cartilage explant viability in a dose-dependent manner (p < 0.01 for both). Cartilage explants cultured with MSU crystals had a greater percentage of dead chondrocytes at the articular surface compared to untreated cartilage (p = 0.004). Relative mRNA expression of type II collagen and the cartilage matrix proteins aggrecan and versican was decreased in chondrocytes following culture with MSU crystals (p < 0.05 for all). However, expression of the degradative enzymes ADAMTS4 and ADAMTS5 was increased (p < 0.05 for both). In joints affected by gout, normal cartilage architecture was lost, with empty chondrocyte lacunae observed. CONCLUSION: MSU crystals have profound inhibitory effects on chondrocyte viability and function. Interactions between MSU crystals and chondrocytes may contribute to cartilage damage in gout through reduction of chondrocyte viability and promotion of a catabolic state.

Population-specific influence of SLC2A9 genotype on the acute hyperuricaemic response to a fructose load.

BACKGROUND: SLC2A9 is a strong genetic risk factor for hyperuricaemia and gout. SLC2A9 (GLUT9) is a high capacity urate transporter and reportedly transports glucose and fructose. Intake of fructose-containing beverages is associated with development of hyperuricaemia and gout. OBJECTIVE: To determine whether genetic variation in SLC2A9 influences the acute serum urate response to a fructose load. METHODS: Following an overnight fast, 76 healthy volunteers (25 Maori, 26 Pacific, 25 European Caucasian) drank a solution containing 64 g fructose. Serum and urine were obtained immediately before and then 30, 60, 120 and 180 min after ingestion. The SLC2A9 single nucleotide polymorphism (SNP) rs11942223 was genotyped and data were analysed based on the presence or absence of the gout protective minor allele (C). RESULTS: The rs11942223 C allele was present in 17 participants (22%). In the entire group, fructose intake led to an increase in serum urate, which peaked 60 min following fructose ingestion (analysis of variance p=0.006). The presence of the C allele was associated with an attenuated hyperuricaemic response (p(SNP)<0.0001) and increased fractional excretion of uric acid (FEUA) (p(SNP)<0.0001) following the fructose load. The effects of rs11942223 variants on serum urate and FEUA in response to fructose were present only in Caucasian ancestral subgroups but not in the Maori and Pacific ancestral subgroup. CONCLUSIONS: Variation in SLC2A9 influences acute serum urate and FEUA responses to a fructose load. SLC2A9 genotype may influence the development of gout on exposure to fructose-containing beverages, particularly in European Caucasian populations.

No reduction in circulating preosteoclasts 18 months after treatment with zoledronate: analysis from a randomized placebo controlled trial.

The conventional model that bisphosphonates bind to the bone surface and inhibit mature osteoclasts does not convincingly explain the prolonged duration of action of zoledronate. We hypothesized that zoledronate on the bone surface adjacent to marrow cells impairs osteoclastogenesis, contributing to sustained inhibition of resorption. In this case, numbers of circulating preosteoclasts may be reduced after zoledronate treatment. This study assessed this possibility in subjects from a clinical trial. Twenty-two osteopenic women participating in a randomized, controlled trial comparing zoledronate 5 mg with placebo were recruited, 18 months after administration of study drug. Peripheral blood mononuclear cells were analyzed for the presence of osteoclast precursors using flow cytometry for preosteoclast markers and the ability to form osteoclast-like cells in culture with RANKL and M-CSF. There was no difference in the percentage of CD14(+)/CD11b(+) cells in peripheral blood between the two groups. The numbers of TRAP(+) multinucleated cells in cultures in the absence of RANKL and M-CSF were very low in both groups, but a significantly higher number of these cells was observed in the zoledronate group compared with the placebo group (p = 0.01). The number of TRAP(+) multinucleated cells and resorption pits following culture with RANKL and M-CSF did not differ between the two groups. Serum P1NP was reduced 53 % at 18 months in the zoledronate group but unchanged in the placebo group. These results do not support the hypothesis that the inhibitory action of zoledronate contributes to its prolonged action on preosteoclasts within bone marrow.