Influence of knee osteoarthritis on exercise capacity and quality of life in obese adults

Objective: The objective was to determine whether knee osteoarthritis (OA) reduces exercise ambulatory capacity and impairs quality of life (QOL) in obese individuals. Research Methods and Procedures: There were 56 subjects, with and without knee OA, who were obese. The subjects were evaluated with anthropometric measurements, a body composition assessment, maximal cardiopulmonary exercise test, 6‐minute walk test (6‐MWT), perceived exertion (RPE), self‐reported disability [Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC)], and the Medical Outcomes Study Short Form 36 (SF‐36). Results: VO_2peak was significantly higher in the controls when compared with the patients (mean ± standard deviation, 1.584 ± 0.23 L/kg per min vs. 0.986 ± 0.20 L/kg per min; p < 0.001). Obese subjects without knee OA walked a significantly longer distance in the 6‐MWT than obese patients with knee OA (p < 0.001). We also observed significant negative correlation between Vo ₂max and RPE, WOMAC pain and physical limitation, and bodily pain and general health domains of short‐form 36. Discussion: Knee OA reduces exercise and ambulatory capacity and impairs QOL in obese individuals. RPE, WOMAC pain, and SF‐36 items might provide information about exercise capacity in the obese subjects with knee OA. Our study confirms that exercise capacity and QOL might be improved by energetic and intensive treatment of pain resulting from knee OA.     

Association of chemerin levels in synovial fluid with the severity of knee osteoarthritis

Context: Chemerin has been implicated to be correlated with inflammation.

Objective: This study aims to determine the association of chemerin levels in serum and synovial fluid (SF) with the disease severity of patients with knee Osteoarthritis (OA).

Methods: 124 patients with knee OA and 76 healthy controls were enrolled in this study.

Results: Chemerin levels in serum were significant higher with regard to paired SF. Chemerin levles in SF of knee OA patients were correlated with disease severity evaluated by KL grading criteria.

Conclusion: Chemerin levels may be involved in the pathophysiology of the development and progression of OA.     

Quadriceps activity and physical activity profiles over long durations in patients with osteoarthritis of the knee and controls

Exercise is recommended as a non-pharmacological, non-invasive intervention for osteoarthritis (OA) of the knee. Physiological data concerning the duration and intensity of muscle activity or physical activity profiles during normal daily activity for this population is lacking. Our aim was to explore this using surface Electromyography (EMG) and accelerometer-based activity monitoring. Thirty-four patients with knee OA, mean (SD) age 63.2 (9.8) years and 30 aged-matched asymptomatic controls 64.1 (10.9) years participated. The duration of recording was similar in knee OA and control groups – median (IRQ range) 12:34 (10:11–14:17) h and 13:10 (12:02–14:56) h, respectively (p = 0.514). VM and VL were quiescent for 81.8 (75.3–91.0) to 89.2 (81.5–94.7)% of the time. VM was active for significantly longer durations than VL in the highest intensity band for those with knee OA (p = 0.00), and for longer durations in those with knee OA compared to controls (p = 0.027). The median (IRQ range) percentage of the total recording time spent in an upright posture was 32.4 (28.3–43.9)% and 38.8 (33.6–45.8)%, and time spent stepping or walking was 12.7 (9.7–16.4)% and 16.0 (11.9–19.6)% for those with knee OA and controls respectively. These novel data may prove useful for designing therapeutic exercise programmes and lifestyle changes for those with knee OA.     

A novel approach to measure the contribution of matrix metalloproteinase in the overall net proteolytic activity present in synovial fluids of patients with arthritis

