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:INTRODUCTION: MicroRNAs (miRNAs), endogenous small noncoding RNAs regulating the activities of target mRNAs and cellular processes, are present in human plasma in a stable form. In this study, we investigated whether miRNAs are also stably present in synovial fluids and whether plasma and synovial fluid miRNAs could be biomarkers of rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: We measured concentrations of miR-16, miR-132, miR-146a, miR-155 and miR-223 in synovial fluid from patients with RA and OA, and those in plasma from RA, OA and healthy controls (HCs) by quantitative reverse transcription-polymerase chain reaction. Furthermore, miRNAs in the conditioned medium of synovial tissues, monolayer fibroblast-like synoviocytes, and mononuclear cells were examined. Correlations between miRNAs and biomarkers or disease activities of RA were statistically examined. RESULTS: Synovial fluid miRNAs were present and as stable as plasma miRNAs for storage at -20 degrees C and freeze-thawing from -20 degrees C to 4 degrees C. In RA and OA, synovial fluid concentrations of miR-16, miR-132, miR-146a, and miR-223 were significantly lower than their plasma concentrations, and there were no correlation between plasma and synovial fluid miRNAs. Interestingly, synovial tissues, fibroblast-like synoviocytes, and mononuclear cells secreted miRNAs in distinct patterns. The expression patterns of miRNAs in synovial fluid of OA were similar to miRNAs secreted by synovial tissues. Synovial fluid miRNAs of RA were likely to originate from synovial tissues and infiltrating cells. Plasma miR-132 of HC was significantly higher than that of RA or OA with high diagnosability. Synovial fluid concentrations of miR-16, miR-146a miR-155 and miR-223 of RA were significantly higher than those of OA. Plasma miRNAs or ratio of synovial fluid miRNAs to plasma miRNAs, including miR-16 and miR-146a, significantly correlated with tender joint counts and 28-joint Disease Activity Score. CONCLUSIONS: Plasma miRNAs had distinct patterns from synovial fluid miRNAs, which appeared to originate from synovial tissue. Plasma miR-132 well differentiated HCs from patients with RA or OA, while synovial fluid miRNAs differentiated RA and OA. Furthermore, plasma miRNAs correlated with the disease activities of RA. Thus, synovial fluid and plasma miRNAs have potential as diagnostic biomarkers for RA and OA and as a tool for the analysis of their pathogenesis.

Project description:High tibial osteotomy (HTO) is a well-established treatment for medial compartmental knee osteoarthritis. Several microRNAs (miRNAs) are involved in osteoarthritis progression and are useful as osteoarthritis-related biomarkers. In this prospective study, we investigated differentially expressed microRNAs in the synovial fluid (SF) before and after HTO in patients with medial compartmental knee osteoarthritis to identify microRNAs that can be used as prognostic biomarkers. We used miRNA-PCR arrays to screen for miRNAs in SF samples obtained preoperatively and 6 months postoperatively from 6 patients with medial compartmental knee osteoarthritis who were treated with medial open wedge HTO. Differentially expressed miRNAs identified in the profiling stage were validated by real-time quantitative PCR in 22 other patients who had also been treated with HTO. All patients radiographically corresponded to Kellgren-Lawrence grade II or III with medial compartmental osteoarthritis. These patients were clinically assessed using a visual analogue scale and Western Ontario McMaster Universities scores. Mechanical axis changes were measured on standing anteroposterior radiographs of the lower limbs assessed preoperatively and at 6 months postoperatively. Among 84 miRNAs known to be involved in the inflammatory process, 14 were expressed in all SF specimens and 3 (miR-30a-5p, miR-29a-3p, and miR-30c-5p) were differentially expressed in the profiling stage. These 3 miRNAs, as well as 4 other miRNAs (miR-378a-5p, miR-140-3p, miR-23a-3p, miR-27b-3p), are related to osteoarthritis progression. These results were validated in the SF from 22 patients. Clinical and radiological outcomes improved after HTO in all patients, and only 2 miRNAs (miR-30c-5p and miR-23a-3p) were significantly differentially expressed between preoperative and postoperative 6-month SF samples (p = 0.006 and 0.007, respectively). Of these two miRNAs, miR-30c-5p correlated with postoperative pain relief. This study provides potential prognostic miRNAs after HTO and further investigations should be considered to determine clinical implications of these miRNAs.

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:BackgroundOsteoarthritis (OA) is the most common chronic degenerative joint disease and is mainly characterized by cartilage degeneration, subcartilage bone hyperplasia, osteophyte formation and joint space stenosis. Recent studies showed that synovitis might also be an important pathological change of OA. However, the molecular mechanisms of synovitis in OA are still not well understood.ObjectiveThis study was designed to identify key biomarkers and immune infiltration in the synovial tissue of osteoarthritis by bioinformatics analysis.Materials and methodsThe gene expression profiles of GSE12021, GSE55235 and GSE55457 were downloaded from the GEO database. The differentially expressed genes (DEGs) were identified by the LIMMA package in Bioconductor, and functional enrichment analyses were performed. A protein-protein interaction network (PPI) was constructed, and module analysis was performed using STRING and Cytoscape. The CIBERSORT algorithm was used to analyze the immune infiltration of synovial tissue between OA and normal controls.ResultsA total of 106 differentially expressed genes, including 68 downregulated genes and 38 upregulated genes, were detected. The PPI network was assessed, and the most significant module containing 14 hub genes was identified. Gene Ontology analysis revealed that the hub genes were significantly enriched in immune cell chemotaxis and cytokine activity. KEGG pathway analysis showed that the hub genes were significantly enriched in the rheumatoid arthritis signaling pathway, IL-17 signaling pathway and cytokine-cytokine receptor interaction signaling pathway. The immune infiltration profiles varied significantly between osteoarthritis and normal controls. Compared with normal tissue, OA synovial tissue contained a higher proportion of memory B cells, naive CD4+ T cells, regulatory T cells, resting dendritic cells and resting mast cells, while naive CD4+ T cells, activated NK cells, activated mast cells and eosinophils contributed to a relatively lower portion (P > 0.05). Finally, the expression levels of 11 hub genes were confirmed by RT-PCR.ConclusionThe hub genes and the difference in immune infiltration in synovial tissue between osteoarthritis and normal controls might provide new insight for understanding OA development.

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.

Project description:Pancreatic cancer (PC) is one of the most lethal tumors, due to late diagnosis and limited surgical strategies. It has been reported that serum exosomal microRNAs (S-Exo-miRNAs) play a pivotal role as signaling molecules and serve as noninvasive diagnosis methods for PC. The combination of S-Exo-miRNAs with the corresponding target also plays an important role in the tumor microenvironment.Here we investigated S-Exo-miRNAs involved in PC. The gene expression profile was downloaded from the Gene Expression Omnibus (GEO) database. The analysis was carried out using GEO2R. The targets of differentially expressed serum exosomal miRNAs (DE-S-Exo-miRNAs) were predicted by 4 bioinformatic algorithms (miRanda, miRDB, miRWalk, and Targetscan). Further analysis with gene ontology (GO) and Kyoto Encyclopedia of Genomes pathway (KEGG) enrichment analyses were performed with Cytoscape software version 3.4.0. Subsequently, the interaction regulatory network of target genes was performed with the Search Tool for the Retrieval of Interacting Genes (STRING) database (http://www.string-db.org/) and visualized using Cytoscape software.We downloaded the gene expression profile GSE50632, which was based on an Agilent microarray GPL17660 platform containing 4 eligible samples. In total 467 DE-S-Exo-miRNAs were obtained, including 7 overexpressed miRNAs (1.50%), and 460 remaining underexpressed miRNAs (98.50%). The databases miRWalk, miRDB, miRanda, and TargetScan were used to predict their potential targets, which were subsequently submitted to Cytoscape software version 3.4.0 (www.cytoscape.org). Next the functional and pathway enrichment analysis were used for the KEGG pathway and GO categories analysis. The enrichment analysis identified the genes involved in such processes as developmental and negative regulation of multicellular organismal processes, regulation of anatomical structure morphogenesis, regulation of cell death, apoptotic processes and mitogen-activated protein kinase (MAPK) signaling pathway, transforming growth factor - beta (TGF -β) signaling pathway, cyclic adenosine monophosphate (cAMP) signaling pathway, and the phosphatidylinositol-3 kinases/Akt (PI3K-Akt) signaling pathway. Subsequently according to the protein-protein interaction (PPI) network, the top 10 genes were obtained. The enrichment analyses of the genes involved in a significant module revealed that these genes were related to the TGF-β signaling pathway. After reviewing the literature, we identified the apoptosis genes, and their corresponding miRNAs that have a relationship with apoptosis of the tumor.This analysis provides a comprehensive understanding of the roles of S-Exo-miRNAs and the related targets in the development of PC. Additionally, the present study provides promising candidate targets for early diagnosis and therapeutic intervention. However, these predictions require further experimental validation in future studies.

Project description:BACKGROUND:Rheumatoid arthritis and osteoarthritis are two common musculoskeletal disorders that affect the joints. Despite high prevalence rates, etiological factors involved in these disorders remain largely unknown. Dissecting the molecular aspects of these disorders will significantly contribute to improving their diagnosis and clinical management. In order to identify proteins that are differentially expressed between these two conditions, a quantitative proteomic profiling of synovial fluid obtained from rheumatoid arthritis and osteoarthritis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis. RESULTS:We have identified 575 proteins out of which 135 proteins were found to be differentially expressed by ?3-fold in the synovial fluid of rheumatoid arthritis and osteoarthritis patients. Proteins not previously reported to be associated with rheumatoid arthritis including, coronin-1A (CORO1A), fibrinogen like-2 (FGL2), and macrophage capping protein (CAPG) were found to be upregulated in rheumatoid arthritis. Proteins such as CD5 molecule-like protein (CD5L), soluble scavenger receptor cysteine-rich domain-containing protein (SSC5D), and TTK protein kinase (TTK) were found to be upregulated in the synovial fluid of osteoarthritis patients. We confirmed the upregulation of CAPG in rheumatoid arthritis synovial fluid by multiple reaction monitoring assay as well as by Western blot. Pathway analysis of differentially expressed proteins revealed a significant enrichment of genes involved in glycolytic pathway in rheumatoid arthritis. CONCLUSIONS:We report here the largest identification of proteins from the synovial fluid of rheumatoid arthritis and osteoarthritis patients using a quantitative proteomics approach. The novel proteins identified from our study needs to be explored further for their role in the disease pathogenesis of rheumatoid arthritis and osteoarthritis.Sartaj Ahmad and Raja Sekhar Nirujogi contributed equally to this article.

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Identification and analysis of key microRNAs derived from osteoarthritis synovial fluid exosomes.

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Identification and analysis of key microRNAs derived from osteoarthritis synovial fluid exosomes.

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