Role of Advanced Glycation End Products as New Biomarkers in Systemic Lupus Erythematosus.

Advanced glycation end-products (AGEs) may play a relevant role as inducers in the chronic inflammatory pathway present in immune-mediated diseases, such as systemic lupus erythematosus (SLE). AGEs concentrations have been associated, with discrepant results to date, with some parameters such as disease activity or accrual damage, suggesting their potential usefulness as biomarkers of the disease. Our objectives are to confirm differences in AGEs levels measured by cutaneous autofluorescence between SLE patients and healthy controls (HC) and to study their correlation with various disease parameters. Cross-sectional study, where AGEs levels were measured by skin autofluorescence, and SLE patients' data were compared with those of sex- and age-matched HC in a 1:3 proportion through a multiple linear regression model. Associations of AGEs levels with demographic and clinical data were analyzed through ANOVA tests. Both analyses were adjusted for confounders. AGEs levels in SLE patients were significantly higher than in HC (p < 0.001). We found statistically significant positive associations with SLE disease activity index (SLEDAI) and damage index (SDI), physician and patient global assessment, C-reactive protein, leukocyturia, complement C4, IL-6 and oral ulcers. We also found a negative statistically significant association with current positivity of anti-nuclear and anti-Ro60 antibodies. AGEs seem to have a contribution in LES pathophysiology, being associated with activity and damage and having a role as a new management and prognosis biomarker in this disease. The association with specific antibodies and disease manifestations may indicate a specific clinical phenotype related to higher or lower AGEs levels.

Serum Advanced Glycation End Products and Their Soluble Receptor as New Biomarkers in Systemic Lupus Erythematosus.

It has been postulated that advanced glycation end products (AGEs) and their soluble receptor (sRAGE) may play a relevant role as inducers in the chronic inflammatory pathway in various conditions, among them, in immune-mediated diseases such as systemic lupus erythematosus (SLE). However, previous studies show conflicting results about their association with SLE characteristics and their usefulness as disease biomarkers. We aimed to study the association of specific serum AGEs (pentosidine, Nξ-(carboxymethyl)lysine (CML), Nξ-(carboxyethyl)lysine (CEL)), sRAGE levels and AGEs (specific serum AGEs and skin AGEs) to sRAGE ratios with various disease parameters, in order to clarify their potential as new biomarkers in SLE and to study their relationship with cardiovascular disease (CVD). To this aim, serum pentosidine, CML, CEL and sRAGE were measured via ELISA, and skin AGEs levels were measured by skin autofluorescence. Correlations of pentosidine levels with demographic and clinical data, indexes of activity, accrual damage and patient-reported outcomes were analyzed through multiple linear regression models, while correlations of the rest of the AGEs, sRAGE and AGE to sRAGE ratios (non-normal) were analyzed using both an OLS regression model and a GML. All of the analyses were adjusted for confounders. A total of 119 SLE patients were recruited. Serum AGEs and sRAGEs were significantly associated with SLE activity indexes and/or demographic or disease characteristics: pentosidine with pulmonary manifestations; CML with anti-dsDNA antibodies, IL-6, disease duration and non-Caucasian ethnicities; CEL with anti-dsDNA antibodies, IL-6 and accumulated number of manifestations; and sRAGE with male gender, photosensitivity and being on specific immunosuppressants. These results suggest that the AGE-sRAGE axis may serve as a novel biomarker for managing and prognosticating this disease. Its correlation with certain antibodies, demographics and disease presentations may indicate a distinct clinical phenotype associated with varying levels of AGEs and/or sRAGE. The significance of specific AGE/sRAGE ratios, introduced in this study for the first time, warrants additional investigation in forthcoming research. Our study did not confirm the link between serum AGEs and CVD, which merits further exploration through studies designed for this specific purpose.

Left ventricular dysfunction and arrhythmias in asymptomatic patients with systemic sclerosis / Disfunción ventricular izquierda y arritmias en pacientes asintomáticos con esclerosis sistémica

Introduction and aims Cardiac involvement in systemic sclerosis (SS) is frequently silent and a major cause of mortality in these patients. This work aims to study the prevalence and associations of left ventricular dysfunction (LVD) and arrhythmias in SS. Methods and results Prospective study of SS patients (n=36), excluding those with symptoms of (or) cardiac disease, pulmonary arterial hypertension or cardiovascular risk factors (CVRF). A clinical, analytical, electrocardiogram (EKG), Holter, and echocardiogram with global longitudinal strain (GLS) assessment were performed. Arrhythmias were classified into clinically significant arrhythmias (CSA) and non-significant.Twenty-eight percent had left ventricular diastolic dysfunction (LVDD), 22% LV systolic dysfunction (LVSD) according to the GLS, 11.1% both, and 16.7% cardiac dysautonomia. Fifty percent presented alterations by EKG (44% CSA), 55.6% by Holter (75% CSA) and 8.3% CSA by both. An association was found between the elevation of troponin T (TnTc) and CSA and between the elevation of both NT-proBNP and TnTc with LVDD. Conclusions We found a higher prevalence of LVSD than in the literature, detected by GLS and being 10 times higher than that detected by LVEF, which justifies the need to incorporate this technique in the routine evaluation of these patients. The association of TnTc and NT-proBNP with LVDD suggests that they can be used as minimally invasive biomarkers of this affectation. The absence of correlation between LVD and CSA indicates that the arrhythmias could be due, not only to a supposed structural alteration of the myocardium, but to an independent and early cardiac involvement, which should be actively investigated even in asymptomatic patients without CVRF. (AU)

Introducción y objetivosLa afectación cardiaca en la esclerosis sistémica (ES) es frecuentemente asintomática y se asocia con una mortalidad importante. Este trabajo tiene como objetivo estudiar la prevalencia y las asociaciones de la disfunción ventricular izquierda (DVI) y las arritmias en la ES. Métodos y resultados Estudio prospectivo de pacientes con ES (n = 36), excluyendo aquellos con síntomas o enfermedad cardiaca, hipertensión arterial pulmonar o factores de riesgo cardiovascular (FRCV). Se les realizó una evaluación clínica, analítica, con electrocardiograma (ECG), Holter y ecocardiograma con strain longitudinal global (SLG). Las arritmias se clasificaron en arritmias clínicamente significativas (ACS) y no significativas.De los pacientes estudiados, 27,8% presentaba disfunción diastólica del ventrículo izquierdo (DDVI), 22% disfunción sistólica del VI (DSVI) según el SLG, 11,1% ambas y 16,7% disautonomía cardiaca; 50% presentó alteraciones por ECG (44% ACS), 55,6% por Holter (75% ACS) y 8,3% ACS por ambos. Se encontró una asociación entre la elevación de troponina T (TnTc) y ACS y entre la elevación NT-proBNP y TnTc con la DDVI. Conclusiones Encontramos una prevalencia de DSVI mayor que en la literatura, detectada por SLG y siendo 10 veces superior a la detectada por FEVI, lo que justifica la necesidad de incorporar esta técnica en la evaluación rutinaria de estos pacientes. La asociación de TnTc y NT-proBNP con DDVI sugiere que pueden ser utilizados como biomarcadores mínimamente invasivos de esta afectación. La ausencia de correlación entre DVI y ACS indica que las arritmias podrían deberse, no solo a una supuesta alteración estructural del miocardio, sino a un compromiso cardiaco independiente y temprano, que debe investigarse activamente incluso en pacientes asintomáticos sin FRCV. (AU)

Evaluation of a single-shot of a high-density viscoelastic solution of hyaluronic acid in patients with symptomatic primary knee osteoarthritis: the no-dolor study.

BACKGROUND: Pronolis®HD mono 2.5% is a novel, one-shot, high-density sterile viscoelastic solution, recently available in Spain, which contains a high amount of intermediate molecular weight hyaluronic acid (HA), highly concentrated (120 mg in 4.8 mL solution: 2.5%). The objective of the study was to analyze the efficacy and safety of this treatment in symptomatic primary knee osteoarthritis (OA). METHODS: This observational, prospective, multicenter, single-cohort study involved 166 patients with knee OA treated with a single-shot of Pronolis®HD mono 2.5% and followed up as many as 24 weeks. RESULTS: Compared with baseline, the score of the Western Ontario and McMaster Universities Arthritis Osteoarthritis Index (WOMAC) pain subscale reduced at the 12-week visit (primary endpoint, median: 9 interquartile range [IQR]: 7-11 versus median: 4; IQR: 2-6; p < 0.001). The percentage of patients achieving > 50% improvement in the pain subscale increased progressively from 37.9% (at 2 weeks) to 66.0% (at 24 weeks). Similarly, WOMAC scores for pain on walking, stiffness subscale, and functional capacity subscale showed significant reductions at the 12-week visit which were maintained up to the 24-week visit. The EuroQol visual analog scale score increased after 12 weeks (median: 60 versus 70). The need for rescue medication (analgesics/nonsteroidal anti-inflammatory drugs) also decreased in all post-injection visits. Three patients (1.6%) reported local adverse events (joint swelling) of mild intensity. CONCLUSIONS: In conclusion, a single intra-articular injection of the high-density viscoelastic gel of HA was associated with pain reduction and relief of other symptoms in patients with knee OA. TRIAL REGISTRATION: ClinicalTrial# NCT04196764.

