A human meniscus explant model for studying early events in osteoarthritis development by proteomics.

Degenerative meniscus lesions have been associated with both osteoarthritis etiology and its progression. We, therefore, sought to establish a human meniscus ex vivo model to study the meniscal response to cytokine treatment using a proteomics approach. Lateral menisci were obtained from five knee-healthy donors. The meniscal body was cut into vertical slices and further divided into an inner (avascular) and outer region. Explants were either left untreated (controls) or stimulated with cytokines. Medium changes were conducted every 3 days up to Day 21 and liquid chromatography-mass spectrometry was performed at all the time points for the identification and quantification of proteins. Mixed-effect linear regression models were used for statistical analysis to estimate the effect of treatments versus control on protein abundance. Treatment by IL1ß increased release of cytokines such as interleukins, chemokines, and matrix metalloproteinases but a limited catabolic effect in healthy human menisci explants. Further, we observed an increased release of matrix proteins (collagens, integrins, prolargin, tenascin) in response to oncostatin M (OSM) + tumor necrosis factor (TNF) and TNF+interleukin-6 (IL6) + sIL6R treatments, and analysis of semitryptic peptides provided additional evidence of increased catabolic effects in response to these treatments. The induced activation of catabolic processes may play a role in osteoarthritis development.

Tissue catabolism and donor-specific dexamethasone response in a human osteochondral model of post-traumatic osteoarthritis.

BACKGROUND: Post-traumatic osteoarthritis (PTOA) does not currently have clinical prognostic biomarkers or disease-modifying drugs, though promising candidates such as dexamethasone (Dex) exist. Many challenges in studying and treating this disease stem from tissue interactions that complicate understanding of drug effects. We present an ex vivo human osteochondral model of PTOA to investigate disease effects on cartilage and bone homeostasis and discover biomarkers for disease progression and drug efficacy. METHODS: Human osteochondral explants were harvested from normal (Collins grade 0-1) ankle talocrural joints of human donors (2 female, 5 male, ages 23-70). After pre-equilibration, osteochondral explants were treated with a single-impact mechanical injury and TNF-α, IL-6, and sIL-6R ± 100 nM Dex for 21 days and media collected every 2-3 days. Chondrocyte viability, tissue DNA content, and glycosaminoglycan (sGAG) percent loss to the media were assayed and compared to untreated controls using a linear mixed effects model. Mass spectrometry analysis was performed for both cartilage tissue and pooled culture medium, and the statistical significance of protein abundance changes was determined with the R package limma and empirical Bayes statistics. Partial least squares regression analyses of sGAG loss and Dex attenuation of sGAG loss against proteomic data were performed. RESULTS: Injury and cytokine treatment caused an increase in the release of matrix components, proteases, pro-inflammatory factors, and intracellular proteins, while tissue lost intracellular metabolic proteins, which was mitigated with the addition of Dex. Dex maintained chondrocyte viability and reduced sGAG loss caused by injury and cytokine treatment by 2/3 overall, with donor-specific differences in the sGAG attenuation effect. Biomarkers of bone metabolism had mixed effects, and collagen II synthesis was suppressed with both disease and Dex treatment by 2- to 5-fold. Semitryptic peptides associated with increased sGAG loss were identified. Pro-inflammatory humoral proteins and apolipoproteins were associated with lower Dex responses. CONCLUSIONS: Catabolic effects on cartilage tissue caused by injury and cytokine treatment were reduced with the addition of Dex in this osteochondral PTOA model. This study presents potential peptide biomarkers of early PTOA progression and Dex efficacy that can help identify and treat patients at risk of PTOA.

Novel missense ACAN gene variants linked to familial osteochondritis dissecans cluster in the C-terminal globular domain of aggrecan.

