Journal: Biomedicines

Article Title: Fibroblast Activation Protein (FAP)-Mediated Cleavage of Type III Collagen Reveals Serum Biomarker Potential in Non-Small Cell Lung Cancer and Spondyloarthritis

doi: 10.3390/biomedicines12030545

Figure Lengend Snippet: Assay specificity: ( a ) Standard curves of signal inhibition relative to background (B/B0) with increasing peptide concentrations for 30aa selection peptide (std) (AGPAGAPGPAGSRGAPGPQGPRGDKGETGE), 10aa selection peptide (AGPAGAPGPA), deselection peptide (De) 1, 2, and 3 (AGPAGAAGQP, PGPAGAPGPA, and AGPQGAPGPA, respectively), elongated peptide (PAGPAGAPGPA), and truncated peptide (_GPAGAPGPA). Dotted lines indicate measurement range. ( b , c ) Biomarker measurements of C3F ( b ) and C3M ( c ) in solution of recombinant type III collagen (COL3) after incubation with/without matrix metalloproteinases 9 (MMP9) or fibroblast activation protein (FAP), with buffer as control. ( d , e ) Biomarker measurements of C3F ( d ) and C3M ( e ) in solution of recombinant type III collagen pre-cleaved with MMP13 (pre-cleaved COL3) followed by incubation with/without MMP9 or FAP, with buffer as control. Dotted lines indicate lower limit measurement range (LLMR) for C3F ( b , d ) and upper limit measurement range (ULMR) for C3M ( c , e ).

Article Snippet: In addition, we incubated recombinant type III collagen (Sigma-Aldrich, St. Louis, MO, USA, cat. #CC054) with MMP9 (Bio-Techne, Minneapolis, MN, USA, cat. #911-MP) or MMP13 (bio-techne cat. #511-MM) for 24 h and stopped the reaction with EDTA.

Techniques: Inhibition, Selection, Biomarker Assay, Recombinant, Incubation, Activation Assay

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: Proposed action mechanism of ERMs@siM13 in the early-stage posttraumatic osteoarthritis (PTOA) mouse model. (A) Schematic illustration of preparation procedure of ERMs@siM13 and its matrix metalloproteinase 13 (MMP13)-mediated activation. (B) After intraarticular injection into the joints with early-stage PTOA, ERMs@siM13 can be activated by MMP13 overexpressed surrounding diseased chondrocytes, detaching the Cy5-bearing PEG protective shell. Accordingly, cRGD is exposed to promote the siRNA silencing MMP13 (siM13)-loaded micelle uptake by the diseased cells for on-demand MMP13 downregulation, and Cy5 fluorescence is restored to report the diseased state of cartilage tissues.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: Activation Assay, Injection, Fluorescence

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: Preparation and characterization of ERMs@siM13. (A) High-performance liquid chromatography (HPLC) spectra of Cy5-C(PEG 2000 )-GPLGVRGK-NH 2 (EP) and Cy5-C(PEG 2000 )-GplgvrGK-NH 2 (nEP) before and after incubation with matrix metalloproteinase 13 (MMP13). (B) Representative flow cytometry histogram showing the uptake of coumarin 6 (C6)-labeled RMs with various cRGD modification densities by diseased chondrocytes and (C) the corresponding mean fluorescence intensity (MFI) of C6. (D) Representative flow cytometry histogram showing the internalization of nERMs@C6 with various ratios of cRGD: PEG by diseased chondrocytes and (E) the corresponding C6 MFI. (F) Representative fluorescence spectra and fluorescence images of nERMs containing various molar ratios of BHQ3-to-Cy5. (G) Representative transmission electron microscopy (TEM) images of ERMs@siM13. Scale bar: 50 nm. (H) Representative fluorescence spectra and fluorescence images of ERMs and nERMs before and after incubation with MMP13. (I) Serum stability of siM13 loaded by RMs, ERMs, and nERMs determined through agarose gel electrophoresis. Free siM13 served as the controls. “E” represents MMP13. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: High Performance Liquid Chromatography, Incubation, Flow Cytometry, Labeling, Modification, Fluorescence, Transmission Assay, Electron Microscopy, Agarose Gel Electrophoresis, Two Tailed Test

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: ERMs@siM13 delivery at cellular levels. (A) Schematic illustration of MMP13-triggered diseased chondrocyte-specific delivery and fluorescence imaging of ERMs@siM13. (B) Fold change of MMP13 expression in culture medium was determined by enzyme-linked immunosorbent assay (ELISA) after chondrocytes were induced by IL-1β for different intervals. (C) Representative fluorescence images and the corresponding fluorescence intensity of normal or diseased chondrocytes after incubation with ERMs or nERMs for 2 h. (D) Representative flow cytometry histogram showing the uptake of coumarin 6 (C6)-labeled micelles by the diseased chondrocytes after 2 h of incubation and the corresponding mean fluorescence intensity (MFI) of C6. (E) Representative confocal images of IL-1β-induced diseased chondrocytes after 2 h of incubation with C6-labeled various micelles. Scale bar: 50 μm. (F) Intracellular delivery of ERMs@FAM-siRNA in IL-1β-induced diseased chondrocytes. Late endosomes/lysosomes are labeled by LysoTracker Red. Scale bar: 25 μm. (G) Pearson's correlation coefficients calculated from Panel F. Data are presented as mean ± SD (n = 3). ** P < 0.01, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: Fluorescence, Imaging, Expressing, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry, Labeling, Two Tailed Test

