Thiazide diuretics and risk of knee replacement surgery among patients with knee osteoarthritis: a general population-based cohort study.

OBJECTIVE: Thiazide diuretic use is associated with higher bone mineral density (BMD) and possibly lower serum magnesium levels than loop diuretic use, and both high BMD and low serum magnesium have been linked to high prevalent knee osteoarthritis. This study aimed to compare the risk of a clinically relevant endpoint, knee replacement (KR) surgery, among initiators of thiazide and loop diuretics. DESIGN: Among patients aged ≥50 years with a diagnosis of knee osteoarthritis in The Health Improvement Network (THIN) in United Kingdom, we conducted a propensity score-matched cohort study to examine the relation of thiazide diuretic initiation vs loop diuretic initiation to the risk of KR over 5 years. RESULTS: Among thiazide and loop diuretic initiators (n = 3,488 for each group; mean age: 73 years; female ratio: 59%), 359 (28.6/1,000 person-years) and 283 (24.1/1,000 person-years) KRs occurred during the follow-up period, respectively. The hazard ratio (HR) of KR for thiazide diuretic initiation vs loop diuretic initiation was 1.26 (95% confidence interval [CI]: 1.08-1.47). The adherence-adjusted HR of KR for continuous use of thiazide diuretics was 1.44 (95% CI: 1.21-1.72). CONCLUSIONS: In this population-based cohort of patients with knee osteoarthritis, thiazide diuretic use was associated with a higher risk of KR than loop diuretic use. This association may potentially be due to thiazide diuretics' effect on BMD and serum magnesium.

Activation of AMPK-SIRT3 signaling is chondroprotective by preserving mitochondrial DNA integrity and function.

OBJECTIVE: In osteoarthritis (OA), articular chondrocytes manifest mitochondrial damage, including mitochondrial DNA 4977-bp (mtDNA4977) deletion that impairs mitochondrial function. OA chondrocytes have decreased activity of AMPK, an energy biosensor that promotes mitochondrial biogenesis. Here, we tested if pharmacologic AMPK activation, via downstream activation of predominately mitochondrially localized sirtuin 3 (SIRT3), reverses existing decreases in mitochondrial DNA (mtDNA) integrity and function in human OA chondrocytes and limits mouse knee OA development. DESIGN: We assessed mtDNA integrity and function including the common mtDNA4977 deletion and mtDNA content, mitochondrial reactive oxygen species (mtROS) generation, oxygen consumption and intracellular ATP levels. Phosphorylation of AMPKα, expression and activity of SIRT3, acetylation and expression of the mitochondrial antioxidant enzyme SOD2 and DNA repair enzyme 8-oxoguanine glycosylase (OGG1), and expression of subunits of mitochondrial respiratory complexes were examined. We assessed effect of pharmacologic activation of AMPK on age-related spontaneous mouse knee OA. RESULTS: The mtDNA4977 deletion was detected in both OA chondrocytes and menadione-treated normal chondrocytes, associated with increased mtROS, decreased SIRT3, and increased acetylation of SOD2 and OGG1. AMPKα1 deficient chondrocytes exhibited significantly reduced SIRT3 activity. AMPK pharmacologic activation attenuated existing mtDNA4977 deletion and improved mitochondrial functions in OA chondrocytes via SIRT3 by reducing acetylation and increasing expression of SOD2 and OGG1, and limited aging-associated mouse knee OA development and progression. CONCLUSIONS: AMPK activation, via SIRT3, limits oxidative stress and improves mtDNA integrity and function in OA chondrocytes. These effects likely contribute to chondroprotective effects of AMPK activity.

Prospective associations of C-reactive protein (CRP) levels and CRP genetic risk scores with risk of total knee and hip replacement for osteoarthritis in a diverse cohort.

OBJECTIVE: To examine associations of high-sensitivity C-reactive protein (CRP) levels and polygenic CRP genetic risk scores (GRS) with risk of end-stage hip or knee osteoarthritis (OA), defined as incident total hip (THR) or knee replacement (TKR) for OA. DESIGN: This study included a cohort of postmenopausal white, African American, and Hispanic women from the Women's Health Initiative. Women were followed from baseline to date of THR or TKR, death, or December 31, 2014. Medicare claims data identified THR and TKR. Hs-CRP and genotyping data were collected at baseline. Three CRP GRS were constructed: 1) a 4-SNP GRS comprised of genetic variants representing variation in the CRP gene among European populations; 2) a multilocus 18-SNP GRS of genetic variants significantly associated with CRP levels in a meta-analysis of genome-wide association studies; and 3) a 5-SNP GRS of genetic variants significantly associated with CRP levels among African American women. RESULTS: In analyses conducted separately among each race and ethnic group, there were no significant associations of ln hs-CRP with risk of THR or TKR, after adjusting for age, body mass index, lifestyle characteristics, chronic diseases, hormone therapy use, and non-steroidal anti-inflammatory drug use. CRP GRS were not associated with risk of THR or TKR in any ethnic group. CONCLUSIONS: Serum levels of ln hs-CRP and genetically-predicted CRP levels were not associated with risk of THR or TKR for OA among a diverse cohort of women.

