par2 antagonist i-191  (Axon Medchem LLC)

Journal: Cells

Article Title: Neutrophil Elastase Increases Vascular Permeability and Leukocyte Transmigration in Cultured Endothelial Cells and Obese Mice

doi: 10.3390/cells11152288

Figure Lengend Snippet: NE regulated VE-cadherin, actomyosin cytoskeleton, and permeability in hECs through activating protease-activated receptor 2 (PAR2). ( A ) Immunofluorescence images of PAR2 (green), VE-cadherin (green), F-actin (red), or p-MLC (pSer19, red) and nuclei counterstaining DAPI (blue). hEC cells were treated with NE (20 nM) or PAR2 agonist (PAR2-AP, 7.5 μM) in the presence or absence of PAR2 inhibitor (1 μM) for 16 h. Images were taken at 20x using a fluorescence microscope; scale bar: 100 μm. ( B ) Graph showing percentage permeability of endothelial monolayer treated with 20 nM NE, 7.5 μM PAR2 agonist, and 1 μM PAR2 inhibitor. Permeability assay was carried out with Lucifer Yellow dye. All data are expressed as mean ± SD of three independent experiments. ** p < 0.01 vs. vehicle control group, # p < 0.05 vs. NE group. NE: neutrophil elastase, PAR2 Inh: protease-activated receptor 2 inhibitor (I191), PAR2 AP: protease-activated receptor 2 agonist (PAR2 (I-6) amide trifluroacetate salt). See also .

Article Snippet: PAR2 antagonist I-191, Alexa-Fluor 568 goat anti-rabbit IgG and Alexa-Flour 488 chicken anti-goat IgG, transwell inserts (1.13 cm 2 culture area, 0.4 μm pore size polycarbonate filter), and pierce TM ECL Western blotting chemiluminescence substrate solution were obtained from Thermo Scientific (Waltham, MA, USA).

Techniques: Permeability, Immunofluorescence, Fluorescence, Microscopy

Journal: Cells

Article Title: Neutrophil Elastase Increases Vascular Permeability and Leukocyte Transmigration in Cultured Endothelial Cells and Obese Mice

doi: 10.3390/cells11152288

Figure Lengend Snippet: Regulation of NE’s effects by PAR2 downstream signaling components ROCK and MLCK. ( A ) hECs were treated with ROCK inhibitor (Y27632, 5 μM) and MLCK inhibitor (peptide 18, 5 μM) with or without NE (20 nM) for 16 h. Images (20x) were taken after immunofluorescence staining of VE-cadherin (green) and p-MLC (pSer19, red), F-actin staining with phalloidin (red), and nuclei staining with DAPI (blue). Images are representatives of three independent experiments (scale bar, 100 μm). ( B ) Western blot analysis confirming the changes of VE-cadherin, β-catenin, p120-catenin, p-MYPT1 (pThr853), and p-MLC (pSer19) in hECs. GAPDH was used as an internal control for protein loading. ( C ) Confluent hECs on transwells (with 0.4 μm pore size) were treated with or without NE (20 nM) in the presence or absence of ROCK or MLCK inhibitor for 16 h. Lucifer Yellow was used to measure cell permeability, and the results were expressed as percentage of control (transwells without cells). All data are expressed as mean ± SD of three independent experiments. ** p < 0.01 vs. vehicle control group, # p < 0.05 vs. NE group. NE: neutrophil elastase, ROCK Inh: ROCK inhibitor, MLCK Inh: MLCK inhibitor. See also .

Article Snippet: PAR2 antagonist I-191, Alexa-Fluor 568 goat anti-rabbit IgG and Alexa-Flour 488 chicken anti-goat IgG, transwell inserts (1.13 cm 2 culture area, 0.4 μm pore size polycarbonate filter), and pierce TM ECL Western blotting chemiluminescence substrate solution were obtained from Thermo Scientific (Waltham, MA, USA).

