Dataset Information

Synovial Fluid Derived from Human Knee Osteoarthritis Increases the Viability of Human Adipose-Derived Stem Cells through Upregulation of FOSL1.

ABSTRACT: Knee osteoarthritis (Knee OA) is an irreversible condition that causes bone deformity and degeneration of the articular cartilage that comprises the joints, resulting in chronic pain and movement disorders. The administration of cultured adipose-derived stem cells (ADSCs) into the knee joint cavity improves the clinical symptoms of Knee OA; however, the effect of synovial fluid (SF) filling the joint cavity on the injected ADSCs remains unclear. In this study, we investigated the effect of adding SF from Knee OA patients to cultured ADSCs prepared for therapeutic use in an environment that mimics the joint cavity. An increase in the viability of ADSCs was observed following the addition of SF. Gene expression profiling of SF-treated ADSCs using DNA microarrays revealed changes in several genes involved in cell survival. Of these genes, we focused on FOSL1, which is involved in the therapeutic effect of ADSCs and the survival and proliferation of cancer stem cells. We confirmed the upregulation of FOSL1 mRNA and protein expression using RT-PCR and western blot analysis, respectively. Next, we knocked down FOSL1 in ADSCs using siRNA and observed a decrease in cell viability, indicating the involvement of FOSL1 in the survival of ADSCs. Interestingly, in the knockdown cells, ADSC viability was also decreased by SF exposure. These results suggest that SF enhances cell viability by upregulating FOSL1 expression in ADSCs. For therapy using cultured ADSCs, the therapeutic effect of ADSCs may be further enhanced if an environment more conducive to the upregulation of FOSL1 expression in ADSCs can be established.

SUBMITTER: Kitajima H

PROVIDER: S-EPMC9856741 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Synovial Fluid Derived from Human Knee Osteoarthritis Increases the Viability of Human Adipose-Derived Stem Cells through Upregulation of FOSL1.

Kitajima Hironori H Sakamoto Takuya T Horie Tetsuhiro T Kuwano Ayane A Fuku Atsushi A Taki Yasuhiko Y Nakamura Yuka Y Tanida Ikuhiro I Sunami Hiroshi H Hirata Hiroaki H Tachi Yoshiyuki Y Yamamoto Naoki N Iida Yasuo Y Ishigaki Yasuhito Y Yamada Sohsuke S Shimodaira Shigetaka S Shimizu Yusuke Y Ichiseki Toru T Kaneuji Ayumi A Osawa Satoshi S Kawahara Norio N

Cells 20230115 2

Knee osteoarthritis (Knee OA) is an irreversible condition that causes bone deformity and degeneration of the articular cartilage that comprises the joints, resulting in chronic pain and movement disorders. The administration of cultured adipose-derived stem cells (ADSCs) into the knee joint cavity improves the clinical symptoms of Knee OA; however, the effect of synovial fluid (SF) filling the joint cavity on the injected ADSCs remains unclear. In this study, we investigated the effect of addin ...[more]

PMID: 36672268

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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: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:Macrophage polarization is a steering factor of osteoarthritis (OA) progression. Synovial fluid (SF) obtained from OA patients with different Kellgren-Lawrence grades (KL grades) holds several proinflammatory factors and was hypothesized to induce macrophage differentiation and polarization by providing the needed microenvironment. U937 cells and peripheral-blood-mononuclear-cell-derived monocytes (PBMC-derived CD14+ cells) were induced with SFs of progressive KL grades for 48 h, and the status of the differentiated cells was evaluated by cell surface markers representing M1 and M2 macrophage phenotypes. Functional viability assessment of the differentiated cells was performed by cytokine estimation. The fraction of macrophages and their phenotypes were estimated by immunophenotyping of SF-isolated cells of different KL grades. A grade-wise proteome analysis of SFs was performed in search of the factors which are influential in macrophage differentiation and polarization. In the assay on U937 cells, induction with SF of KL grade III and IV showed a significant increase in M1 type (CD86+). The percentage of M2 phenotype (CD163+) was significantly higher after the induction with SF of KL grade II. A Significantly higher M1/M2 ratio was estimated in the cells induced with KL grade III and IV. The cell differentiation pattern in the assay on PBMC-derived CD14+ cells showed a grade-wise decline in both M1 (CD11C+, CD86+) and M2 phenotype (CD163+). Cytokine estimation specific to M1 (TNF-α, IL-6, IL-1β, IFN-γ) and M2 (IL-4 and IL-10) macrophages corelated with the differentiation pattern in the U937 cell assay, while it did not reveal any significant changes in the PBMC-derived CD14+ cells assay. SF cells' immunophenotyping showed the highest percentage of CD14+ macrophages in KL grade II; CD86+ and CD163+ cells were minimal in all KL grades' SFs. The proteome analysis revealed significantly expressed MIF, CAPG/MCP, osteopontin, and RAS-related RAB proteins in KL grade III and IV samples, which are linked with macrophages' movement, polarization, and migration-behavior. In conclusion, this study demonstrated that SF in OA joints acts as a niche and facilitates M1 phenotype polarization by providing a proinflammatory microenvironment.

Dataset Information

Synovial Fluid Derived from Human Knee Osteoarthritis Increases the Viability of Human Adipose-Derived Stem Cells through Upregulation of FOSL1.

Publications

Synovial Fluid Derived from Human Knee Osteoarthritis Increases the Viability of Human Adipose-Derived Stem Cells through Upregulation of FOSL1.

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