Systematic review and quality analysis of emerging diagnostic measures for calcium pyrophosphate crystal deposition disease.

OBJECTIVES: Calcium pyrophosphate crystal deposition disease (CPPD) is common, yet prevalence and overall clinical impact remain unclear. Sensitivity and specificity of CPPD reference standards (conventional crystal analysis (CCA) and radiography (CR)) were meta-analysed by EULAR (published 2011). Since then, new diagnostic modalities are emerging. Hence, we updated 2009-2016 literature findings by systematic review and evidence grading, and assessed unmet needs. METHODS: We performed systematic search of full papers (PubMed, Scopus/EMBASE, Cochrane 2009-2016 databases). Search terms included CPPD, chondrocalcinosis, pseudogout, ultrasound, MRI, dual energy CT (DECT). Paper selection, data abstraction, EULAR evidence level, and Quality Assessment of Diagnostic Accuracy Studies (QUADAS)-2 bias and applicability grading were performed independently by 3 authors. RESULTS: We included 26 of 111 eligible papers, which showed emergence in CPPD diagnosis of ultrasound (U/S), and to lesser degree, DECT and Raman spectroscopy. U/S detected CPPD crystals in peripheral joints with sensitivity >80%, superior to CR. However, most study designs, though analytical, yielded low EULAR evidence level. DECT was marginally explored for CPPD, compared with 35 published DECT studies in gout. QUADAS-2 grading indicated strong applicability of U/S, DECT and Raman spectroscopy, but high study bias risk (in ∼30% of papers) due to non-controlled designs, and non-randomised subject selection. CONCLUSIONS: Though CCA and CR remain reference standards for CPPD diagnosis, U/S, DECT and Raman spectroscopy are emerging U/S sensitivity appears to be superior to CR. We identified major unmet needs, including for randomised, blinded, controlled studies of CPPD diagnostic performance and rigorous analyses of 4 T MRI and other emerging modalities.

Health care utilization in patients with gout.

OBJECTIVE: To study health care utilization patterns in patients with gout. METHODS: In a gout population from primary care and rheumatology clinics in 3 U.S. metropolitan cities, we collected data on gout-related utilization (primary care, rheumatology, urgent care, emergency room, and other) in the past year. We evaluated the association of comorbidities, age, gender, gout characteristics (time since last gout attack and tophi), and gout severity ratings (mean of serum uric acid, patient-rated, and physician-rated gout severity) and with emergency/urgent care and primary care utilization using regression and correlation analyses. RESULTS: Of the 296 patients who reported visiting at least 1 type of health practitioner for gout in the past year, the percentage of patients utilizing the service at least once and annual utilization rates among utilizers were as follows: primary care physician, 60%, 3.0 ± 3.4; nurse practitioner/physician assistant, 26%, 2.7 ± 2.5; rheumatologist, 51%, 3.7 ± 5.7; urgent care, 23%, 2.1 ± 2.2; emergency room, 20%, 2.0 ± 1.7; and hospitalization, 7%, 2.1 ± 1.4. Higher overall gout severity was associated with greater use of each resource type and with overall gout-related utilization. Nonemergency/nonurgent care utilization (primary care physician, nurse practitioner, physician's assistant, and rheumatologist for gout) was the strongest predictor of gout-related emergency/urgent care utilization. Patients with more comorbidities had greater gout-related primary care utilization. CONCLUSIONS: Overall gout severity was associated with all types of gout-related utilization. This may help to screen high utilizers for targeted behavioral and therapeutic interventions. Having a higher number of comorbid conditions was a risk factor for higher gout-related primary care utilization.

The interleukin 1 inhibitor rilonacept in treatment of chronic gouty arthritis: results of a placebo-controlled, monosequence crossover, non-randomised, single-blind pilot study.