Relationship Between the Choice of Clinical Treatment, Gait Functionality and Kinetics in Patients With Comparable Knee Osteoarthritis.

Objective: The objective of this study was to investigate the relationship between the choice of clinical treatment, gait functionality, and kinetics in patients with comparable knee osteoarthritis. Design: This was an observational case-control study. Setting: The study was conducted in a university biomechanics laboratory. Participants: Knee osteoarthritis patients were stratified into the following groups: clinical treatment (conservative/total knee replacement (TKR) planned), sex (male/female), age (60-67/68-75), and body mass index (BMI) (<30/≥30). All patients had a Kellgren-Lawrence score of 2 or 3 (N = 87). Main Outcome Measures: All patients underwent gait analysis, and two groups of dependent variables were extracted: • Spatiotemporal gait variables: gait velocity, stride time, and double-support time, which are associated with patient functionality. • Kinetic gait variables: vertical, anterior-posterior, and mediolateral ground reaction forces, vertical free moment, joint forces, and moments at the ankle, knee, and hip. Multifactorial and multivariate analyses of variance were performed. Results: Functionality relates to treatment decisions, with patients in the conservative group walking 25% faster and spending 24% less time in the double-support phase. However, these differences vary with age and are reduced in older subjects. Patients who planned to undergo TKR did not present higher knee forces, and different joint moments between clinical treatments depended on the age and BMI of the subjects. Conclusions: Knee osteoarthritis is a multifactorial disease, with age and BMI being confounding factors. The differences in gait between the two groups were mitigated by confounding factors and risk factors, such as being a woman, elderly, and obese, reducing the variability of the gait compression loads. These factors should always be considered in gait studies of patients with knee osteoarthritis to control for confounding effects.

Regulatory network-based model to simulate the biochemical regulation of chondrocytes in healthy and osteoarthritic environments.

In osteoarthritis (OA), chondrocyte metabolism dysregulation increases relative catabolic activity, which leads to cartilage degradation. To enable the semiquantitative interpretation of the intricate mechanisms of OA progression, we propose a network-based model at the chondrocyte level that incorporates the complex ways in which inflammatory factors affect structural protein and protease expression and nociceptive signals. Understanding such interactions will leverage the identification of new potential therapeutic targets that could improve current pharmacological treatments. Our computational model arises from a combination of knowledge-based and data-driven approaches that includes in-depth analyses of evidence reported in the specialized literature and targeted network enrichment. We achieved a mechanistic network of molecular interactions that represent both biosynthetic, inflammatory and degradative chondrocyte activity. The network is calibrated against experimental data through a genetic algorithm, and 81% of the responses tested have a normalized root squared error lower than 0.15. The model captures chondrocyte-reported behaviors with 95% accuracy, and it correctly predicts the main outcomes of OA treatment based on blood-derived biologics. The proposed methodology allows us to model an optimal regulatory network that controls chondrocyte metabolism based on measurable soluble molecules. Further research should target the incorporation of mechanical signals.

Fibronectin-coating enhances attachment and proliferation of mesenchymal stem cells on a polyurethane meniscal scaffold.

