Dataset Information

Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis.

ABSTRACT: Osteoarthritis is a common and progressive joint disorder. Despite its widespread, in clinical practice only late phases of osteoarthritis that are characterized by severe joint damage are routinely detected. Since osteoarthritis cannot be cured but relatively well managed, an early diagnosis and thereby early onset of disease management would lower the burden of osteoarthritis. Here we evaluated if biophysical parameters of small synovial fluid samples extracted by single molecule microscopy can be linked to joint damage. In healthy synovial fluid (ICRS-score??2) (4.5??m(2)/s, N?=?16). More strikingly, the diffusion coefficient of hyaluronan in healthy synovial fluid was on average 30% slower than expected by sample viscosity. This effect was diminished or missing in samples from patients with joint damage. Since single molecule microscopy needs only microliters of synovial fluid to extract the viscosity and the specific diffusion coefficient of hyaluronan this method could be of use as diagnostic tool for osteoarthritis.

SUBMITTER: Kohlhof H

PROVIDER: S-EPMC4751503 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis.

Kohlhof Hendrik H Gravius Sascha S Kohl Sandro S Ahmad Sufian S SS Randau Thomas T Schmolders Jan J Rommelspacher Yorck Y Friedrich Max M Kaminski Tim P TP

Scientific reports 20160212

Osteoarthritis is a common and progressive joint disorder. Despite its widespread, in clinical practice only late phases of osteoarthritis that are characterized by severe joint damage are routinely detected. Since osteoarthritis cannot be cured but relatively well managed, an early diagnosis and thereby early onset of disease management would lower the burden of osteoarthritis. Here we evaluated if biophysical parameters of small synovial fluid samples extracted by single molecule microscopy ca ...[more]

PMID: 26868769

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Project description:BACKGROUND:Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography. RESULTS:We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples. CONCLUSIONS:We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.

Project description:Surgical transection of the anterior cruciate ligament (ACL) in the porcine model leads to posttraumatic osteoarthritis if left untreated. However, a recently developed surgical treatment, bridge-enhanced ACL repair, prevents further cartilage damage. Since the synovial fluid bathes all the intrinsic structures of knee, we reasoned that a comparative analysis of synovial fluid protein contents could help to better understand the observed chondroprotective effects of the bridge-enhanced ACL repair. We hypothesized that post-surgical changes in the synovial fluid proteome would be different in the untreated and repaired knees, and those changes would correlate with the degree of cartilage damage. Thirty adolescent Yucatan mini-pigs underwent unilateral ACL transection and were randomly assigned to either no further treatment (ACLT, n = 14) or bridge-enhanced ACL repair (BEAR, n = 16). We used an isotopically labeled high resolution LC MS/MS-based proteomics approach to analyze the protein profile of synovial fluid at 6 and 12 months after ACL transection in untreated and repaired porcine knees. A linear mixed effect model was used to compare the normalized protein abundance levels between the groups at each time point. Bivariate linear regression analyses were used to assess the correlations between the macroscopic cartilage damage (total lesion area) and normalized abundance levels of each of the identified secreted proteins. There were no significant differences in cartilage lesion area or quantitative abundance levels of the secreted proteins between the ACLT and BEAR groups at 6 months. However, by 12 months, greater cartilage damage was seen in the ACLT group compared to the BEAR group (p = 0.005). This damage was accompanied by differences in the abundance levels of secreted proteins, with higher levels of Vitamin K-dependent protein C (p = 0.001), and lower levels of Apolipoprotein A4 (p = 0.021) and Cartilage intermediate layer protein 1 (p = 0.049) in the ACLT group compared to the BEAR group. There were also group differences in the secreted proteins that significantly changed in abundance between 6 and 12 months in ACLT and BEAR knees. Increased concentration of Ig lambda-1 chain C regions and decreased concentration of Hemopexin, Clusterin, Coagulation factor 12 and Cartilage intermediate layer protein 1 were associated with greater cartilage lesion area. In general, ACLT knees had higher concentrations of pro-inflammatory proteins and lower concentrations of anti-inflammatory proteins than BEAR group. In addition, the ACLT group had a lower and declining synovial concentrations of CILP, in contrast to a consistently high abundance of CILP in repaired knees. These differences suggest that the knees treated with bridge-enhanced ACL repair may be maintaining an environment that is more protective of the extracellular matrix, a function which is not seen in the ACLT knees.