Despite decades of research, only a very limited number of matrix metalloproteinase (MMP) inhibitors have been successful in clinical trials of arthritis. One of the central problems associated with this failure may be our inability to monitor the local activity of proteases in the joints since the integrity of the extracellular matrix results from an equilibrium between noncovalent, 1:1 stoichiometric binding of protease inhibitors to the catalytic site of the activated forms of the enzymes. In the present work, we have measured by flow cytometry the net proteolytic activity in synovial fluids (SF) collected from 95 patients with osteoarthritis and various forms of inflammatory arthritis, including rheumatoid arthritis, spondyloarthropathies, and chronic juvenile arthritis. We found that SF of patients with inflammatory arthritis had significantly higher levels of proteolytic activity than those of osteoarthritis patients. Moreover, the overall activity in inflammatory arthritis patients correlated positively with the number of infiltrated leukocytes and the serum level of C-reactive protein. No such correlations were found in osteoarthritis patients. Members of the MMP family contributed significantly to the proteolytic activity found in SF. Small-molecular-weight MMP inhibitors were indeed effective for inhibiting proteolytic activity in SF, but their effectiveness varied greatly among patients. Interestingly, the contribution of MMPs decreased in patients with very high proteolytic activity, and this was due both to a molar excess of tissue inhibitor of MMP-1 and to an increased contribution of other proteolytic enzymes. These results emphasize the diversity of the MMPs involved in arthritis and, from a clinical perspective, suggest an interesting alternative for testing the potential of new protease inhibitors for the treatment of arthritis.     

Plasma and synovial fluid inflammatory cytokine profiles in primary knee osteoarthritis

Objective: The objective of this study is to compare inflammatory cytokine levels in primary knee osteoarthritis (OA) patients and healthy controls.

Methods: A total of 32 knee OA patients and 14 healthy controls were enrolled. A multiplex immunoassay was utilized for 10 cytokines in plasma and synovial fluid.

Results: Plasma IL-2, IL-4, and IL-6 concentrations were significantly greater in knee OA patients than controls. Moreover, both plasma IL-4 and IL-6 were positively correlated with the radiographic severity of knee OA.

Conclusions: Plasma IL-4 and IL-6 may serve as biomarkers reflecting the severity of OA.     

Proteomic profiling and functional characterization of early and late shoulder osteoarthritis

Introduction

The development of effective treatments for osteoarthritis (OA) has been hampered by a poor understanding of OA at the cellular and molecular levels. Emerging as a disease of the ''whole joint’, the importance of the biochemical contribution of various tissues, including synovium, bone and articular cartilage, has become increasingly significant. Bathing the entire joint structure, the proteomic analysis of synovial fluid (SF) from osteoarthritic shoulders offers a valuable ''snapshot’ of the biologic environment throughout disease progression. The purpose of this study was to identify differentially expressed proteins in early and late shoulder osteoarthritic SF in comparison to healthy SF.

Methods

A quantitative ¹⁸O labeling proteomic approach was employed to identify the dysregulated SF proteins in early (n = 5) and late (n = 4) OA patients compared to control individuals (n = 5). In addition, ELISA was used to quantify six pro-inflammatory and two anti-inflammatory cytokines.

Results

Key results include a greater relative abundance of proteins related to the complement system and the extracellular matrix in SF from both early and late OA. Pathway analyses suggests dysregulation of the acute phase response, liver x receptor/retinoid x receptor (LXR/RXR), complement system and coagulation pathways in both early and late OA. The network related to lipid metabolism was down-regulated in both early and late OA. Inflammatory cytokines including interleukin (IL) 6, IL 8 and IL 18 were up-regulated in early and late OA.

Conclusions

The results suggest a dysregulation of wound repair pathways in shoulder OA contributing to the presence of a ''chronic wound’ that progresses irreversibly from early to later stages of OA. Protease inhibitors were downregulated in late OA suggesting uncontrolled proteolytic activity occurring in late OA. These results contribute to the theory that protease inhibitors represent promising therapeutic agents which could limit proteolytic activity that ultimately leads to cartilage destruction.     

Neuromuscular alterations during walking in persons with moderate knee osteoarthritis.

This paper compared the neuromuscular responses during walking between those with early-stage knee osteoarthritis (OA) to asymptomatic controls. The rationale for studying those with mild to moderate knee OA was to determine the alterations in response to dynamic loading that might be expected before severe pain, joint space narrowing and joint surface changes occur. We used pattern recognition techniques to explore both amplitude and shape changes of the surface electromyograms recorded from seven muscles crossing the knee joint of 40 subjects with knee OA and 38 asymptomatic controls during a walking task. The principal patterns for each muscle grouping explained over 83% of the variance in the waveforms. This result supported the notion that the main neuromuscular patterns were similar between asymptomatic controls and those with OA, reflecting the specific roles of the major muscles during walking. ANOVA revealed significant (p<0.05) differences in the principal pattern scores reflecting both amplitude and shape alterations in the OA group and among muscles. These differences captured subtle changes in the neuromuscular responses of the subjects with OA throughout different phases of the gait cycle and most likely reflected changes in the mechanical environment (joint loading, instability) and pain. The subjects with OA attempted to increase activity of the lateral sites and reduce activity in the medial sites, having minimal but prolonged activity during late stance. Therefore, alterations in neuromuscular responses were found even in this high functioning group with moderate knee OA.     

