Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Expression of CD163 on PB monocytes was measured by FACS after 3 days in no-contact co-cultures with HT-1080-Luc-shPAI1 or HT-1080-Luc-scPAI1 cells, in the absence or presence of doxycycline. The MFIs from 2 independent experiments were normalized to the values obtained in co-cultures with HT1080-Luc-scPAI1 cells in the absence of doxycycline and averaged. Tests of difference in means between groups comprising independent, non-matched observations were based on analysis of variance (*p<0.5); B. MFIs of CD163, CD86 and CD80 on PB monocytes from 4 healthy blood donors cultured in the presence of rPAI-1, rPAI-1 R76E I91L and rPAI-1 T333R A335R (100 nM). The data represent the MFI value obtained for each independent experiment; C. Levels of IL-10 and IL-12 produced by PB monocytes from (B). D. Levels of IL-6 in same conditions as in (C). The data represent the mean (± SD) concentration in technical duplicate samples from four independent experiments. D-E. Analysis of GEO datasets (E) and RNA-seq TCGA datasets (F) for PAI-1 and IL-6 genes for indicated tumors. A two-way t-test was used to establish the significance of the correlation coefficients.

Article Snippet: The antibodies used for western blotting, flow cytometry and immunohistochemistry are listed in the . rPAI-1, rPAI-1 T333R A335R, rPAI-1 R76E I91L and RAP proteins were purchased from Molecular Innovations and used in experiments at a final concentration of 40 nM and 100 nM.

Techniques: Expressing, Cell Culture, Produced, Concentration Assay, RNA Sequencing Assay

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Phospho-Kinase Array of PB monocytes exposed to rPAI-1 and rPAI-1 T333R A335R (100 nM) for 10 min (left panel). The intensity of the signal was quantified as a fold change in signal intensity in rPAI-1-treated cells over rPAI-1 T333R A335R-treated cells (right panel); B. Top panel: western blot analysis of p38MAPK phosphorylation in lysates of PB monocytes obtained 10 min after exposure to rPAI-1 and rPAI-1 T333R A335R (100 nM). The data are representative of one of 3 independent experiments showing similar results. Lower panel: mean (± 95% CI) fold change in the ratio pp38MAPK:p38MAPK from 3 independent experiments. P values are based on analysis of variance of log10 ratios. C. Western blot analysis of phosphorylation of p38MAPK in lysates of PB monocytes 10 min after exposure to CM from HT1080-Luc-shPAI1 and HT1080-Luc-scPAI1 cultured in the presence or absence of doxycycline. Ratios of pp38MAPK:p38MAPK obtained by scanning densitometry are shown at the bottom of the gel. The data are representative of one of 2 independent experiments showing similar results; D. Western blot analysis of p38MAPK, pSTAT3 and STAT3 in PB monocytes transduced with p38MAPK siRNA and scrambled control siRNA and treated for 7 h with rPAI-1 (100 nM); E. Top panel: western blot analysis of pSTAT3 in PB monocytes exposed for 7 h to 100 nM rPAI-1, DMSO, or SB203580 (20 μM; 1 h preincubation) or their combination. Lower panel: IL-6 protein levels in the medium of PB monocytes under the conditions described in the top panel. The data represent the mean (± SD) of technical duplicates.

Article Snippet: The antibodies used for western blotting, flow cytometry and immunohistochemistry are listed in the . rPAI-1, rPAI-1 T333R A335R, rPAI-1 R76E I91L and RAP proteins were purchased from Molecular Innovations and used in experiments at a final concentration of 40 nM and 100 nM.

Techniques: Western Blot, Cell Culture, Transduction

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A-B. PAI-1 expression in HT-1080-Luc-shPAI1 (A), and A549-Luc-shPAI1 (B) cell lines after 3 days of doxycycline treatment. C-D. Migration of human monocytes towards conditioned medium generated from HT-1080-Luc-shPAI1 (C) and A549-Luc-shPAI1 (D) was determined using the Boyden migration assay; E-F. Migration of human monocytes in presence of rPAI-1, rPAI-1 R76E I91L, rPAI-1 T333R A335R (E) and in presence or absence of RAP (F). Data represent the mean number (± SD) of migrated cells in 9 random fields per filter in technical triplicate samples. Two-sided Student t test was applied for comparisons between the groups. The data from a biological duplicate experiment for E and F is presented in Figure S1.