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: Diagnostic and therapeutic effects of ERMs@siM13 on diseased chondrocytes. (A) The diseased chondrocytes were treated with the indicated formulations, followed by 2 h of incubation with ERMs to diagnose the chondrocyte status. Normal chondrocytes served as a control. (B) Mean fluorescence intensity (MFI) of Cy5 calculated from Panel (A). (C) Mmp1 3 mRNA levels of diseased chondrocytes relative to that of the PBS control after treated with RMs@siM13, nERMs@siM13, and ERMs@siM13. (D) Representative Western blot images showing MMP13, Col 2, and GAPDH protein levels after treatment mentioned above and the corresponding mean protein expression of MMP13 and Col 2. (E–F) Immunofluorescence staining and semiquantitative analysis of (E) MM13 and (F) Col 2 for diseased chondrocytes after treatment mentioned above. (G) Proliferation assay of diseased chondrocytes that were treated as mentioned above, as determined via EdU staining. EdU + cell ratios relative to the PBS control were calculated. Scale bar: 100 μm. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: Diagnostic Assay, Incubation, Control, Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining, Proliferation Assay, Two Tailed Test

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: In vivo delivery and diagnostic effects of ERMs@siM13 in the murine posttraumatic osteoarthritis (PTOA) model. (A) Representative fluorescence images of the PTOA mouse joints at various time points after intraarticular injection with ERMs@DiR and nERMs@DiR. (B) The fluorescence signal from Panel (A) was qualified. (C) Representative confocal images showing the ERMs@C6 and nERMs@C6 penetration into healthy and PTOA cartilage. (D) Mean fluorescence intensity (MFI) of C6 calculated from Panel (C). (E) The PTOA mice were treated with ERMs@siM13 and nERMs@siM13 every 5 days for 9 cycles. ERMs were intraarticularly injected at preset time points to monitor the PTOA progression. (F) Cy5 MFI calculated from Panel (E). (G) The PTOA mice were treated as described above. The joints were collected at preset time points and processed for immunofluorescence staining of MMP13. (H) MMP13 MFI calculated from Panel (G). Scale bar: 100 μm. Data are presented as mean ± SD. * P < 0.05, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: In Vivo, Diagnostic Assay, Fluorescence, Injection, Immunofluorescence, Staining, Two Tailed Test

Journal: Bioactive Materials

Article Title: MMP13-targeted siRNA-loaded micelles for diagnosis and treatment of posttraumatic osteoarthritis

doi: 10.1016/j.bioactmat.2024.04.010

Figure Lengend Snippet: Immunohistochemical (IHC) staining of sham or posttraumatic osteoarthritis (PTOA) mouse knee joints after 4 and 8 weeks of treatment. (A) Representative IHC staining images of MMP13 in the mouse cartilages after treatment with the indicated formulations. The sham group served as a normal control. (B) The MMP13-positive (MMP13 + ) area (%) calculated from Panel (A). (C) Representative IHC staining images of Col 2 in the mouse cartilages posttreatment with the indicated formulations. (D) The Col 2-positive (Col 2 + ) area calculated from Panel (C). (E) Representative IHC staining images of aggrecan (ACAN) in the mouse cartilages posttreatment with the indicated formulations. (F) The ACAN-positive (ACAN + ) area calculated from Panel (E). Scale bar: 100 μm. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns, no significant difference among the marked groups using nonparametric two-tailed analysis of variance.

Article Snippet: Recombinant human MMP13 proenzyme (511-MM-010) was ordered from the R&D Systems Co., Ltd. (Minnesota, USA).

Techniques: Immunohistochemical staining, Immunohistochemistry, Control, Two Tailed Test

Journal: Oncotarget

Article Title: Melatonin inhibits Sirt1-dependent NAMPT and NFAT5 signaling in chondrocytes to attenuate osteoarthritis

doi: 10.18632/oncotarget.18356

Figure Lengend Snippet: Chondrocytes were pretreated with 100 μM Sirt1 inhibitor (EX527) for 30 min or 100 nM siSirt1 for 1 h and then stimulated with or without 10 ng/ml IL-1β for 30 min, followed by 10 ng/ml melatonin for 24 h. A. qPCR and B. and C. Western blot analyses were performed to determine the mRNA and protein expression of MMP-3 and MMP-13, respectively. GAPDH and β-actin were used as internal controls. D. MMP-3 and MMP-13 levels in the media were measured by ELISAs. Each value represents mean ± SD of 3 replicates or representative of 3 independent experiments. * P < 0.05 compared with control; # P < 0.05 compared with IL-1β-treated group; § P < 0.05 compared with Siscrb group. Mel: melatonin.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human or rat TNF-α, IL-1β, PGE 2 , MMP-3, and MMP-13 were supplied by R&D systems (Minneapolis, MN, USA).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Melatonin inhibits Sirt1-dependent NAMPT and NFAT5 signaling in chondrocytes to attenuate osteoarthritis