An in vivo investigation of the initiation and progression of subchondral cysts in a rodent model of secondary osteoarthritis

Introduction  

Subchondral bone cysts (SBC) have been identified in patients with knee osteoarthritis (OA) as a cause of greater pain, loss of cartilage and increased chance of joint replacement surgery. Few studies monitor SBC longitudinally, and clinical research using three-dimensional imaging techniques, such as magnetic resonance imaging (MRI), is limited to retrospective analyses as SBC are identified within an OA patient cohort. The purpose of this study was to use dual-modality, preclinical imaging to monitor the initiation and progression of SBC occurring within an established rodent model of knee OA.     

Identification of psoriatic arthritis mediators in synovial fluid by quantitative mass spectrometry

Background

Synovial fluid (SF) is a dynamic reservoir for proteins originating from the synovial membrane, cartilage, and plasma, and may therefore reflect the pathophysiological conditions that give rise to arthritis. Our goal was to identify and quantify protein mediators of psoriatic arthritis (PsA) in SF.

Methods

Age and gender-matched pooled SF samples from 10 PsA and 10 controls [early osteoarthritis (OA)], were subjected to label-free quantitative proteomics using liquid chromatography coupled to mass spectrometry (LC-MS/MS), to identify differentially expressed proteins based on the ratios of the extracted ion current of each protein between the two groups. Pathway analysis and public database searches were conducted to ensure these proteins held relevance to PsA. Multiplexed selected reaction monitoring (SRM) assays were then utilized to confirm the elevated proteins in the discovery samples and in an independent set of samples from patients with PsA and controls.

Results

We determined that 137 proteins were differentially expressed between PsA and control SF, and 44 were upregulated. The pathways associated with these proteins were acute-phase response signalling, granulocyte adhesion and diapedesis, and production of nitric oxide and reactive oxygen species in macrophages. The expression of 12 proteins was subsequently quantified using SRM assays.

Conclusions

Our in-depth proteomic analysis of the PSA SF proteome identified 12 proteins which were significantly elevated in PsA SF compared to early OA SF. These proteins may be linked to the pathogenesis of PsA, as well serve as putative biomarkers and/or therapeutic targets for this disease.     

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.     

Analyzing protease specificity and detecting in vivo proteolytic events using tandem mass spectrometry

Although trypsin remains the most commonly used protease in MS, other proteases may be employed for increasing peptide coverage or generating overlapping peptides. Knowledge of the accurate specificity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when label‐free MS is used to discover in vivo proteolytic cleavages. Since in vivo cleavages are inferred by subtracting digestion‐induced cleavages from all observed cleavages, it is important to ensure that the specificity rule used to identify digestion‐induced cleavages are broad enough to capture even minor cleavages produced in digestion, to avoid erroneously identifying them as in vivo cleavages. In this study, we describe MS‐Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage using label‐free MS. The tool is used in conjunction with digestion by trypsin and three other proteases, whose specificity rules are revised and extended before inferring proteolytic cleavages. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome‐wide scale.     

OPN gene polymorphisms influence the risk of knee OA and OPN levels in synovial fluid in a Chinese population

Introduction

A body of studies suggests the role of osteopontin (OPN) in onset and development of osteoarthritis (OA), however, the association between OPN polymorphisms and OA susceptibility as well as its clinical features has not been reported.

Methods

A total of 750 patients with primary knee OA and 794 healthy volunteer were enrolled as controls. Both OA and control groups were interviewed to obtain demographic and clinical data. Three polymorphisms of OPN gene, namely, -156GG/G, -443C/T and -66T/G were determined. The levels of the full length and the thrombin-cleaved OPN in synovial fluid (SF) from OA subjects were measured.