BACKGROUND: Recent studies suggest that blockade of the NLRP3 (cryopyrin) inflammasome interleukin 1beta (IL1beta) pathway may offer a new treatment strategy for gout. OBJECTIVE: To explore the potential utility of rilonacept (IL1 Trap) in patients with chronic active gouty arthritis in a proof-of-concept study. METHODS: This 14-week, multicentre, non-randomised, single-blind, monosequence crossover study of 10 patients with chronic active gouty arthritis included a placebo run-in (2 weeks), active rilonacept treatment (6 weeks) and a 6-week post-treatment follow-up. RESULTS: Rilonacept was generally well tolerated. No deaths and no serious adverse events occurred during the study. One patient withdrew owing to an injection-site reaction. Patients' self-reported median pain visual analogue scale scores significantly decreased from week 2 (after the placebo run-in) to week 4 (2 weeks of rilonacept) (5.0 to 2.8; p<0.049), with sustained improvement at week 8 (1.3; p<0.049); 5 of 10 patients reported at least a 75% improvement. Median symptom-adjusted and severity-adjusted joint scores were significantly decreased. High-sensitivity C-reactive protein levels fell significantly. CONCLUSIONS: This proof-of-concept study demonstrated that rilonacept is generally well tolerated and may offer therapeutic benefit in reducing pain in patients with chronic refractory gouty arthritis, supporting the need for larger, randomised, controlled studies of IL1 antagonism such as with rilonacept for this clinical indication.

Hyperalgesia, synovitis and multiple biomarkers of inflammation are suppressed by interleukin 1 inhibition in a novel animal model of gouty arthritis.

BACKGROUND: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 beta (IL1beta) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis. OBJECTIVE: To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1alpha and IL1beta) on pain, synovitis and systemic inflammatory biomarkers. METHODS: MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation. RESULTS: Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling. CONCLUSIONS: IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.

Transglutaminase 2 is a marker of chondrocyte hypertrophy and osteoarthritis severity in the Hartley guinea pig model of knee OA.

OBJECTIVE: The transglutaminase (TG) isoenzyme TG2, which catalyzes protein cross-linking via transamidation, influences healing phenotype in multiple forms of tissue injury. Moreover, TG2 knockout suppresses cartilage destruction but promotes osteophyte formation in instability-induced mouse knee osteoarthritis (OA). TG2 is marker of growth plate chondrocyte hypertrophy. Moreover, TG2 secreted by chondrocytes acts in part by promoting chondrocyte maturation to hypertrophy, a differentiation state linked with MMP-13 expression and disease progression in OA. Moreover, glucosamine, which is currently under investigation as an OA therapy, binds and inhibits TG2. Here, we examined TG2 as a potential marker of cartilage hypertrophy in the spontaneous guinea pig model of OA. METHODS: Synovial fluid ELISA and cartilage Immunohistochemistry and quantitative Reverse transcription-polymerase chain reaction (RT-PCR), were used to examine TG2 expression and TG transamidation-catalyzed isopeptide bonds. RESULTS: TG isopeptide bonds and TG2 were most abundant in articular cartilage in early knee OA. TG2 expression was robust at sites of early but not established osteophytes. Synovial fluid TG2 correlated with knee OA total histological score (r=0.47, P=0.01), as did medial tibial plateau cartilage TG2 mRNA (r=1.0, P=0.003). At 12 months of age, medial tibial plateau cartilage TG2 mRNA expression rose markedly in association with elevated type X collagen, as well as ADAMTS-5, and MMP-13 expression, changes not shared in age-matched Strain 13 guinea pigs that are less susceptible to knee OA. CONCLUSION: Hartley guinea pig knee TG2 expression associates with enhanced articular chondrocyte hypertrophy and is a biomarker of OA severity.

Calcification of human articular knee cartilage is primarily an effect of aging rather than osteoarthritis.