doi: 10.18632/oncotarget.18356

Figure Lengend Snippet: Chondrocytes were pretreated with anti-TNF-α antibody (50 μg/ml), anti-IL-1β antibody (50 μg/ml), celecoxib (5 μM), or 1400W (50 μM) for 1 h and then stimulated with or without 10 ng/ml IL-1β for 30 min, followed by 10 ng/ml melatonin for 24 h. A. qPCR and B. and C. Western blot analyses were performed to determine the mRNA and protein expression of MMP-3 and MMP-13, respectively. GAPDH and β-actin were used as internal controls. D. Levels of MMP-3 and MMP-13 in the media were measured by ELISAs. Each value represents means ± SD of 3 replicates or representative of 3 independent experiments. * P < 0.05 compared with control; # P < 0.05 compared with IL-1β-treated group. Mel: melatonin; Cel: celecoxib.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human or rat TNF-α, IL-1β, PGE 2 , MMP-3, and MMP-13 were supplied by R&D systems (Minneapolis, MN, USA).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Melatonin inhibits Sirt1-dependent NAMPT and NFAT5 signaling in chondrocytes to attenuate osteoarthritis

doi: 10.18632/oncotarget.18356

Figure Lengend Snippet: ACLT was performed in male Lewis rats. Animals were treated with PBS or IL-1 receptor antagonist (IL-1 ra) or melatonin (n = 6 in each group). A. HE analysis and staining evaluation was performed for synovitis scoring in PBS- or melatonin- or IL-1 ra-treated groups. (100 × magnification) B. ELISA assays were conducted to determine the SF levels of TNF-α, IL-1β, PGE 2 , MMP-3, and MMP-13 in three groups. C. Western blot analyses were performed to determine the protein expression of Sirt1, NFAT5, NAMPT, MMP-3 and MMP-13 in ACLT rat chondrocyte with or without melatonin or IL-1 ra treatment. β-actin was used as internal control. Each value represents means ± SD of 3 replicates or representative of 3 independent experiments. * P < 0.05, ** P < 0.01 compared with control group; # P < 0.05 compared with ACLT + PBS group. ACLT: anterior cruciate ligament transaction; Mel: melatonin; IL-1 ra: IL-1 receptor antagonist.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human or rat TNF-α, IL-1β, PGE 2 , MMP-3, and MMP-13 were supplied by R&D systems (Minneapolis, MN, USA).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Oncotarget

Article Title: Melatonin inhibits Sirt1-dependent NAMPT and NFAT5 signaling in chondrocytes to attenuate osteoarthritis

doi: 10.18632/oncotarget.18356

Figure Lengend Snippet: Melatonin reduced IL-1β-induced MMP-3 and MMP-13 production through Sirt1-dependent NAMPT and NFAT5 signaling in chondrocytes, suggesting melatonin as an efficient therapeutic agent to hinder OA progression.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) kits for human or rat TNF-α, IL-1β, PGE 2 , MMP-3, and MMP-13 were supplied by R&D systems (Minneapolis, MN, USA).

Techniques:

Journal: Journal of biomedical materials research. Part B, Applied biomaterials

Article Title: Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

doi: 10.1002/jbm.b.34339

Figure Lengend Snippet: Scaffold Resistance to ADAMTS-5 Degradation. Graphs depicting A) mass loss and resultant B) stiffness, C) storage modulus, and D) loss modulus of scaffold study groups tested at various frequencies following incubation in recombinant MMP-13. Lines connecting study groups indicate statistical differences (p≤0.05), and † indicates statistical difference (p≤0.05) from pre-digested values. (NQ: not quantifiable).

Article Snippet: 46 Samples (n=3/enzyme/group) were exposed to 1xTris-buffered saline containing 0.05% Brij-35, 5 mM CaCl 2 and either recombinant human ADAMTS-5 (Fisher Scientific) at 0.20485 µg/mL or recombinant human MMP-13 (Fisher Scientific) at 0.02215 µg/mL.

Techniques: Incubation, Recombinant

Journal: Journal of biomedical materials research. Part B, Applied biomaterials

Article Title: Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

doi: 10.1002/jbm.b.34339

Figure Lengend Snippet: Scaffold Resistance to MMP-13 Degradation. Graphs depicting A) mass loss and resultant B) stiffness, C) storage modulus, and D) loss modulus of scaffold study groups tested at various frequencies following incubation in recombinant MMP-13. Lines connecting study groups indicate statistical differences (p≤0.05), and † indicates statistical difference (p≤0.05) from pre-digested values. (ND: none detected; NQ: not quantifiable)

Article Snippet: 46 Samples (n=3/enzyme/group) were exposed to 1xTris-buffered saline containing 0.05% Brij-35, 5 mM CaCl 2 and either recombinant human ADAMTS-5 (Fisher Scientific) at 0.20485 µg/mL or recombinant human MMP-13 (Fisher Scientific) at 0.02215 µg/mL.

Techniques: Incubation, Recombinant