Article Snippet: The antibodies used for western blotting, flow cytometry and immunohistochemistry are listed in the . rPAI-1, rPAI-1 T333R A335R, rPAI-1 R76E I91L and RAP proteins were purchased from Molecular Innovations and used in experiments at a final concentration of 40 nM and 100 nM.

Techniques: Expressing, Migration, Generated

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Upper panel: IL-6 mRNA expression level measured by qRT-PCR at indicated times in PB monocytes exposed to rPAI-1 (100 nM). The data represent levels normalized against GAPDH analyzed using 2−ΔCt method. Lower panel: corresponding IL-6 protein levels determined by ELISA on aliquots of the medium collected at indicated times. The data represent the mean (± SD) of technical duplicates; B. Western blot analysis of STAT3 phosphorylation in cell lysates of PB monocytes at indicated times after treatment with 40 nM rPAI-1; C. Western blot analysis of STAT3 phosphorylation (Y705) in cell lysates of PB monocytes co-cultured (no-contact) for 3 days with HT1080-Luc-shPAI1 cells in presence and absence of doxycycline. The bar diagram under each lane represents ratio of pSTAT3:STAT3; D. Western blot analysis of STAT3 phosphorylation of PB monocytes exposed to a PAI-1 blocking antibody (5 μg/mL) or isotype antibody and rPAI-1 (40 nM) for 5 h. Lower panel: IL-6 levels measured by ELISA in the medium of PB monocytes treated as indicated in the top panel. The data represent the mean IL-6 concentrations (± SD) of technical duplicates; E. Western blot analysis of STAT3 phosphorylation in lysates from PB monocytes cultured for 4 days in the presence of rPAI-1 and rPAI-1 mutants (40 nM). When indicated, rPAI-1 was heated for 60 min at 95°C. The numbers under each lane represent ratio of pSTAT3:STAT3; F. Western blot analysis of STAT3 phosphorylation in lysates of PB monocytes obtained after 6 days of treatment with rPAI-1 (40 nM), ruxolitinib (1 μM), tocilizumab (20 μg/mL), IgG (20 μg/mL) or DMSO or their combination. The numbers under each lane represent ratio of pSTAT3:STAT3. Lower panel: corresponding CD163 MFIs of PB monocytes treated as indicated in the upper panel. The data from an independent biological replicate of this experiment is shown in Figure S3; G. CD163 MFIs in PB monocytes after 3 days in no-contact co-cultures with HT1080 cells in the presence of DMSO, IgG, ruxolitinib or tocilizumab. The data represent the mean values of 2 independent experiments. Tests of difference in means between groups comprising independent, non-matched observations were based on analysis of variance.

Article Snippet: The antibodies used for western blotting, flow cytometry and immunohistochemistry are listed in the . rPAI-1, rPAI-1 T333R A335R, rPAI-1 R76E I91L and RAP proteins were purchased from Molecular Innovations and used in experiments at a final concentration of 40 nM and 100 nM.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Blocking Assay

Journal: Cell reports

Article Title: Plasminogen activator inhibitor-1 promotes the recruitment and polarization of macrophages in cancer