Results

We found the polymorphisms of the -443C/T and the -66/T/G were significantly associated with the OA risk and the radiographic severity. The -443TT and -66GG showed protective effect against developing OA and were associated with lower Kellgren-Lawrence grade. Besides, the polymorphisms of -443C/T and -66T/G significantly affected the thrombin-cleaved OPN levels in SF from OA subjects. Subjects with -443TT and -66GG genotypes had lower thrombin-cleaved OPN levels in SF. The thrombin-cleaved OPN levels in SF were positively correlated to the radiographic severity of OA.

Conclusions

Our findings suggest that certain OPN gene polymorphisms may be used as molecular markers for the susceptibility and severity of OA.     

Influence of BMI on Health‐related Quality of Life: Comparison Between an Obese Adult Cohort and Age‐matched Population Norms

The aim of this study was to determine health‐related quality of life and fatigue measures in obese subjects and to compare scores with age‐ and gender‐matched population norms. A total of 163 obese subjects were recruited from laparoscopic‐adjustable gastric banding or exercise and diet weight loss programs between March 2006 and December 2007. All subjects completed the Medical Outcomes Study Short Form 36 (SF‐36), Assessment of Quality of Life (AQoL), and Multidimensional Assessment of Fatigue (MAF) questionnaires. One‐sample t‐tests were used to compare transformed scores with age‐ and gender‐matched population norms and controls. Obese subjects have significantly lower SF‐36 physical and emotional component scores, significantly lower AQoL utility scores and significantly higher fatigue scores compared to age‐matched population norms. Within the study cohort, the SF‐36 physical functioning, role physical and bodily pain scores, and AQoL utility index were even lower in subjects with clinical knee osteoarthritis (OA). However, obese individuals without OA still had significantly lower scores compared to population norms. Obesity is associated with impaired health‐related quality of life and disability as measured by the SF‐36, AQoL, and fatigue score (MAF) compared to matched population norms.     

Matrix assisted laser desorption ionization mass spectrometry imaging identifies markers of ageing and osteoarthritic cartilage

Introduction

Cartilage protein distribution and the changes that occur in cartilage ageing and disease are essential in understanding the process of cartilage ageing and age related diseases such as osteoarthritis. The aim of this study was to investigate the peptide profiles in ageing and osteoarthritic (OA) cartilage sections using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI).

Methods

The distribution of proteins in young, old and OA equine cartilage was compared following tryptic digestion of cartilage slices and MALDI-MSI undertaken with a MALDI SYNAPT™ HDMS system. Protein identification was undertaken using database searches following multivariate analysis. Peptide intensity differences between young, ageing and OA cartilage were imaged with Biomap software. Analysis of aggrecanase specific cleavage patterns of a crude cartilage proteoglycan extract were used to validate some of the differences in peptide intensity identified. Immunohistochemistry studies validated the differences in protein abundance.

Results

Young, old and OA equine cartilage was discriminated based on their peptide signature using discriminant analysis. Proteins including aggrecan core protein, fibromodulin, and cartilage oligomeric matrix protein were identified and localised. Fibronectin peptides displayed a stronger intensity in OA cartilage. Age-specific protein markers for collectin-43 and cartilage oligomeric matrix protein were identified. In addition potential fibromodulin and biglycan peptides targeted for degradation in OA were detected.

Conclusions

MALDI-MSI provided a novel platform to study cartilage ageing and disease enabling age and disease specific peptides in cartilage to be elucidated and spatially resolved.     

Lack of association of a variable number of aspartic acid residues in the asporin gene with osteoarthritis susceptibility: case-control studies in Spanish Caucasians