OBJECTIVE: Pathologic calcification of articular cartilage in human knees is often associated with advanced age and conditions of osteoarthritis (OA). Coincidently, most studies that have characterized calcification in joint cartilage have examined populations that are aged and presenting with clinical symptoms. Generally, these studies rely upon relatively insensitive plain radiographs or synovial fluid crystal analyses to quantify calcium levels. The purpose of this study was to examine the relationship between cartilage calcification and aging in an unselected donor population of diverse age using highly sensitive calcification imaging. METHODS: A group of 106 knee blocks were obtained from 56 individual donors (25 females and 31 males, aged 12-74, avg. 50.3 years). Condylar surfaces were graded on a 4-point OA grading scale for cartilage degeneration. The condyles were cut into approximately 7-10mm thick slabs. Using a Faxitron radiography system, high-resolution images were taken of the slabs to specifically image calcification in cartilage. The quantified calcification areas were then analyzed and correlations with both OA grade and age were assessed. RESULTS: Every knee presented some measurable calcification. The relative calcium deposition had a significant positive correlation with age. This same positive correlation was seen between condyles showing grade 1 and 2 changes. OA grades higher than 2 did not present any further significant increase in calcium levels. CONCLUSION: These observations indicate that age rather than OA is the predominant factor driving progressive pathologic calcification in articular cartilage.

Lack of association between chondrocalcinosis and increased risk of cartilage loss in knees with osteoarthritis: results of two prospective longitudinal magnetic resonance imaging studies.

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 > or = 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.

Leukocyte transglutaminase 2 expression limits atherosclerotic lesion size.

OBJECTIVE: Transglutaminase 2 (TG2), a broadly expressed regulator of protein cross-linking, wound healing, and tissue fibrosis, mediates apoptotic cell ingestion and transforming growth factor-beta release by macrophages and thereby can limit leukocyte-mediated inflammation. In atherosclerosis, oxidative stress and accumulation of unesterified cholesterol stimulate atherosclerotic lesion cell apoptosis. Cell death in advanced atherosclerotic lesions promotes lesion expansion and vulnerable plaques prone to rupture. Hence, we tested the hypothesis that leukocyte TG2 expression limits atherosclerosis. METHODS AND RESULTS: We transplanted TG2-/- or TG2+/+ bone marrow into lethally irradiated low-density lipoprotein receptor (LDLR)-/- mice and evaluated diet-induced atherosclerosis after 16 weeks. We subsequently studied cultured TG2-/- and congenic TG2+/+ mouse macrophages for selected atherogenesis regulatory functions. Atherosclerotic aortic valve lesions in LDLR-/- recipients of TG2-/- bone marrow were larger and more subintimal lesional macrophage penetration than in TG2+/+ marrow recipients. Lesion intimal TG2 expression appeared robust in TG2+/+ but not TG2-/- marrow recipients. Cultured TG2-/- macrophages demonstrated diminished phagocytosis of apoptotic leukocytes, unaltered endocytosis, and degradation of oxidized LDL but decreased retinoic acid induction of the reverse cholesterol transport and apoptotic cell uptake mediator ABCA1. CONCLUSIONS: We conclude that macrophage TG2 expression promotes both apoptotic cell clearance and ABCA1 expression in vitro and limits atherosclerotic lesion size in vivo.

Chondrocyte calcium-sensing receptor expression is up-regulated in early guinea pig knee osteoarthritis and modulates PTHrP, MMP-13, and TIMP-3 expression.

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 (1.8 mM). 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 (3 mM). 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.

Calcification in calcium pyrophosphate dihydrate (CPPD) crystalline deposits in the knee: anatomic, radiographic, MR imaging, and histologic study in cadavers.

OBJECTIVE: To demonstrate and determine the frequency and location of calcification within cadaveric knees with or without calcification typical of calcium pyrophosphate dihydrate (CPPD), utilizing histologic, radiographic and MR imaging techniques. DESIGN AND PATIENTS: Ten cadaveric knees of elderly individuals that demonstrated no radiographic evidence of prior surgery or trauma were studied with MR imaging and subsequently sectioned in planes corresponding to those obtained with MR imaging. The slices were imaged with high-resolution radiography. Two musculoskeletal radiologists correlated the anatomic, MR and radiographic findings. Three of the knees, which did not demonstrate calcifications, were utilized as controls. Histologic sections were obtained from four knees that contained calcifications and from the three controls, and analyzed with special histologic stains that demonstrate phosphorus and calcium. RESULTS: Radiographic imaging and histologic analysis demonstrated widespread CPPD crystal deposition in four of the 10 knee specimens (40%). MR imaging demonstrated some calcifications only within the articular cartilage of the femoral condyles in three of the four (75%) specimens that had CPPD deposits. In all four specimens radiographs and histologic analysis were more sensitive than MR imaging. Histologic analysis demonstrated no evidence of CPPD crystals in the control specimens. CONCLUSION: MR imaging is insensitive to the presence of CPPD deposits in the knee, even when such deposits are widespread. Our study suggests that the sensitivity of MR imaging was significantly better in detecting CPPD deposits in the hyaline cartilage of the femoral condyles when compared with other internal structures, even when such structures contained a higher amount of calcification.