INTRODUCTION: Partial meniscectomy is one of the most common surgical strategy for a meniscal injury, but sometimes, patients complain of knee pain due to an overload in the ablated compartment. In these cases, implantation of tissue engineering scaffold could be indicated. Currently, two commercial scaffolds, based on collagen or polycaprolactone-polyurethane (PCL-PU), are available for meniscus scaffolding. In short term follow-up assessments, both showed clinical improvement and tissue formation. However, long-term studies carried out in PCL-PU showed that the new tissue decreased in volume and assumed an irregular shape. Moreover, in some cases, the scaffold was totally reabsorbed, without new tissue formation.Mesenchymal stem cells (MSCs) combined with scaffolds could represents a promising approach for treating meniscal defects because of their multipotency and self-renewal. In this work, we aimed to compare the behaviour of MSCs and chondrocytes on a PCL-PU scaffold in vitro. MSCs express integrins that binds to fibronectin (FN), so we also investigate the effect of a FN coating on the bioactivity of the scaffold. METHODS: We isolated rabbit bone marrow MSCs (rBM-MSCs) from two skeletally mature New Zealand white rabbits and stablished the optimum culture condition to expand them. Then, they were seeded over non-coated and FN-coated scaffolds and cultured in chondrogenic conditions. To evaluate cell functionality, we performed an MTS assay to compare cell proliferation between both conditions. Finally, a histologic study was performed to assess extracellular matrix (ECM) production in both samples, and to compare them with the ones obtained with rabbit chondrocytes (rCHs) seeded in a non-coated scaffold. RESULTS: A culture protocol based on low FBS concentration was set as the best for rBM-MSCs expansion. The MTS assay revealed that rBM-MSCs seeded on FN-coated scaffolds have more cells on proliferation (145%; 95% CI: 107%-182%) compared with rBM-MSCs seeded on non-coated scaffolds. Finally, the histologic study demonstrated that rCHs seeded on non-coated scaffolds displayed the highest production of ECM, followed by rBM-MSCs seeded on FN-coated scaffolds. Furthermore, both cell types produced a comparable ECM pattern. CONCLUSION: These results suggest that MSCs have low capacity attachment to PCL-PU scaffolds, but the presence of integrin alpha5beta1 (FN-receptor) in MSCs allows them to interact with the FN-coated scaffolds. These results could be applied in the design of scaffolds, and might have important clinical implications in orthopaedic surgery of meniscal injuries.

Influence of anti-osteoporosis treatments on the incidence of COVID-19 in patients with non-inflammatory rheumatic conditions.

Coronavirus disease 19 (COVID-19) is currently a global pandemic that affects patients with other pathologies. Here, we investigated the influence of treatments for osteoporosis and other non-inflammatory rheumatic conditions, such as osteoarthritis and fibromyalgia, on COVID-19 incidence. To this end, we conducted a cross-sectional study of 2,102 patients being treated at the Rheumatology Service of Hospital del Mar (Barcelona, Spain). In our cohort, COVID-19 cumulative incidence from March 1 to May 3, 2020 was compared to population estimates for the same city. We used Poisson regression models to determine the adjusted relative risk ratios for COVID-19 associated with different treatments and comorbidities. Denosumab, zoledronate and calcium were negatively associated with COVID-19 incidence. Some analgesics, particularly pregabalin and most of the studied antidepressants, were positively associated with COVID-19 incidence, whereas duloxetine presented a negative association. Oral bisphosphonates, vitamin D, thiazide diuretics, anti-hypertensive drugs and chronic non-steroidal anti-inflammatory drugs had no effect on COVID-19 incidence in the studied population. Our results provide novel evidence to support the maintenance of the main anti-osteoporosis treatments in COVID-19 patients, which may be of particular relevance to elderly patients affected by the SARS-CoV-2 pandemic.

COVID-19 Incidence in Patients With Immunomediated Inflammatory Diseases: Influence of Immunosuppressant Treatments.

The effect of immunosuppressant treatments on the incidence of coronavirus disease (COVID-19) remains largely unknown. We studied the association between the pre-exposure to disease-modifying antirheumatic drugs (DMARDs) that decrease immunological responses and the incidence of COVID-19 to explore the possible effects of these treatments in early manifestations of the disease. For this purpose, we performed a cross-sectional study including 2,494 patients with immunomediated inflammatory diseases (IMIDs) recruited at the outpatient Rheumatology, Dermatology and Gastroenterology services of Hospital del Mar. The primary outcome was the clinical diagnosis of COVID-19 performed by a physician at the hospital or at the primary care center, from the March 1-29, 2020. Multivariable Poisson regression models were fitted to estimate COVID-19 relative risk (RR) adjusted by comorbidities. We revealed that biological (RR = 0.46, CI 95% = 0.31-0.67) and synthetic (RR = 0.62, CI 95% = 0.43-0.91) DMARDs used in IMIDs diminished the incidence of COVID-19. Striking sex differences were revealed with anti-TNFα compounds (RR = 0.50, CI 95% = 0.33-0.75) with higher effects in women (RR = 0.33, CI 95% = 0.17-0.647). Treatment with low glucocorticoid doses also revealed sex differences decreasing the incidence of COVID-19 predominantly in women (RR = 0.72, CI 95% = 0.42-1.22). Our results report a decreased incidence of COVID-19 in patients receiving specific DMARDs with different immunodepressor mechanisms with striking sex differences. These results underline the interest of repurposing specific DMARDs for the possibility of minimizing the severity of disease progression in the early stages of COVID-19.