The cartilage aggrecan proteoglycan is crucial for both skeletal growth and articular cartilage function. A number of aggrecan (ACAN) gene variants have been linked to skeletal disorders, ranging from short stature to severe chondrodyplasias. Osteochondritis dissecans is a disorder where articular cartilage and subchondral bone fragments come loose from the articular surface. We previously reported a missense ACAN variant linked to familial osteochondritis dissecans, with short stature and early onset osteoarthritis, and now describe three novel ACAN gene variants from additional families with this disorder. Like the previously described variant, these are autosomal dominant missense variants, resulting in single amino acid residue substitutions in the C-type lectin repeat of the aggrecan G3 domain. Functional studies showed that neither recombinant variant proteins, nor full-length variant aggrecan proteoglycan from heterozygous patient cartilage, were secreted to the same level as wild-type aggrecan. The variant proteins also showed decreased binding to known cartilage extracellular matrix ligands. Mapping these and other ACAN variants linked to hereditary skeletal disorders showed a clustering of osteochondritis dissecans-linked variants to the G3 domain. Taken together, this supports a link between missense ACAN variants affecting the aggrecan G3 domain and hereditary osteochondritis dissecans.

The leucine-rich repeat protein PRELP binds fibroblast cell-surface proteoglycans and enhances focal adhesion formation.

PRELP (proline/arginine-rich end leucine-rich repeat protein) is a member of the leucine-rich repeat (LRR) family of extracellular matrix proteins in connective tissue. In contrast with other members of the family, the N-terminal domain of PRELP has a high content of proline and positively charged amino acids. This domain has previously been shown to bind chondrocytes and to inhibit osteoclast differentiation. In the present study, we show that PRELP mediates cell adhesion by binding to cell-surface glycosaminoglycans (GAGs). Thus, rat skin fibroblasts (RSFs) bound to full-length PRELP and to the N-terminal part of PRELP alone, but not to truncated PRELP lacking the positively charged N-terminal region. Cell attachment to PRELP was inhibited by addition of soluble heparin or heparan sulfate (HS), by blocking sulfation of the fibroblasts or by treating the cells with a combination of chondroitinase and heparinase. Using affinity chromatography, we identified syndecan-1, syndecan-4 and glypican-1 as cell-surface proteoglycans (PGs) binding to the N-terminal part of PRELP. Finally, we show that the N-terminal domain of PRELP in combination with the integrin-binding domain of fibronectin, but neither of the fragments alone, induced fibroblast focal adhesion formation. These findings provide support for a role of the N-terminal region of PRELP as an important regulator of cell adhesion and behaviour, which may be of importance in pathological conditions.

Is there a bidirectional link between insomnia and burnout? A prospective study in the Swedish workforce.

BACKGROUND: Insomnia and burnout have been suggested to form a bidirectional association. PURPOSE: The aim of this study was to investigate whether there is a bidirectional relationship between insomnia and burnout over the course of a year among individuals in the workforce. METHOD: This study employed a prospective design, where a randomly selected sample from the general population (20-60 year; N = 1,812) filled out a survey on insomnia and burnout. In employed participants (n = 1,258), the associations between insomnia and three dimensions of burnout (Maslach Burnout Inventory-General Survey) were examined while controlling for age, gender, anxiety, and depression. RESULTS: The bivariate correlations between insomnia and the three burnout dimensions were significant at a low level (η 0.12-0.29). The longitudinal analyses demonstrated that insomnia was not associated with the incidence of burnout and vice versa. However, insomnia was demonstrated to increase the risk for the persistence of emotional exhaustion (OR = 3.02). Further, insomnia was not associated with the persistence of professional efficacy and cynicism, and burnout was not related to the persistence of insomnia. CONCLUSION: In summary, this investigation demonstrated that insomnia and burnout are not bidirectionally related in the working population. While insomnia was linked to the maintenance of the central part of burnout, burnout was not related to future insomnia.

A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans.

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo.

Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization.

The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10-12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10-12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).

A bidirectional relationship between anxiety and depression, and insomnia? A prospective study in the general population.