doi: 10.1016/j.celrep.2018.10.082

Figure Lengend Snippet: A. Immunofluorescence (IF) analysis of PB monocytes treated with 100 nM rPAI-1 and rPAI-1 T333R A335R for 2 h. When indicated PB monocytes were preincubated with SB203580 (20 μM) for 1 h (or DMSO as a control) and treated with rPAI-1 for 2 h. Cells were analyzed by confocal microscopy for NF-κB. NF-κB: green, DAPI: blue. Scale bar: 20 μm. The histogram represents the mean (± SD) % of nuclei staining positive for NF- κB from a total of 450 nuclei per sample in triplicate (*p<0.05; **p<0.01; ***p<0.001). B. Western blot analysis of p38MAPK and NF-κB p65 and their phosphorylated forms in lysates of PB monocytes exposed to 100 nM rPAI-1 or rPAI-1 T333R A335R for the indicated amount of time; C. Western blot analysis of NF-κB p65 and its phosphorylated form in lysates of PB monocytes exposed for 7 h to rPAI-1, DMSO or SB203580 alone or indicated combinations; D. Western blot analysis of p65 NF-κB, and STAT3 and its phosphorylated form in lysates of PB monocytes in which p65 was downregulated with siRNA and exposed for 7 h to 100 nM rPAI-1; E. Top panel: western blot analysis of STAT3 and its phosphorylated form in lysates of PB monocytes exposed for 7 h to rPAI-1, DMSO, BMS-345541 (5 μM; 1 h pre-incubation) or their combination. Lower panel: IL-6 levels in the medium of PB monocytes treated as indicated in the top panel. The data represent the mean concentrations (± SD) of technical duplicates.

Article Snippet: The antibodies used for western blotting, flow cytometry and immunohistochemistry are listed in the . rPAI-1, rPAI-1 T333R A335R, rPAI-1 R76E I91L and RAP proteins were purchased from Molecular Innovations and used in experiments at a final concentration of 40 nM and 100 nM.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Western Blot, Incubation

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome

doi: 10.1161/ATVBAHA.119.313775

Figure Lengend Snippet: Ldlr−/− mice were fed WD containing or lacking PAI-1 inhibitor for 12 weeks, after which aortic root atherosclerotic plaque composition was assessed. (A) PAI-039 decreases fibrous cap macrophage content. Quantified data (n=5/group; *P<0.05) and representative images demonstrating macrophage invasion (brown color) are shown. (B) MDI-2268 decreases fibrous cap macrophage content. Quantified data (n=6–7/group; *P<0.05) and representative images are shown. (C) MDI-2268 does not significantly affect intimal SMC content, assessed by SMC-α actin immunostaining (brown color). Quantified data (n=4–6/group, difference between groups did not achieve statistical significance; P>0.6) and representative images are shown. (D) MDI-2268 does not significantly affect plaque collagen content, assessed by picrosirius red (PSR) staining. Quantified data (n=6–7/group, difference between groups did not achieve statistical significance; P>0.08) and representative images are shown. L, lumen.

Article Snippet: 11 Recombinant human PAI-1 (Cat. #PAI-A) was from Molecular Innovations, Novi, MI.

Techniques: Immunostaining, Staining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome

doi: 10.1161/ATVBAHA.119.313775

Figure Lengend Snippet: (A) SMCs were incubated for 24 hours with recombinant PAI-1 (10 μg/mL), recombinant PAI-1 and PAI-039 (25 μM), or vehicle control, after which SA-βGal expression (% positive cells) was measured; n= 5–7/group; *P<0.05 vs. other groups. (B) SMCs (passage number 6–8) were incubated 12 hours with or without anti-LRP1 antibody (at indicated concentrations [μg/mL]), after which PAI-1 (1 μg/mL, “+”) or vehicle control (“-”) was added. Cells were incubated an additional 24 hours, after which SA-βGal expression was measured; n=5/group, *P<0.05 vs. control (untreated SMCs). (C) SMCs (passage number 7) were incubated 24 hours with PAI-1-I91L (recombinant PAI-1 mutant with increased stability, normal anti-protease activity, and normal LRP1-binding affinity, concentration 1 μg/mL), PAI-1-I91L,K80/207A (recombinant PAI-1 mutant with increased stability, normal anti-protease activity, and a greater than 20-fold reduction in LRP1-binding affinity; concentration 1 μg/mL), or vehicle control, after which SA-βGal expression was measured; n=4/group; *P<0.001 vs. other groups.