Project description:Changes in cellular metabolism have been implicated in mediating the activated fibroblast phenotype in a number of chronic inflammatory disorders, including pulmonary fibrosis, renal disease and rheumatoid arthritis. The aim of this study was therefore to characterise the metabolic profile of synovial joint fluid and synovial fibroblasts under both basal and inflammatory conditions in a cohort of obese and normal-weight hip OA patients. Furthermore, we sought to ascertain whether modulation of a metabolic pathway in OA synovial fibroblasts could alter their inflammatory activity. Synovium and synovial fluid was obtained from hip OA patients, who were either of normal-weight or obese and were undergoing elective joint replacement surgery. The synovial fluid metabolome was determined by 1H NMR spectroscopy. The metabolic profile of isolated synovial fibroblasts in vitro was characterised by lactate secretion, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF Analyser. The effects of a small molecule pharmacological inhibitor and siRNA targeted at glutaminase-1 (GLS1) were assessed to probe the role of glutamine metabolism in OA synovial fibroblast function. Obese OA patient synovial fluid (n = 5) exhibited a different metabotype, compared to normal-weight patient fluid (n = 6), with significantly increased levels of 1, 3-dimethylurate, N-Nitrosodimethylamine, succinate, tyrosine, pyruvate, glucose, glycine and lactate, and enrichment of the glutamine-glutamate metabolic pathway, which correlated with increasing adiposity. In vitro, isolated obese OA fibroblasts exhibited greater basal lactate secretion and aerobic glycolysis, and increased mitochondrial respiration when stimulated with pro-inflammatory cytokine TNFα, compared to fibroblasts from normal-weight patients. Inhibition of GLS1 attenuated the TNFα-induced expression and secretion of IL-6 in OA synovial fibroblasts. These findings suggest that altered cellular metabolism underpins the inflammatory phenotype of OA fibroblasts, and that targeted inhibition of glutamine-glutamate metabolism may provide a route to reducing the pathological effects of joint inflammation in OA patients who are obese.

Project description:The lack of early diagnostic maneuvers and effective pharmacotherapy for osteoarthritis (OA) is predominantly attributed to current limited understanding of its pathogenesis. In the present study, the alteration of synovial fluid (SF) proteome in OA were analyzed using SWATH‑mass spectrometry (SWATH‑MS) to further elucidate the pathogenesis of OA. SF samples were collected from 10 OA and 10 rheumatoid arthritis (RA) patients undergoing arthroplasty and 10 patients undergoing arthroscopy for traumatic arthritis (meniscus injury without cartilage lesion). According to the Kellgren‑Lawrence (KL) radiographic grading criteria, all the OA and RA patients were classified as KL grade 4. SWATH‑MS was applied to identify differentially expressed proteins specifically regulated in OA. Differentially expressed proteins identified by SWATH‑MS were subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. Proteins of interest were quantified by enzyme‑linked immunosorbent assay (ELISA) following identification. With the use of SWATH‑MS, 131 proteins were identified to be differentially expressed in OA, of which 93 corresponded to upregulation and 38 to downregulation. Complement C1r was the most significantly upregulated protein in OA. Twenty‑eight out of the 131 proteins were specifically regulated in OA, of which 17 were upregulated and 11 were downregulated. Dickkopf‑related protein 2 (DKK2) was one of the proteins specifically upregulated in OA. Furthermore, KEGG pathway annotation indicated that differentially expressed proteins in OA were enriched in complement and coagulation cascades. ELISA indicated that OA severity was positively correlated with the levels of complement C1r (r=0.549; P<0.001) and DKK2 (r=0.79; P<0.001) in the SF. The results indicate that complement and coagulation cascades are involved in the pathogenesis of OA. Differentially expressed proteins, including complement C1r and DKK2 may be used as potential biomarkers or drug targets, which may facilitate with intervention of OA.

Dataset Information

Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis.

Publications

Single Molecule Microscopy Reveals an Increased Hyaluronan Diffusion Rate in Synovial Fluid from Knees Affected by Osteoarthritis.

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