In vivo effect of opticin deficiency in cartilage in a surgically induced mouse model of osteoarthritis.

The SLRP opticin (OPTC) has been demonstrated to be produced and degraded in osteoarthritic (OA) human cartilage. Here, we investigated the in vivo effect of OPTC deficiency in OA cartilage. OA was induced in 10-week-old Optc -/- and Optc +/+ mice. Ten weeks post-surgery, cartilage was processed for histology and immunohistochemistry. SLRP expression was determined in non-operated mouse cartilage. OA Optc -/- demonstrated significant protection against cartilage degradation. Data revealed that in non-operated Optc -/- cartilage, expression of SLRPs lumican and epiphycan was up-regulated at day 3 and in 10-week-olds (p ≤ 0.039), and fibromodulin down-regulated in 10-week-olds (p = 0.001). Immunohistochemistry of OA mice showed a similar pattern. In OA Optc -/- cartilage, markers of degradation and complement factors were all down-regulated (p ≤ 0.038). In OA Optc -/- cartilage, collagen fibers were thinner and better organized (p = 0.038) than in OA Optc +/+ cartilage. The protective effect of OPTC deficiency during OA results from an overexpression of lumican and epiphycan, known to bind and protect collagen fibers, and a decrease in fibromodulin, contributing to a reduction in the complement activation/inflammatory process. This work suggests that the evaluation of the composition of the different SLRPs in OA cartilage could be applied as a new tool for OA prognosis classification.

Efecto del condroitín sulfato en la sinovitis de pacientes con artrosis de rodilla / Effect of chondroitin sulphate on synovitis of knee osteoarthritic patients

Objetivo: Evaluar mediante ecografía el efecto del condroitín sulfato (CS) en la sinovitis de pacientes con artrosis (OA) de rodilla, y colaborar en el conocimiento de los mecanismos bioquímicos involucrados en la inflamación sinovial. Métodos: Estudio controlado, aleatorizado, ciego simple de 70 pacientes con OA de rodilla tratados durante 6 meses con CS o paracetamol (PCT). Los pacientes fueron visitados a tiempo basal, a las 6 semanas, y a los 3 y 6 meses para valorar el estado de su OA según los siguientes parámetros: sinovitis evaluada mediante ecografía (según definición de expertos OMERACT); dolor y función, mediante la escala visual analógica y el índice de Lequesne; y concentración de mediadores inflamatorios en suero y líquido sinovial, mediante ELISA. Resultados: El tratamiento con CS redujo en un 50% el número de individuos que presentaban sinovitis; sin embargo, se observó un incremento de un 123% en el grupo tratado con PCT. En los pacientes sin sinovitis inicial, se observó el establecimiento de esta en un 85,71 y 25% de los casos tratados con PCT y CS, respectivamente. Ambas terapias mejoraron la función articular, pero únicamente el tratamiento con CS produjo una mejora significativa del dolor al final del tratamiento. Se observó una asociación entre el tratamiento con CS y los cambios en la concentración de RANTES y UCN en el líquido sinovial. Conclusiones: El tratamiento con CS tiene un efecto mantenido beneficioso, previniendo la aparición de sinovitis o disminuyendo su presencia, así como reduciendo los síntomas de la artrosis. El PCT también mejora los síntomas clínicos, pero no tiene ningún efecto sobre la inflamación. Las variaciones observadas en la concentración de RANTES y UCN podrían estar relacionadas con el efecto antiinflamatorio asociado al tratamiento con CS (AU)

Objective: To evaluate by ultrasonography the effect of chondroitin sulfate (CS) on synovitis in patients with knee osteoarthritis (KOA). To collaborate in the understanding of the biochemical mechanisms involved in the synovial inflammation process. Methods: Randomized, single-blind, controlled trial involving 70 patients with primary KOA treated for 6 months with CS or acetaminophen (ACT). Evaluation of KOA status at baseline, 6 weeks, 3 and 6 months included: ultrasonography to assess synovitis (following the OMERACT expertise group definition), visual analogue scale and Lequesne index to measure pain and function, and ELISA to quantify inflammatory mediators in serum and synovial fluid. Results: Synovitis presence was reduced by 50% in the CS group while a 123% increase was observed in ACT group. Conversely, patients without initial synovitis and treated with ACT reached 85.71% synovitis onset, but only 25% in CS group. Both therapies improved articular function, but only CS resulted in significant pain improvement at the end of the treatment. Changes in RANTES and UCN synovial fluid concentration were associated with CS treatment. Conclusions: Treatment with CS had a sustained beneficial effect, preventing synovitis onset or reducing its presence as well as reducing KOA symptoms. ACT ameliorated clinical symptoms but had no effect on inflammation. The CS anti-inflammatory effect could be related to the observed changes in RANTES and UCN concentration (AU)

Effect of chondroitin sulphate on synovitis of knee osteoarthritic patients. / Efecto del condroitín sulfato en la sinovitis de pacientes con artrosis de rodilla.

OBJECTIVE: To evaluate by ultrasonography the effect of chondroitin sulfate (CS) on synovitis in patients with knee osteoarthritis (KOA). To collaborate in the understanding of the biochemical mechanisms involved in the synovial inflammation process. METHODS: Randomized, single-blind, controlled trial involving 70 patients with primary KOA treated for 6 months with CS or acetaminophen (ACT). Evaluation of KOA status at baseline, 6 weeks, 3 and 6 months included: ultrasonography to assess synovitis (following the OMERACT expertise group definition), visual analogue scale and Lequesne index to measure pain and function, and ELISA to quantify inflammatory mediators in serum and synovial fluid. RESULTS: Synovitis presence was reduced by 50% in the CS group while a 123% increase was observed in ACT group. Conversely, patients without initial synovitis and treated with ACT reached 85.71% synovitis onset, but only 25% in CS group. Both therapies improved articular function, but only CS resulted in significant pain improvement at the end of the treatment. Changes in RANTES and UCN synovial fluid concentration were associated with CS treatment. CONCLUSIONS: Treatment with CS had a sustained beneficial effect, preventing synovitis onset or reducing its presence as well as reducing KOA symptoms. ACT ameliorated clinical symptoms but had no effect on inflammation. The CS anti-inflammatory effect could be related to the observed changes in RANTES and UCN concentration.

Healthy and Osteoarthritic Synovial Fibroblasts Produce a Disintegrin and Metalloproteinase with Thrombospondin Motifs 4, 5, 7, and 12: Induction by IL-1ß and Fibronectin and Contribution to Cartilage Damage.

Current description of osteoarthritis includes the involvement of synovial inflammation. Studies contributing to understanding the mechanisms of cross-talk and feedback among the joint tissues could be relevant to the development of therapies that block disease progression. During osteoarthritis, synovial fibroblasts exposed to anomalous mechanical forces and an inflammatory microenvironment release factors such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) metalloproteinases that mediate tissue damage and perpetuate inflammation. We therefore studied the production of ADAMTS by synovial fibroblasts and their contribution to cartilage degradation. Moreover, we analyzed the implication of two mediators present in the osteoarthritis joint, IL-1ß as proinflammatory cytokine, and 45-kDa fibronectin fragments as products of matrix degradation. We reported that synovial fibroblasts constitutively express and release ADAMTS 4, 5, 7, and 12. Despite the contribution of both mediators to the stimulation of Runx2 and Wnt/ß-catenin signaling pathways, as well as to ADAMTS expression, promoting the degradation of aggrecan and cartilage oligomeric matrix protein from cartilage, fibronectin fragments rather than IL-1ß played the major pathological role in osteoarthritis, contributing to the maintenance of the disease. Moreover, higher levels of ADAMTS 4 and 7 and a specific regulation of ADAMTS-12 were observed in osteoarthritis, suggesting them as new potential therapeutic targets. Therefore, synovial fibroblasts provide the biochemical tools to the chronicity and destruction of the osteoarthritic joints.

Characterization of opticin digestion by proteases involved in osteoarthritis development.