Techniques: Immunofluorescence, Staining, Western Blot, Permeability

Journal: Cells

Article Title: Neutrophil Elastase Increases Vascular Permeability and Leukocyte Transmigration in Cultured Endothelial Cells and Obese Mice

doi: 10.3390/cells11152288

Figure Lengend Snippet: Inhibition of proteasome activity reversed NE-induced VE-cadherin degradation and permeability in hECs. Cells were treated with or without proteasome inhibitor MG132 (5 μM) for 2 h before adding NE (20 nM) or PAR2 agonist (Par2-AP, 7.5 μM) for 16 h, as indicated. Then, cells were used for immunofluorescence imaging of VE-cadherin and Western blot analysis. ( A ) VE-cadherin fluorescence images (green) are representative of three independent experiments (scale bar, 100 μm). ( B ) Western blot images and quantification showing VE-cadherin, β-catenin, and p120-catenin in hECs. ( C ) Gene expression of VE-cadherin in hECs treated with NE (20 nM) or PAR2 agonist (Par2-AP, 7.5 μM) in the presence or absence of ROCK inhibitor (Y27632, 5 μM) for 16 h. RT-PCR was used for quantifying gene expression levels of VE-cadherin normalized to 36B4 for each sample. All data are expressed as mean ± SD of three independent experiments. ** p < 0.01 vs. vehicle control, # p < 0.05 vs. NE group, $ p < 0.05 vs. PAR2-AP. PAR2-AP: PAR2 agonist (PAR2 (I-6) amide trifluroacetate salt) (7.5 μM), ROCK Inh: ROCK inhibitor. See also .

Article Snippet: PAR2 antagonist I-191, Alexa-Fluor 568 goat anti-rabbit IgG and Alexa-Flour 488 chicken anti-goat IgG, transwell inserts (1.13 cm 2 culture area, 0.4 μm pore size polycarbonate filter), and pierce TM ECL Western blotting chemiluminescence substrate solution were obtained from Thermo Scientific (Waltham, MA, USA).

Techniques: Inhibition, Activity Assay, Permeability, Immunofluorescence, Imaging, Western Blot, Fluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: Neutrophil Elastase Increases Vascular Permeability and Leukocyte Transmigration in Cultured Endothelial Cells and Obese Mice

doi: 10.3390/cells11152288

Figure Lengend Snippet: Interaction of endothelial cells with NE or isolated neutrophils stimulated monocyte transendothelial migration via activation of the PAR2 pathway. ( A ) Confluent hECs grown on collagen-coated transwell inserts (8 μm pore size) were treated with NE (20 nM) or PAR2 agonist (PAR2-AP, 7.5 μM) in the presence or absence of NE inhibitor (NEI, 1 μM), PAR2 inhibitor (PAR2 Inh, 1 μM), ROCK inhibitor (ROCK Inh, 5 μM), and MLCK inhibitor (MLCK Inh, 5 μM) for 16 h. hECs were washed with normal media before adding BCECF-AM labeled THP-1 cells (1 × 10 5 ) for 6 h. ( B ) Confluent hECs on transwell inserts were treated with freshly isolated neutrophils (1 × 10 6 cells/well) for 16 h in the presence or absence of NE inhibitor (NEI, 1 μM) or PAR2 inhibitor (I191, 1 μM). hEC monolayer on the top chamber was washed with cell media to remove neutrophils. Then, BCECF-AM labeled THP-1 cells (1 × 10 5 cells/well) were used to assess transmigration for 2 h. Representative images show fluorescence-labeled THP-1 cells transmigrated through the hEC monolayer were captured ( A , B ). For quantification, monocytes were counted in five random microscopic fields per well. The values are expressed as mean ± SD of three independent experiments. Top panel: ** p < 0.01 vs. vehicle control group, # p < 0.05 vs. NE group; lower panel: ** p < 0.01 vs. vehicle control group, # p < 0.05 vs. neutrophil (Neut) group.

Article Snippet: PAR2 antagonist I-191, Alexa-Fluor 568 goat anti-rabbit IgG and Alexa-Flour 488 chicken anti-goat IgG, transwell inserts (1.13 cm 2 culture area, 0.4 μm pore size polycarbonate filter), and pierce TM ECL Western blotting chemiluminescence substrate solution were obtained from Thermo Scientific (Waltham, MA, USA).

Techniques: Isolation, Migration, Activation Assay, Labeling, Transmigration Assay, Fluorescence