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

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) sCD13 showed significant correlations with Cit-H3 ( n = 172) and MPO-DNA ( n = 172) in patients with COVID-19 while S100 A8/A9 ( n = 172) showed a positive slope but not a statistically significant correlation. ( B ) Representative images of neutrophils isolated from peripheral blood and analyzed after stimulation with PBS or sCD13. Panels show merged images of NETs in which neutrophil elastase was stained green by immunofluorescence and DNA was stained blue by Hoechst 33342. n = 3 technical replicates. Scale bar: 100 μm. ( C ) sCD13 blocked the staining of PAR4-5F10 antibodies, which recognize only the inactivated/uncleaved form of PAR4 yet had no effect on PAR4-FITC antibodies, which recognize both the activated and inactivated forms. n = 3 technical replicates, repeated 2 times. Original magnification, ×1,000. ( D ) sCD13-induced NETosis was blocked by B1R inhibitor SSR-240612 and PAR4 inhibitor BMS-986120 (all n = 3). ( E ) PMA-induced NETosis was not impacted by the B1R or PAR4 inhibitors (all n = 3). Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Significance was determined by Spearman’s test ( A ) and 1-way ANOVA ( D and E ).

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Isolation, Staining, Immunofluorescence

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: Single-cell RNA-Seq results of nasopharyngeal/pharyngeal swabs, bronchial brushings and bronchial lavages were generated from patients with COVID-19 ( n = 19) and healthy controls ( n = 5). Data are extracted from Chua et al. . ( A ) Both ANPEP (codes for CD13) and MMP14 are expressed on various epithelial cells and macrophages. ANPEP is also expressed on neutrophils while MMP14 is expressed on mast cells. BDKRB1 (codes for B1R) is expressed on some epithelial cells with the highest expression on secretory cells and ciliated cells. F2RL3 (codes for PAR4) was barely detected in this dataset but seemed to be expressed by epithelial cells. Cell abbreviations are defined in . ( B ) Cellular ANPEP expression in nasopharyngeal cells is elevated in patients with COVID-19 (moderate COVID-19 n = 8, severe COVID-19 n = 11) compared with healthy controls ( n = 5). ( C ) Using the median expression levels of ANPEP in patients, patients with COVID-19 were divided into 2 populations: ANPEP -high and ANPEP -low. Differentially expressed genes in neutrophils from these 2 groups were shown in the volcano plot ( P < 1 × 10 –20 , |log 2 (fold-change)| < 0.25). ( D ) Pathway analysis of the differentially expressed genes in ANPEP -high and ANPEP -low neutrophils showed pathways related to neutrophil degranulation, leukocyte activation, and inflammation. ( E ) The neutrophils from ANPEP -high patients with COVID-19 ( n = 10) showed a gene signature of immature-like neutrophils characterized by the overexpression of genes coding for several granule-content proteins (healthy controls n = 5, ANPEP -low n = 9). ( F ) The score for neutrophil immaturity was higher in critically ill patients with COVID-19 ( n = 11) compared with moderate patients (left, n = 8). The median neutrophil-immaturity score of neutrophils in ANPEP -high patients ( n = 10) was lower than that in ANPEP -low patients (right, n = 9). ( G ) The neutrophil-immaturity signature was most prominent in ANPEP -high patients ( n = 10) as developing neutrophils versus mature neutrophils from the peripheral blood were examined (healthy controls n = 5, ANPEP -low n = 9). Results are expressed as mean ± SD. **** P < 0.0001. Significance was determined by 1-way ANOVA ( B and G ) and Mann-Whitney test ( F ).

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Single Cell, RNA Sequencing, Generated, Expressing, Analysis, Activation Assay, Over Expression, MANN-WHITNEY, Single Cell RNA-Seq

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) Gating scheme of flow cytometry analysis on monocytes and neutrophils. ( B ) CD13, B1R, and PAR4 were expressed on monocytes and neutrophils, while MMP14 showed minimal expression on these cells ( n = 3). ( C ) Histograms showing the changes in CD13, PAR4, and B1R expression in neutrophils and monocytes with or without IL-1β, TNF-α, or IL-6 stimulation. ( D ) Quantification of relative expression of CD13, PAR4, and B1R on neutrophils and monocytes after stimulation with IL-1β, TNF-α, or IL-6 compared with unstimulated control from 3 healthy donors. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001. Significance was determined by 1-way ANOVA. FMO, fluorescence minus 1 (gating control); NT, not treated.

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Flow Cytometry, Analysis, Expressing, Control, Fluorescence, FACS

Journal: JCI Insight

Article Title: Soluble CD13 is a potential mediator of neutrophil-induced thrombogenic inflammation in SARS-CoV-2 infection

doi: 10.1172/jci.insight.184975

Figure Lengend Snippet: ( A ) Reanalysis of single-cell RNA-Seq results generated from circulating human neutrophils revealed that immature neutrophils have higher ANPEP expression. Dot plot of ANPEP , IFN, and neutrophil maturity genes for each neutrophil cluster, showing the average expression level and the percentage of cells expressing the gene in each cluster. ( B ) Gating scheme of flow cytometry analysis on mature and immature neutrophils isolated from whole blood. CD10 + CD16 hi defines mature neutrophils while CD10 – CD16 lo defines immature neutrophils. ( C ) Significantly higher expression of CD13 and lower expression of PAR4 were observed in mature neutrophils compared with immature neutrophils while B1R expression showed similar levels. Data generated from 5–10 healthy controls. Results are expressed as mean ± SD. ** P < 0.01. Significance was determined by Wilcoxon’s test.