OBJECTIVE: Opticin is a class III member of the small leucine-rich repeat proteoglycan (SLRP) family, produced in articular joint tissues. In normal and osteoarthritic (OA) cartilage, opticin is degraded. This study aimed to assess whether human cartilage opticin is degraded by the main proteases involved in OA pathophysiology, and to determine the protease cleavage sites of this SLRP. METHODS: We analyzed the proteolytic activity of matrix metalloproteinases (MMPs)-1, -2, -3, -7, -8 and -9, and ADAMTS-4 and -5 on proteoglycan extracts from normal and moderately fibrillated OA human cartilage, and on recombinant human opticin. Opticin degradation was analyzed by Western blotting and cleavage sites were determined by sequence analysis. RESULTS: All eight proteases digested opticin from proteoglycan extracts from both normal and OA samples, as well as recombinant human opticin, MMP-2 and MMP-7 are the proteases that degrade recombinant human opticin most efficiently. The opticin cleavage site determined for these MMPs was between the glycosylation and leucine-rich repeat domains. MMP-7 had two additional digestion sites near the N-terminal end of opticin. CONCLUSION: Opticin is a substrate for several MMPs and aggrecanases involved during OA cartilage degradation, and seems to be a preferential substrate for MMP-7. The role of opticin in cartilage degeneration could be related to decreased levels of intact opticin, followed by its proteolytic degradation, which in turn may stimulate some of the modifications observed in the OA cartilage, such as neovascularisation and changes in the extracellular matrix.

Reduced proliferation and osteocalcin expression in osteoblasts of male idiopathic osteoporosis.

Osteoporosis is characterized by low bone mineral density (BMD), resulting in increasing susceptibility to bone fractures. In men, it has been related to some diseases and toxic habits, but in some instances the cause of the primary--or idiopathic--osteoporosis is not apparent. In a previous study, our group compared histomorphometric measurements in cortical and cancellous bones from male idiopathic osteoporosis (MIO) patients to those of control subjects and found reduced bone formation without major differences in bone resorption. To confirm these results, this study analyzed the etiology of this pathology, examining the osteoblast behavior in vitro. We compared two parameters of osteoblast activity in MIO patients and controls: osteoblastic proliferation and gene expression of COL1A1 and osteocalcin, in basal conditions and with vitamin D(3) added. All these experiments were performed from a first-passage osteoblastic culture, obtained from osteoblasts that had migrated from the transiliac explants to the plate. The results suggested that the MIO osteoblast has a slower proliferation rate and decreased expression of genes related to matrix formation, probably due to a lesser or slower response to some stimulus. We concluded that, contrary to female osteoporosis, in which loss of BMD is predominantly due to increased resorption, low BMD in MIO seems to be due to an osteoblastic defect.

Drosophila proteins interacting with metallothioneins: a metal-dependent recognition.

Metallothioneins (MTs) are ubiquitous, low-molecular weight, cysteine-rich proteins. Despite a well-established protective role in metal excess detoxification, there is little data about their putative physiological functions, commonly assumed to be metal homeostasis and redox equilibrium. Protein-protein interactions should have provided useful information to unveil unsuspected functions, but reports on MT interactions are scarce. This is probably due to the MT metal-dependent 3D structure, a fact that has been seldom taken into account when performing proteomic interaction assays. In the present work, we have detected that the two major D. melanogaster isoforms (MtnA and MtnB) interact with the peroxiredoxin (Prx) encoded by the gene Jafrac1, both in a clear metal-dependent pattern. The MT-Prx interaction is further confirmed in Saccharomyces cerevisiae by assaying both yeast MTs (Crs5p and Cup1p) versus Tsa1p and Tsa2p, the Jafrac1 homologous Prxs in this organism. Thus, a new methodological approach to detect MT-interacting proteins in different proteomes is established on the basis of assaying MTs in the form of different metal complexes. Furthermore, new perspectives to investigate the often hypothesized contribution of MTs to the redox physiological networks are open.

The Zn- and Cd-clusters of recombinant mammalian MT1 and MT4 metallothionein domains include sulfide ligands.