Article Snippet: 11 Recombinant human PAI-1 (Cat. #PAI-A) was from Molecular Innovations, Novi, MI.

Techniques: Incubation, Recombinant, Expressing, Mutagenesis, Activity Assay, Binding Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: Effect of AZ3976 on clot lysis in human plasma with added PAI-1. Before addition of plasma, 1.7 nm PAI-1 in buffer was preincubated briefly at 37 °C with 0 to 0.22 mm AZ3976. The reaction was started by dilution with plasma containing tPA to final concentration of 50% plasma, 1.2 nm tPA and 0.65 nm PAI-1. Fibrin formation was monitored from the change in A405 in a microplate reader at 2-min intervals at 37 °C for up to 10 h. A, example of plasma clot lysis experiment: A405 is plotted versus time. Curves are shown for PAI-1 with 82.5 (b), 41.25 (c), 20.6 (d), 10.3 (e), 5.16 (f), and 0 μm (g) AZ3976 and for tPA only (a), without added PAI-1. B, the IC50 determination. The clot lysis time was defined as the time to halve the amplitude on the negative slope minus the time to halve the amplitude on the positive slope where the maximal effect (100%) was the time without added PAI-1 and minimal effect (0%) the time without added compound. The line depicts the best fit using Grafit IC50 fit. The mean of four experiments is plotted ± S.E.

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: Lysis, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: Titration of glycosylated active and latent PAI-1 by isothermal calorimetry with AZ3976. Titration of 45 μm active PAI-1 with specific activity ∼75%, or 50 μm glycosylated latent PAI-1 in 50 mm sodium phosphate (pH 7.4), 100 mm NaCl, and 2% DMSO was achieved by injecting 20 × 0.5 μl and 20 × 1 μl aliquots, respectively, of 1 mm AZ3976 at 35 °C with 90 s waiting time between subsequent injections. The line depicts the best fit according to a single site binding model used to extract the thermodynamic properties of the interactions, which are shown in Table 2.

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: Titration, Activity Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: The structure of latent PAI-1 in complex with AZ3976. A, comparison of the latent PAI-1·AZ3976 holostructure with the latent PAI-1 apoform (PDB code 1DVN) (18). PAI-1 is colored by sequence from blue (N-terminal) to red (C-terminal). The inhibitor is shown in stick representation. The structure of the latent PAI-1 apostructure is shown as a gray ribbon representation. B, comparison of the AZ3976 binding site in ligand bound and free latent PAI-1. C, AZ3976 binding site in latent PAI-1. The Fo − Fc map calculated using the refined AZ3979-bound structure of latent PAI-1 is contoured at 3σ. D–F show the surface of the ligand binding site in three different forms of PAI-1. D, latent ligand complexed (this work); E, latent (PDB code 1DVN) (18); and F, active (PDB code 1DVM) (18). The magenta-colored surface describes the solvent accessible area in the region of AZ3979 binding. The nomenclature for the secondary structure is adopted from Stein and Chotia (50).

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: Sequencing, Binding Assay, Ligand Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: Interaction of PAI-1 with immobilized VN and effect of AZ3976. A, sensorgram overlay of the association and dissociation in real time after injection of 10 nm PAI-1 with 10 μm AZ3976 (a), incubated for increasing time intervals (arrow from 0 to 215 min) at 20 °C >900 RU immobilized VN, at change to buffer in the presence of 10 μm AZ3976 (b), and at injection of 20 μl of 25 nm tPA in the presence of 10 μm AZ3976 (c), followed by running buffer (d). The sensorgram of buffer only (dashed line) and reference, 10 nm PAI-1 in buffer without preincubation (marked ref) are included, whereas the sensorgrams of PAI-1 incubated in buffer are not shown for clarity. The ΔRU between the channel with immobilized VN, flow channel 2, and the channel without VN, flow channel 1, is shown. B, data are from the experiment shown in A. Initial binding rate to VN, measured after 10 s, as a function of the incubation time for 10 nm PAI-1 in buffer and for 10 nm PAI-1 with AZ3976 are shown. The lines are fitted to the data assuming single exponential decay, k = 0.0007 (buffer) and 0.01 min−1 (AZ3976), respectively.