Upregulated ank expression in osteoarthritis can promote both chondrocyte MMP-13 expression and calcification via chondrocyte extracellular PPi excess.

OBJECTIVE: In idiopathic chondrocalcinosis and in osteoarthritis (OA), increased extracellular PP(i) (ecPP(i)) promotes calcification. In chromosome 5p-associated familial chondrocalcinotic degenerative arthropathy, certain mutations in the membrane protein ANK may chronically raise ecPP(i) via enhanced PP(i) channeling. Therefore, we assessed if dysregulated wild-type ANK expression could contribute to pathogenesis of idiopathic degenerative arthropathy through elevated ecPP(i). DESIGN: Using cells with genetic alterations in expression of ANK and the PP(i)-generating nucleotide pyrophosphatase phosphodiestrase (NPP) PC-1, we examined how increased ANK expression elevates ecPPI, testing for codependent effects with PC-1. We also evaluated the effects of ANK expression on chondrocyte growth, matrix synthesis, and MMP-13 expression and we immunohistochemically examined ANK expression in situ in human knee OA cartilages. RESULTS: Using cells expressing defective ANK, as well as PC-1 knockout cells, we demonstrated that ANK required PC-1 (and vice versa) to raise ecPP(i) and that the major ecPP(i) regulator TGFbeta required both ANK and PC-1 to elevate ecPP(i). Upregulation of wild-type ANK by transfection in normal chondrocytes not only raised ecPP(i) 5-fold to approximately 100nM but also directly stimulated matrix calcification and inhibited collagen and sulfated proteoglycans synthesis. In addition, upregulated ANK induced chondrocyte MMP-13, an effect that also was stimulated within 2h by treatment of chondrocytes with 100nM PP(i) alone. Finally, ANK expression was upregulated in situ in human knee OA cartilages. CONCLUSION: Elevation of ecPP(i) by ANK critically requires the fraction of cellular PP(i) generated by PC-1. The upregulation of ANK expression in OA cartilage and the capacity of increased ANK expression to induce MMP-13 and to promote matrix loss suggest that increased ANK expression and ecPP(i) exert noxious effects in degenerative arthropathies beyond stimulation of calcification.

Src family protein tyrosine kinase signaling mediates monosodium urate crystal-induced IL-8 expression by monocytic THP-1 cells.

Neutrophil-dependent inflammation dependent on monosodium urate (MSU) crystal-induced IL-8 expression occurs in gout. MSU crystals activate phagocyte Src family tyrosine kinases and the serine/threonine kinase p70s6k. Thus, using monocytic THP-1 cells, we assessed the potential for Src family kinases and p70s6k to mediate MSU-induced IL-8 expression. MSU crystals induced phosphorylation of p70s6k and the Src kinases c-Src, Lyn, Hck, and Fyn. IL-8 expression was attenuated more by the Src kinase inhibitor PP1 than by the p70s6k inhibitor rapamycin. PP1 inhibited crystal-induced phosphorylation of ERK1/2 and IkappaBalpha and suppressed IkappaB kinase (IKK) activation and NF-kappaB binding to the IL-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 IkappaBalpha phosphorylation and IL-8 expression. We conclude that Src family tyrosine kinase signaling plays a significant role in MSU crystal-induced IL-8 expression via stimulation of ERK1/2 pathway and NF-kappaB activation.

Comparative tissue distribution of the processing enzymes "prohormone thiol protease," and prohormone convertases 1 and 2, in human PTHrP-producing cell lines and mammalian neuroendocrine tissues.

Peptide hormones are generated by proteolytic processing of their respective protein precursors by several prohormone processing proteases. The peptide hormone PTHrP is widely expressed in normal and malignant tissues, where proPTHrP undergoes proteolytic processing to generate PTHrP peptides with distinct biological actions. In this study, the tissue distribution of the prohormone processing enzymes PTP, PC1, and PC2 were compared by immunohistochemistry in human PTHrP-producing cancer cell lines, and in mammalian neuroendocrine and other tissues from rat and bovine that contain peptide hormones. PTP, PC1, and PC2 were prominently expressed in PTHrP-expressing human cancer cell lines originating from tumors of the breast, lung, prostate, as well as lymphoma. These processing enzymes also showed significant expression in normal mammalian neuroendocrine tissues from bovine and rat, including pituitary, hypothalamus, adrenal medulla, pancreas, and other tissues. Most neuroendocrine tissues contained prominent levels of at least two of the three processing enzymes examined, and all tissues contained at least one of these three enzymes. Differential expression of processing enzyme proteins was also demonstrated by Western blots. The differential expression of PTP, PC1, and PC2 observed in certain cancer and normal neuroendocrine cell types postulates selective roles for these processing enzymes in different tissues for generating biologically active peptide hormones. These results support the importance of these processing enzymes in their hypothesized roles in prohormone processing.