OBJECTIVE: The purpose of this study was to examine whether there is a bidirectional relationship between, on one hand, anxiety and depression and, on the other hand, insomnia over the course of a year. METHODS: A randomly selected sample of 3000 participants from the general population filled out a baseline survey (N=1812) and a 1-year follow-up survey (N=1498) on anxiety, depression, and insomnia. RESULTS: On cross-sectional analyses, bivariate correlations showed that anxiety, depression, and insomnia were significantly intercorrelated (varphi=.31-.54). On prospective analyses, logistic regression analyses demonstrated that anxiety at baseline [odds ratio (OR)=4.27 (8% of variance)] and depression at baseline [OR=2.28 (2% of variance)] were related to new cases of insomnia on follow-up. Furthermore, insomnia at baseline was related to new episodes of high anxiety and high depression on follow-up [OR=2.30 (2% of variance) and OR=3.51 (4% of variance), respectively]. CONCLUSION: Evidence suggests that there is a bidirectional relationship between, on one hand, anxiety and depression and, on the other hand, insomnia. This suggests that anxiety, depression, and insomnia are intertwined over time, implying implications for theoretical conceptualizations and interventions.

A randomized controlled trial of exposure in vivo for patients with spinal pain reporting fear of work-related activities.

BACKGROUND: Pain-related fear is related to disability in persistent pain conditions. Exposure treatment has been reported to be of great benefit in replicated single case experiments. AIM: To evaluate the effects of exposure in vivo on fear and function in patients with persistent pain and work disability. METHOD: We recruited 46 patients suffering from long-term back pain and reduced function, who also were deemed fearful according to standardized measures. Participants were randomized into either an exposure plus usual treatment or waiting list control plus usual treatment group. After the waiting period the control group crossed over and received the exposure treatment. RESULTS: Between group comparisons showed a significantly better result for the exposure group on function, but not for fear or pain and effect sizes were modest (function=.6; fear=.4; pain=.1). When the control group crossed over to treatment significant treatment effects were noted for fear and function. For all patients treated, the pre to post-treatment effect sizes were large (function=.7; fear=1.1; pain=.9). There were 12 dropouts (8 in exposure and 4 in the control) during the first treatment phase and an additional 4 when the control group crossed over to exposure. CONCLUSIONS: Compared to a group receiving usual treatment and waiting for exposure, the exposure in vivo group demonstrated a significantly larger improvement on function. Overall exposure had moderate effects on function, fear and pain intensity. We conclude that exposure may be important in treatment, but is not recommended as a "stand alone" adjunct to usual treatment.

Chondroitin sulfate perlecan enhances collagen fibril formation. Implications for perlecan chondrodysplasias.

Inactivation of the perlecan gene leads to perinatal lethal chondrodysplasia. The similarity to the phenotypes of the Col2A1 knock-out and the disproportionate micromelia mutation suggests perlecan involvement in cartilage collagen matrix assembly. We now present a mechanism for the defect in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional to the content of the 4,6-disulfated disaccharide in the different cartilage extracts, with growth plate cartilage glycosaminoglycan being the most efficient enhancer. These findings demonstrate a role for perlecan chondroitin sulfate side chains in cartilage extracellular matrix assembly and provide an explanation for the perlecan-null chondrodysplasia.

Burnout in the working population: relations to psychosocial work factors.

This study investigated levels of burnout in the general population irrespective of occupation and relations between burnout and psychosocial work factors. A cross-sectional survey featuring sleep problems, psychological distress, burnout (Maslach Burnout Inventory-General Survey), and psychosocial factors at work, was mailed to a random sample of 3,000 participants, aged 20-60. Response rate was 61%. A high level (18%), a low level (19%), and an intermediate group (63%) for burnout were constructed. The high level group was associated with those who were > 50 years old, women, those experiencing psychological distress, and those with a poor psychosocial work climate. The analyses on variables significant in previous analyses showed that the high level group was strongly related to high demands, low control, lack of social support, and disagreeing about values at the workplace even when accounting for age, gender, and psychological distress. We conclude that psychosocial work factors are important in association to burnout regardless of occupation.