Recombinant (E. coli ) synthesis of mammalian MT1 and MT4 domains as separate peptides in Zn(II) and Cd(II) enriched growth media has rendered metal complexes containing sulfide anions as additional ligands. The Cd preparations show higher sulfide content than the Zn preparations. Also, the betaMT1 and betaMT4 fragments exhibit higher sulfide/peptide ratios than the respective alpha fragments. Titration of Zn3-betaMT1 with Cd(II) followed by addition of several sodium sulfide equivalents shows that the Cd(II)-betaMT1 species can incorporate sulfide ligands in vitro, with a concomitant evolution of their UV-vis and CD fingerprints to those characteristic of the Cd-S2- chromophores. Current results have also provided full understanding of previous data collected by this group in the characterization of the Cd-betaMT1 preparations obtained from large-scale fermentor synthesis by allowing identification of at least 2S2- ligands per Cd-betaMT1 species. Furthermore, the results here presented have revealed that synthesis of betaMT4 in Cd-supplemented cultures yielded Cd,S(2-)-containing clusters instead of the proposed heterometallic Zn,Cd-betaMT4 complexes. Finally, a global evaluation of our results suggests that the higher the Cu-thionein character of a MT peptide, the higher is its tendency to harbor nonproteic ligands (i.e., sulfide anions) when building divalent metal clusters, especially Cd-MT complexes.

Specificity and divergence in the neurobiologic effects of different metallothioneins after brain injury.

Brain injury and neuroinflammation are pathophysiologic contributors to acute and chronic neurologic disorders, which are progressive diseases not fully understood. Mammalian metallothioneins I and II (MT-I&II) have significant neuroprotective functions, but the precise mechanisms underlying these effects are still unknown. To gain insight in this regard, we have evaluated whether a distant, most likely single-domain MT (Drosophila MTN) functions similarly to mammalian MT-I&II (recombinant mouse MT-I and human MT-IIa and native rabbit MT-II) after cryogenic injury to the cortex in Mt1&2 KO mice. All the recombinant proteins showed similar neuroprotective properties to native MT-II, significantly reducing brain inflammation (macrophages, T cells, and pro-inflammatory cytokines), oxidative stress, neurodegeneration, and apoptosis. These results in principle do not support specific protein-protein interactions as the mechanism underlying the neuroprotective effects of these proteins because a non-homologous and structurally unrelated MT such as Drosophila MTN functions similarly to mammalian MTs. We have also evaluated for the first time the neurobiologic effects of exogenous MT-III, a major CNS MT isoform. Human rMT-III, in contrast to human nMT-IIa, did not affect inflammation, oxidative stress, and apoptosis, and showed opposite effects on several growth factors, neurotrophins, and markers of synaptic growth and plasticity. Our data thus highlight specific and divergent roles of exogenous MT-III vs. the MT-I&II isoforms that are consistent with those attributed to the endogenous proteins, and confirm the suitability of recombinant synthesis for future therapeutic use that may become relevant to clinical neurology.

Comparative metal binding and genomic analysis of the avian (chicken) and mammalian metallothionein.

Chicken metallothionein (ckMT) is the paradigm for the study of metallothioneins (MTs) in the Aves class of vertebrates. Available literature data depict ckMT as a one-copy gene, encoding an MT protein highly similar to mammalian MT1. In contrast, the MT system in mammals consists of a four-member family exhibiting functional differentiation. This scenario prompted us to analyse the apparently distinct evolutionary patterns followed by MTs in birds and mammals, at both the functional and structural levels. Thus, in this work, the ckMT metal binding abilities towards Zn(II), Cd(II) and Cu(I) have been thoroughly revisited and then compared with those of the mammalian MT1 and MT4 isoforms, identified as zinc- and copper-thioneins, respectively. Interestingly, a new mechanism of MT dimerization is reported, on the basis of the coordinating capacity of the ckMT C-terminal histidine. Furthermore, an evolutionary study has been performed by means of in silico analyses of avian MT genes and proteins. The joint consideration of the functional and genomic data obtained questions the two features until now defining the avian MT system. Overall, in vivo and in vitro metal-binding results reveal that the Zn(II), Cd(II) and Cu(I) binding abilities of ckMT lay between those of mammalian MT1 and MT4, being closer to those of MT1 for the divalent metal ions but more similar to those of MT4 for Cu(I). This is consistent with a strong functional constraint operating on low-copy number genes that must cope with differentiating functional limitation. Finally, a second MT gene has been identified in silico in the chicken genome, ckMT2, exhibiting all the features to be considered an active coding region. The results presented here allow a new insight into the metal binding abilities of warm blooded vertebrate MTs and their evolutionary relationships.