A recent genetic association study has identified a microsatellite in the coding sequence of the asporin gene as a susceptibility factor for osteoarthritis (OA). Alleles of this microsatellite determine the variable number of aspartic acid residues in the amino-terminal end of the asporin protein. Asporin binds directly to the growth factor transforming growth factor beta and inhibits its anabolic effects in cartilage, which include stimulation of collagen and aggrecan synthesis. The OA-associated allele, with 14 aspartic acid residues, inhibits the anabolic effects of transforming growth factor beta more strongly than other asporin alleles, leading to increased OA liability. We have explored whether the association found in several cohorts of Japanese hip OA and knee OA patients was also present in Spanish Caucasians. We studied patients that had undergone total joint replacement for primary OA in the hip (n = 303) or the knee (n = 188) and patients with hand OA (n = 233), and we compared their results with controls (n = 294) lacking overt OA clinical symptoms. No significant differences were observed in any of the multiple comparisons performed, which included global tests of allele frequency distributions and specific comparisons as well as stratification by affected joint and by sex. Our results, together with reports from the United Kingdom and Greece, indicate that the stretch of aspartic acid residues in asporin is not an important factor in OA susceptibility among European Caucasians. It remains possible that lifestyle, environmental or genetic differences allow for an important effect of asporin variants in other ethnic groups as has been reported in the Japanese, but this should be supported by additional studies.     

Proteomics Profiling of Human Synovial Fluid Suggests Increased Protein Interplay in Early-Osteoarthritis (OA) That Is Lost in Late-Stage OA

The underlying molecular mechanisms in osteoarthritis (OA) development are largely unknown. This study explores the proteome and the pairwise interplay of proteins in synovial fluid from patients with late-stage knee OA (arthroplasty), early knee OA (arthroscopy due to degenerative meniscal tear), and from deceased controls without knee OA. Synovial fluid samples were analyzed using state-of-the-art mass spectrometry with data-independent acquisition. The differential expression of the proteins detected was clustered and evaluated with data mining strategies and a multilevel model. Group-specific slopes of associations were estimated between expressions of each pair of identified proteins to assess the co-expression (i.e., interplay) between the proteins in each group. More proteins were increased in early-OA versus controls than late-stage OA versus controls. For most of these proteins, the fold changes between late-stage OA versus controls and early-stage OA versus controls were remarkably similar suggesting potential involvement in the OA process. Further, for the first time, this study illustrated distinct patterns in protein co-expression suggesting that the interplay between the protein machinery is increased in early-OA and lost in late-stage OA. Further efforts should focus on earlier stages of the disease than previously considered.     

Association Between Single Nucleotide Polymorphisms of Asporin (ASPN) and BMP5 with the Risk of Knee Osteoarthritis in a Chinese Han Population

The aim of this study was to investigate associations between single nucleotide polymorphisms rs13301537 in asporin (ASPN) and rs373444 in the bone morphogenetic protein 5 (BMP5) gene with knee osteoarthritis (OA) susceptibility in a Chinese Han population. ASPN rs13301537 and BMP5 rs373444 polymorphisms were genotyped in patients with knee OA and age- and sex-matched OA-free controls from a Chinese Han population. A total of 510 patients with knee OA and 520 controls were enrolled in the study. CT and CC genotypes of rs13301537, and variant C, were associated with a significantly increased risk of knee OA. On stratification analysis, the association between the risk of OA and rs13301537 CT heterozygotes compared with TT homozygotes was stronger in females and those aged >65 years. In contrast, the CT and CC genotypes of rs373444 in BMP5 were not significantly associated with the risk of knee OA, even after further stratification analysis according to age or sex. Our results showed that ASPN rs13301537 T to C change and variant C genotype may contribute to knee OA risk in a Chinese Han population.     

Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis

An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.     

Association Between Single Nucleotide Polymorphisms of DOT1L Gene and Risk of Knee Osteoarthritis in a Chinese Han Population

To investigate associations between single nucleotide polymorphisms rs12982744 and rs12459350 in the DOT1L gene and knee osteoarthritis (OA) susceptibility in a Chinese Han population. DOT1L rs12982744 and rs12459350 polymorphisms were genotyped in patients with knee OA and age- and sex-matched OA-free controls from a Chinese Han population. A total of 605 patients with knee OA and 615 controls were enrolled in the study. GC and CC genotypes of rs12982744, and variant C, were associated with a significantly increased risk of knee OA. On stratification analysis, the association between the risk of OA and rs12982744 GC heterozygotes compared with GG homozygotes was stronger in females and those aged >65 years. In contrast, the GA and AA genotypes of rs12459350 were not significantly associated with the risk of knee OA, even after further stratification analysis according to age or sex. Our results showed that DOT1L rs12982744 G to C change and variant C genotype may contribute to knee OA risk in a Chinese Han population.