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: Injection, Incubation, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: Interaction of PAI-1 with captured tPA and effect of incubation time with AZ3976. A, initial rate, measured as ΔRU, the change in RU 10 s after injection over tPA, as a function of the incubation time (min) of 10 nm PAI-1 with AZ3976 at different concentrations at 20 °C. Lines are drawn by fitting a single exponential decay to the data. B, rate constants from the data in A (circles) for single exponential decay of PAI-1, or from the data in Fig. 5 (triangles) from binding to VN, versus the concentration of AZ3976. The line was drawn by a linear fit with a slope of 14 m−1·s−1.

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: Incubation, Injection, Binding Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: Decay of PAI-1 activity and concomitant generation of latent PAI-1. A, sensorgram overlay of injection onto the monoclonal antibody H4B3 (specific for non-glycosylated latent PAI-1) captured on immobilized RamFc at 10 nm non-glycosylated active PAI-1 in buffer (dotted line) or with 20 μm AZ3976 for 1 h (solid line), or buffer alone (dashed line) at 20 °C (a). b, flow change to running buffer with 20 μm AZ3976. The ΔRU between the channel with H4B3 and the channel with the reference antibody is shown. B, values in % of ΔRU for 10 nm PAI-1, measured from the sensorgram 70 s after injection, which represent active PAI-1 bound to captured tPA and latent PAI-1 bound to H4B3, without AZ3976 and with 20 μm AZ3976 incubated with PAI-1 for 1 h at 20 °C.

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: Activity Assay, Injection, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: SPR sensorgrams for the interaction of AZ3976 with immobilized glycosylated latent and active PAI-1. Concentrations ranged from 20 μm to 27.4 nm following a 3-fold dilution scheme. The SPR experiments were run in PBSTD buffer at 25 °C, using a flow rate of 30 μl/min. The normalized responses were obtained by dividing the observed response levels with the predicted maximum response levels for a 1:1 binding interaction. The kinetic analysis was performed by fitting the data to a 1:1 binding model (fitted model traces as red dotted line). A, sensorgrams of AZ3976 binding to latent PAI. The normalized responses indicate a ligand binding activity of ∼90%. B, sensorgrams of AZ3976 binding to active PAI. The normalized responses indicate a ligand binding activity of ∼30%.

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: Binding Assay, Ligand Binding Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: Overview of inhibitor binding to PAI-1. A, the structure of active PAI-1 in complex with the somatomedin B domain of VN (PDB code 1OC0). PAI-1 is shown in a gray ribbon representation, except for the RCL, which is colored red (gap due to missing residues in structure). Somatomedin B is shown as a ribbon diagram in magenta. Additional residues implicated in VN binding are shown in stick representation colored in magenta and yellow. Residues implicated in binding of the small molecule PAI-1 inhibitor PAI-039 are shown in cyan and yellow, i.e. residues shown in yellow are implicated in both PAI-039 and VN binding. Glycosylation sites are shown in blue stick representation, and additional residues mentioned in the discussion are shown in green. B, the structure described in this work. The overall structure is colored the same way as in A, highlighting the different topology of the central β-sheet in the two structures. AZ3976 is shown in an orange stick representation and residues within 5 Å of the inhibitor are shown as cyan sticks.

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation *

doi: 10.1074/jbc.M112.371732

Figure Lengend Snippet: Mechanistic scheme of PAI-1 in presence of latency accelerating inhibitor. A represents active PAI-1, pL represents prelatent PAI-1, L represents latent PAI-1, and x indicates inhibitor.

Article Snippet: Active non-glycosylated human PAI-1 expressed in Escherichia coli was purchased from Molecular Innovations, Inc. (Novi, MI) and was shown to have a specific activity of 78%.

Techniques: