Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: The expression of EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) is higher in human osteoarthritis (OA) cartilage compared with normal cartilage. Paraffin tissue sections were established from both pathological and normal cartilage. Safranin O and immunofluorescence staining were performed for the human articular cartilage sections. a) and b) Compared with normal cartilage, OA cartilage demonstrated chondrocyte cell clustering, empty lacunae morphology, and increased EDIL3 fluorescence signal. c) and d) The chondrocyte cluster and EDIL3-positive cells in the articular cartilage were quantified. Data are presented as means and standard errors. **p < 0.05, ***p < 0.001; independent-samples t -test. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) prevents chondrocyte clustering and chondrocyte loss, and maintains enhanced SOX9 expression in human osteoarthritis (OA) cartilage. a) Osteochondral plugs obtained from patients were cultured ex vivo and treated with phosphate-buffered saline (PBS) (vehicle) or recombinant EDIL3 proteins for six days. Representative images of Safranin O staining and magnified images of regions include the superficial zone (SZ), middle zone (MZ), and deep zone (DZ). Compared with the vehicle control group, the EDIL3 protein-treated group demonstrated prevention of chondrocyte clustering, cell loss, and lacunae morphology in SZ, MZ, and DZ regions. b) The percentage of clustered chondrocytes and chondrocyte number in the cartilage were quantified. The EDIL3 protein treatment reduces the severity of chondrocyte clustering and chondrocyte loss. c) and d) SOX9 protein is displayed in green, and 4′,6-diamidino-2-phenylindole-stained nuclei are in blue. The EDIL3 protein treatment increased the expression level of SOX9. Data are presented as means and standard errors. *p < 0.05, **p < 0.01, ***p < 0.001; two-way analysis of variance, followed by Bonferroni's multiple comparison test for selected pairs of groups for multiple comparisons.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Expressing, Cell Culture, Ex Vivo, Saline, Recombinant, Staining, Control, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) has beneficial effects on cartilage maintenance in mice with osteoarthritis (OA). a) Schematic representation of the timeline of the antibody drug or recombinant protein (EDIL3 or CD98) treatment during 16 to 21 weeks of age. STR/ort mice were injected weekly through the tail vein with phosphate-buffered saline (PBS, vehicle) combined with either the antibody or the protein for six consecutive weeks. Mice were killed at 22 weeks. This was followed by paraffin tissue sections and Safranin O and immunofluorescence (IF) staining in knee articular cartilage. b) Representative images include articular cartilage in the tibial plateau knee joints. Arrows indicate representative matrix-producing (red) and matrix-non-producing (black) chondrocytes. c) The total chondrocyte number in the articular cartilage was quantified. EDIL3 protein treatment increased the number of chondrocytes. d) The number and percentage of matrix-non-producing chondrocytes (MNCs) in the articular cartilage were quantified. EDIL3 antibody treatment increased the number of MNCs. e) EDIL3 protein treatments decreased the Osteoarthritis Research Society International (OARSI) score in the LFC. f) Representative images of IF staining (green) in whole articular cartilage obtained from STR/ort mice for the EDIL3; 4',6-diamidino-2-phenylindole (DAPI) (blue) stained nuclei; orange dashed lines define the cartilage region. EDIL3 antibody treatments decreased the EDIL3 contents in the cartilage region. g) The fluorescence of EDIL3 was quantified in the whole articular cartilage region. EDIL3 antibody significantly decreased EDIL3 expression. h) Representative images of IF staining (green) in whole articular cartilage obtained from STR/ort mice for the indicated OA markers; DAPI (blue) stained nuclei; orange dashed lines define the cartilage region. i) Fluorescence was quantified in the whole articular cartilage region. Data are presented as means and standard errors in . Data are presented as mean and maximum with 95% confidence intervals in . CD98 protein significantly increased SOX9 expression, and EDIL3 protein significantly reduced aggrecan fragments. LTP, lateral tibial plateau; MFC, medial femoral condyle; MMP, matrix metalloproteinase; MTP, medial tibial plateau. Data presented in Figures 3c to 3e were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test for selected pairs of groups for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. Data in Figures 3g to 3i were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. Isotype Ab, immunoglobulin G (IgG)1 antibody.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Recombinant, Injection, Saline, Immunofluorescence, Staining, Fluorescence, Expressing, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) treatment prevents subchondral bone plate thickness (PI.Th) and epiphyseal trabecular mineralization. a) Representative cross and horizontal sections of knee joints obtained from STR/ort mice. The 3D reconstruction of a proximal tibia with a plane indicates the location from which the cross and horizontal sections were obtained. The vehicle control mice exhibited uneven trabecular bone distribution; moreover, EDIL3 antibody treatment was observed to worsen this phenomenon. However, EDIL3 and CD98 protein prevented subchondral bone mineralization. b) Trabecular bone morphometric parameters were calculated using micro-CT analysis. The medial and lateral subchondral bone plate and underlying epiphyseal trabecular bone were analyzed separately. Epiphysis was manually selected as representative of subchondral bone. The epiphyseal trabeculae were split from the subchondral bone plate, and trabecular bone morphometric parameters were calculated. EDIL3 protein treatment prevented osteoarthritis (OA)-associated reduction in subchondral bone total porosity (Po(tot)). EDIL3 protein treatment also decreased the subchondral PI.Th, bone volume (BV/TV), and trabecular parameters (trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp)). Analyses were conducted with two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Data are presented as means and standard deviations. *p < 0.05, **p < 0.01; analyzed using a two-way analysis of variance followed by Tukey’s multiple comparison test.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Control, Micro-CT, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) inhibition promotes pro-inflammatory cytokine production in STR/ort mice. a) The variable expressions of 48 cytokines among vehicle control, isotype IgG1, EDIL3 IgG1, recombinant EDIL3 protein, and CD98 protein groups were identified by comparing the expression pattern in the serum of STR/ort mice and those with vehicle control group by heat-map analysis. Arrows indicate representative anti-inflammatory cytokines (red) and pro-inflammatory cytokines (green). b) Quantitation of each cytokine demonstrated serum samples from different groups using Luminex xMAP technology. Six anti-inflammatory cytokines and 20 pro-inflammatory cytokines were identified with the most significant difference between the five groups. c) Notably, many pro-inflammatory cytokines in serum increase with ageing, including monocyte chemotactic protein-3 (MCP-3), RANTES, interleukin (IL)-17A, IL-22, and GRO-alpha. EDIL3 antibody significantly promoted the increase of these pro-inflammatory cytokines. Analyses were conducted with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test for selected pairs of groups. Data are presented as means and standard errors. *p < 0.05, **p < 0.01, ***p < 0.001. Data presented in Figure 5c were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test for selected pairs of groups for multiple comparisons. IgG, immunoglobulin G.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Inhibition, Control, Recombinant, Expressing, Quantitation Assay, Luminex, Comparison

Journal: Bone & Joint Research

Article Title: The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

doi: 10.1302/2046-3758.1212.BJR-2023-0087.R1

Figure Lengend Snippet: EGF-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) prevents chondrocyte loss by inhibiting phosphorylation of glycogen synthase kinase 3 alpha/beta (GSK-3α/β) and phospholipase C gamma 1 (PLC-γ1). a) Chondrocytes were transfected with siEDIL3 or scramble small interfering RNA (siRNA). In comparison with scramble siRNA, siEDIL3 successfully inhibited EDIL3 protein expression in chondrocytes. b) and c) The siRNA-mediated EDIL3 knockdown in chondrocytes attenuated cell viability and increased TUNEL signal. d) Intracellular proteins were collected from chondrocytes and subsequently phosphokinase protein arrays were performed to measure the phosphorylation profiles of the kinases. e) Spots with high-intensity changes were measured by Image J software. EDIL3 attenuated the interleukin (IL)-1β-enhanced phosphokinase protein expression pattern in the chondrocytes, including GSK-3α/β, p38α, PLC-γ1, Src, STAT5ab, and WNK1. f) Hypertrophic chondrocyte-related genes were measured in IL-1β-treated chondrocytes, including SOX9, type II procollagen gene (COL2A1), and COL10A1. EDIL3 restored IL-1β-decreased SOX9 and COL2A1 expression. EDIL3 did not prevent IL-1β-increased type X procollagen gene (COL10A1) expression. Data are presented as means and standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001. Data in c) were analyzed using an independent-samples t -test. Data presented in f) were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test for selected pairs of groups for multiple comparisons. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The next morning, plugs were treated with either vehicle PBS or recombinant EDIL3 protein (2 μg/ml, 6046-ED; R&D Systems, USA).

Techniques: Phospho-proteomics, Transfection, Small Interfering RNA, Comparison, Expressing, Knockdown, TUNEL Assay, Software