Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 3. Proliferative diabetic retinopathy epiretinal membranes. Negative control slide that was treated identically with an irrelevant antibody showing no labeling (A). Immunohistochemical staining for CD31 showing blood vessels positive for CD31 (B). Immunohistochemical staining for protease-activated receptor-1 (PAR1) showing immunoreactivity in the vascular endothelium (arrows), in stromal cells (arrowheads) (C), in intravascular leukocytes (arrowheads) (D), and in stromal spindle-shaped cells (E). Immunohistochemical staining for CD45 showing stromal cells positive for CD45 (F). Immunohistochemical staining for α-smooth muscle actin showing immunoreactivity in spindle-shaped myofibroblasts (G). Double immunohistochemistry for CD45 (brown) and PAR1 (red) showing stromal cells and intravascular leukocytes (arrowheads) co-expressing CD45 and PAR1 (H) (original magnification X40).

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Negative Control, Labeling, Immunohistochemical staining, Staining, Immunohistochemistry, Expressing

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 5. Western blot analysis of protease-activated receptor-1 (PAR1) (A) and thrombin (B) in rat retinas. Significant increases in the ratios of the cleaved PAR1 over the intact protein and of the proteolytically active thrombin over the zymogen prothrombin in the retinas of diabetic rats (D) compared to the nondiabetic control rats (C). Each experiment was repeated three times with fresh samples (n = 7). *p < 0.05 compared to control.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Western Blot, Control

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 4. Human retinal microvascular endothelial cells were left untreated or were treated with either IL-1β (10 ng/ml), TNF-α (30 ng/ml), or a combination of cytokines containing IL-1β (10 ng/ml) plus TNF-α (30 ng/ml) for 24 h. The levels of expression of protease-activated receptor-1 (PAR1) were compared by Western blot analysis. Western blots are representative of at least three independent experiments, each is performed in duplicate and bar graphs are representative of all three experiments. *p < 0.05 compared to untreated control. #p < 0.05 compared to IL-1β.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Western Blot, Control

Journal: Current eye research

Article Title: Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

doi: 10.3109/02713683.2016.1141964

Figure Lengend Snippet: Figure 7. Effects of thrombin (A) and matrix metalloproteinase-1 (MMP-1) (B) on the migration of human retinal microvascular endothelial cells (HRMEC). Thrombin- induced migration of HRMEC was inhibited by the protease-activated receptor-1 (PAR1) inhibitor vorapaxar. HRMECs were left untreated or treated either with 1 nM recombinant thrombin, with 10 µM PAR1 inhibitor vorapaxar for 30 min before addition of 1 nM recombinant thrombin or with 5 nM recombinant MMP-1. Cells were visualized using inverted microscope (Olympus 1X 81, Olympus Corporation, Tokyo, Japan). Three independent experiments were performed. Six independent field images were taken from each treated group for migration analysis, which was performed using Image J software. One image from each group is illustrated and the bar graphs show the analysis of all six images from each group. *p < 0.05 compared to untreated control. #p < 0.05 compared to vorapaxar + thrombin.

Article Snippet: Subsequently, the sections were incubated for 60 min with mouse monoclonal anti-CD31 (ready-touse; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11 + PD7/26; Dako), mouse monoclonal anti-α-smooth muscle actin (ready-to-use; clone 1A4; Dako), and rabbit polyclonal anti-PAR1 antibody (1:200, APR-031, Alomone Labs, Jerusalem, Israel).

Techniques: Migration, Recombinant, Inverted Microscopy, Software, Control