Interleukin-1 induces pro-mineralizing activity of cartilage tissue transglutaminase and factor XIIIa.

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.

Inorganic pyrophosphate generation and disposition in pathophysiology.

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

Up-regulated expression of the phosphodiesterase nucleotide pyrophosphatase family member PC-1 is a marker and pathogenic factor for knee meniscal cartilage matrix calcification.

OBJECTIVE: Elevated cartilage inorganic pyrophosphate (PPi) production and PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity are strongly linked with aging-related cartilage calcification in meniscal and articular cartilages. We hypothesized that there were divergent relationships of 3 NTPPPH isozymes with cartilage matrix calcification and sought to identify them. METHODS: We studied knee medial meniscal expression in situ of 3 NTPPPH isozymes of the phosphodiesterase nucleotide pyrophosphatase (PDNP) family: plasma cell membrane glycoprotein 1 (PC-1, or PDNP1), autotaxin (ATX, or PDNP2), and B10/PDNP3. We also used complementary DNA transfection to assess differential functions in matrix calcification of each NTPPPH isozyme in vitro in meniscal cells. RESULTS: We observed diffuse cell-associated ATX and B10/PDNP3 expression in central (chondrocytic) and, to a lesser degree, peripheral (fibroblastic) regions of normal, degenerative uncalcified, and degenerative calcified menisci. In contrast, PC-1 expression was only robust at sites of apoptotic cells and calcification in central regions of degenerative menisci. Only PC-1 was abundant at the perimeter of meniscal cells and in association with meniscal cell-derived matrix vesicles (MVs). Because each PDNP-family isozyme was expressed by cells near calcifications, we transfected the isozymes in nonadherent knee meniscal cells cultured with ascorbic acid, beta-glycerophosphate, and dexamethasone supplementation to stimulate them to calcify the matrix. PC-1, but not ATX or B10/PDNP3, consistently promoted increased MV NTPPPH, MV-associated PPi, and extracellular PPi. PC-1 also increased matrix calcification (with hydroxyapatite crystals) by meniscal cells. ATX uniquely induced alkaline phosphatase activity, but promoted only moderately increased matrix calcification. CONCLUSION: We identified divergent effects of 3 PDNP-family NTPPPH isozymes on meniscal cell matrix calcification. Increased expression of PC-1 is both a marker and a potential pathogenic factor for knee meniscal cartilage matrix calcification.

High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex-vivo gene therapy to repair articular cartilage defects.

BACKGROUND: Primary perichondrial cells and chondrocytes have been used to repair articular cartilage defects in tissue engineering studies involving various animal models. Transfection of these cells with a gene that induces chondrocytic phenotype may form an ideal method to affect tissue engineering of articular cartilage. DESIGN: A protocol for high-efficiency transfection of primary perichondrial and cartilage cells was optimized. Plasmids carrying the marker beta-galactosidase (beta-gal), PTHrP and TGF-beta1 genes driven by a strong mammalian promoter were transfected into primary perichondrial cells and chondrocytes. A three-step method was used to achieve high efficiency of transfection: (1) permeabilization of primary cells using a mild detergent, (2) association of plasmid DNAs with a polycationic (poly-l-lysine) core covalently linked to a receptor ligand (transferrin), (3) introduction of cationic liposomes to form the quaternary complex. For in-vivo assessment, polylactic acid (PLA) scaffolds seeded with beta-gal transfected perichondrial cells were implanted into experimentally created osteochondral defects in rabbit knees for 1 week. RESULTS: The efficiency of transfection was determined to be over 70%in vitro. The transformed cells continued to express beta-gal, in vivo for the entire test period of 7 days. Furthermore, primary perichondrial cells transfected with TGF-beta1 and PTHrP over-expressed their cognate gene products. CONCLUSION: The ability to transfect autologous primary perichondrial cells and chondrocytes with high efficiency using a non-viral system may form a first step towards tissue engineering with these transformed cells to repair articular cartilage defects.