Article Snippet: White blood cells in the upper phase were washed with PBS containing 2 mM EDTA and 2% FBS and stained with fluorochrome-conjugated antibodies against human CD3 (PE-Cy7, BioLegend 300420), CD13 (PE, BioLegend 301703), CD14 (APC-Cy7, BioLegend 301820) CD16 (PerCPCy5.5, Tonbo 615-1619-T025), CD19 (BV510, BioLegend 302241), CD45 (APC-Cy7, Tonbo 25-0459-T100), CD56 (PE, BioLegend 362508), CD10 (Super Bright 702, Invitrogen 67-010-642), PAR4 (FITC, Alomone Labs APR-034-F), B1R (APC, LS Bio LS-C275585), MMP14 (APC, R&D Systems, Bio-Techne; FAB9181A), and CD66b (Pacific Blue, BioLegend 305111).

Techniques: Single Cell, RNA Sequencing, Generated, Expressing, Flow Cytometry, Analysis, Isolation, FACS

Journal:

Article Title:

doi:

Figure Lengend Snippet: PAR2-activation induces a depression of synaptic transmission at Schaffer collateral-CA1 synapses in the hippocampus. (A) Application of PAR2-agonist (10 μM AC55541) causes LTD. (B) Removal of the PAR2-agonist (10 μM AC55541) following induction of LTD does not affect the stability of synaptic depression. (C) In presence of a PAR2-antagonist (50 μM FSLLRY-NH 2 ) the PAR2-agonist (10 μM AC55541) is not able to induce synaptic depression. (D) Application of PAR2-agonist (10 μM AC55541) at different concentrations results in similar levels of synaptic depression. (E) In a two pathways experimental setting, low frequency stimulation (LFS, 1 Hz, 900 pulses) and PAR2-activation (10 μM AC55541) induce similar levels of LTD. (F) LFS-induced LTD is not blocked by the PAR2-antagonist. (G) In a two pathways experimental setting, the NMDAR-antagonist (50 μM APV) blocks both LFS-induced LTD and PAR2-induced LTD. (H) While the group I mGluR-antagonist MCPG (200 μM) partially affects LFS-LTD it does not influence PAR2-LTD. Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section, n = 12 slices for each experiment, refer to text for statistics.

Article Snippet: The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

Techniques: Activation Assay, Transmission Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: PAR2 and TRPV4 expression in the hippocampus. Immunohistochemistry discloses the expression of PAR2 and TRPV4 in the hippocampus. A comparable expression pattern is observed: high levels of PAR2 and TRPV4 are detected in CA1 stratum pyramidale (pcl, pyramidal cell layer; oriens, stratum oriens; rad, stratum radiatum; la-mol, stratum lacunosum-moleculare). No pronounced colocalization between PAR2 and GFAP was detected. Scale bars: 100 and 10 μm, n = 9 slices out of three animals.

Article Snippet: The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

Techniques: Expressing, Immunohistochemistry

Journal:

Article Title:

doi:

Figure Lengend Snippet: PAR2 induces LTD through the activation of TRPV4. (A) Application of TRPV4-agonist (2 μM RN1747) causes LTD. (B) Removal of the TRPV4-agonist (2 μM RN1747) following induction of LTD does not affect the stability of synaptic depression. (C) In presence of the TRPV4-antagonist (10 μM RN1734) the TRPV4-agonist is not able to induce synaptic depression. (D) In a two pathways experimental setting, low frequency stimulation (LFS, 1 Hz, 900 pulses) and TRPV4-agonist application induce similar levels of LTD. (E) LFS-induced LTD is not blocked by the TRPV4-antagonist. (F) Application of PAR2-agonist (10 μM AC55541) in presence of a TRPV4-antagonist (10 μM RN1734) blocks PAR2-induced LTD. (G) Application of TRPV4-agonist (2 μM RN1747) in presence of PAR2-antagonist (50 μM FSLLRY-NH 2 ) does not affect TRPV4-induced LTD. (H) Once PAR2-agonist mediated LTD is established, the TRPV4-agonist (2 μM RN1747) does not further de-potentiate a second pathway at adjusted response level (upward arrow). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section, n = 12 slices for each experiments, refer to text for statistics.

Article Snippet: The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

Techniques: Activation Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: TRPV4-mediated LTD depends on NMDAR-activity. (A) Similar to PAR2-induced LTD (c.f., Figures ), the NMDAR-antagonist (50 μM APV) blocks TRPV4 (2 μM RN1747)-induced LTD, while (B) application of a TRPV4-agonist (2 μM RN1747) induces LTD in presence of the mGluR-antagonist (200 μM MCGP). Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section.

Article Snippet: The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

Techniques: Activity Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: PAR2-mediated LTD is protein kinase A (PKA)-dependent. (A) PAR2-agonist (10 μM AC55541) in presence of a PKA-inhibitor (2 μM KT5720) KT5720 (2 μM) fails to induce LTD. (B) Application of a protein kinase C (PKC) inhibitor (2 μM GF109203x) does not affect the induction of PAR2-mediated LTD. Averaged EPSP are plotted versus time. Representative traces at indicated times (a, b) are shown on top of each section.

Article Snippet: The following primary antibodies were used for immunodetection: goat anti-PAR2 (sc-8205, Santa Cruz, 1:25), rabbit anti-TRPV4 (ACC-124, Alomone Labs, 1:50), rabbit anti-PAR2 (APR-032, Alomone Labs 1:500) and mouse anti-GFAP (G3893, Sigma-Aldrich, 1:2000).

Techniques: