Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Expression of TIMP-3 in vitreous fluid samples. Equal volumes (15 μl) of vitreous fluid samples from patients with proliferative diabetic retinopathy (PDR) ( n = 12) and non-diabetic patients with rhegmatogenous retinal detachment (RD) ( n = 12) were subjected to gel electrophoresis and the presence of TIMP-3 was detected by Western blot analysis. A representative set of samples is shown.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) attenuates retinal inflammation and prevents diabetes-induced blood-retinal barrier (BRB) breakdown. The BRB breakdown [panel (A) ] was quantified with the fluorescein isothiocyanate-conjugated dextran technique after treatment with intravitreal injection of 350 μM TIMP-3 in 5 μl in one eye and the same volume of phosphate-buffered saline (PBS) in the contralateral eye of rats 10 weeks after induction of diabetes. Results are expressed as mean ± standard deviation of five rats in each group. Evaluation of inflammatory mediators was evaluated shortly after diabetes induction (see section “Materials and Methods”). Western blot analysis of rat retinas was performed to evaluate protein expression levels of phospho-ERK1/2 [panel (B) ], the p65 subunit of NF-κB [panel (C) ], intercellular adhesion molecule-1 (ICAM-1) [panel (D) ], and vascular endothelial growth factor (VEGF) [panel (E) ]. Results are expressed as mean ± standard deviation of 12 rats in each group. One-way ANOVA and independent t -test were used for comparisons between the three and two groups, respectively, panels (A–E) . * p < 0.05 compared with non-diabetic controls. # p < 0.05 compared with PBS-treated diabetic rats.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Injection, Standard Deviation, Western Blot, Expressing

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Müller cells were left untreated or treated with high-glucose (HG, 25 mM) [panel (A) ] cobalt chloride (CoCl 2 ) (300 μM) [panel (B) ] or tumor necrosis factor -α (TNF-α) (50 ng/ml) [panel (C) ] for 24 h or TIMP-3 (100 ng/ml) for 1 h followed by HG, CoCl 2 , or TNF-α. For HG treatment, cultures containing 25 mM mannitol were used as a control. Levels of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were quantified in the culture media by ELISA. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparison between three groups and two groups, respectively. * p < 0.05 compared with values obtained from control cells. # p < 0.05 compared with values obtained from cells treated with HG, CoCl 2 , or TNF-α.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Müller cells were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before increasing the sugar content of the cultures [25 mM of mannitol as control or 25 mM of glucose (HG)]. After 24 h levels of the p65 subunit of NF-κB [panel (A) ], phospho-ERK1/2 [panel (B) ], caspase-3 [panel (C) ], and ADAM17 [panel (D) ] in cell lysates was determined by Western blot analysis. Results are expressed as median (interquartile range) from three different experiments performed in triplicate. Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with values obtained from cells treated with mannitol. # p < 0.05 compared with values obtained from cells treated with HG.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Incubation, Western Blot, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: THP-1 monocytes were left untreated or treated with TIMP-3 (100 ng/ml) for 24 h. Subsequently, the THP-1 cells were fluorescently labeled and adhesion to a human retinal microvascular endothelial cell (HRMEC) monolayer was assessed [panel (A) ]. Results are expressed as median (interquartile range) from two independent experiments (each treatment condition: 6 wells) (* p < 0.05; Mann-Whitney test). Alternatively, HRMECs were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium, tumor necrosis factor-α (TNF-α) (25 ng/ml) [panel (B) ] or vascular endothelial growth factor (VEGF) (50 ng/ml) [panel (C) ] for 24 h. Adhesion of fluorescently labeled monocytic cells to the HRMEC monolayer was assessed. Results are expressed as median (interquartile range) from three independent experiments (each treatment condition: 6 wells). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively (RFU = relative fluorescence unit). HRMECs were left untreated or were stimulated with TNF-α (50 ng/ml) for 24 h with/without a 1-h pre-incubation with TIMP-3 (100 ng/ml). Protein expression of vascular cell adhesion molecule-1 (VCAM-1) [panel (D) ] and intercellular adhesion molecule-1 (ICAM-1) [panel (E) ] was determined by Western blot analysis. Results are expressed as mean ± standard deviation from three independent experiments (each treatment condition: 8 wells). One-way ANOVA and independent t -test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with values obtained from untreated cells. # p < 0.05 compared with values obtained from cells treated with TNF-α or VEGF.

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Labeling, MANN-WHITNEY, Incubation, Fluorescence, Expressing, Western Blot, Standard Deviation

Journal: Frontiers in Physiology

Article Title: Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation

doi: 10.3389/fphys.2021.807747

Figure Lengend Snippet: Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) inhibits vascular endothelial growth factor (VEGF)-mediated human retinal microvascular endothelial cell (HRMEC) migration, chemotaxis and proliferation. A scratch was made in confluent monolayers of overnight starved HRMECs with a micropipette tip. Subsequently, the cultures were pre-incubated with TIMP-3 (100 ng/ml) or dilution medium for 1 h before stimulation with dilution medium or vascular endothelial growth factor (VEGF) (50 ng/ml) for 24 h. Cells were visualized using an inverted microscope. Three independent experiments were performed. Each experiment was done in triplicate and 6–8 independent field images were taken for the migration analysis which was done by using image J software. In the figure one representative image is shown [panel (A) ]. Results are expressed as median (interquartile range). Kruskal-Wallis test and Mann-Whitney test were used for comparisons between three groups and two groups, respectively. * p < 0.05 compared with untreated cells. # p < 0.05 compared with VEGF-treated cells. Chemotaxis and proliferation of HRMECs stimulated with 10 ng/ml VEGF was modulated by a 30-min pre-incubation with TIMP-3 (10 or 100 ng/ml). Cell migration was monitored using the xCELLigence RTCA-DP system. The median (interquartile range) percentage of inhibition of VEGF-induced chemotaxis [panel (B) ] or proliferation [panel (C) ] is shown. In total five chemotaxis experiments were performed and conditions were tested in duplicate or triplicate within 1 experiment. For proliferation, 6 experiments were performed and conditions were tested at least in triplicate within 1 experiment. * p < 0.05; Mann-Whitney test (compared with VEGF).

Article Snippet: The antibodies used for Western blot analysis in the present study were as follows: rabbit monoclonal anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibody (1:1,500, MAB1018, R&D Systems, Minneapolis, MA, United States), mouse monoclonal anti-phospho-p65 subunit of nuclear factor-kappa B (NF-κB) antibody (1:500, sc-136548, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States), rabbit polyclonal anti-phospho vascular endothelial growth factor receptor (VEGFR-2) antibody (1:1,000, ab194806, Abcam, Cambridge, United Kingdom), mouse monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody (1:1,000, sc-8439, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-vascular cell adhesion protein-1 (VCAM-1) antibody (1:1,000, sc-13160, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-VEGF antibody (1:1,500, MAB293, R&D Systems), rabbit polyclonal caspase-3 antibody (1:1,000, sc-7148, Santa Cruz Biotechnology Inc.), rabbit polyclonal ADAM-17 antibody (1:1,000, ab39163, Abcam), and rabbit monoclonal anti-TIMP-3 antibody (1:1,000, ab277794, Abcam).

Techniques: Migration, Chemotaxis Assay, Incubation, Inverted Microscopy, Software, MANN-WHITNEY, Inhibition