1. The vitronectin-binding function of PAI-1 exacerbates lung fibrosis in mice
by Anthony J. Courey, Jeffrey C. Horowitz, Kevin K. Kim, Timothy J. Koh, Margaret L. Novak, Natalya Subbotina, Mark Warnock, Bing Xue, Andrew K. Cunningham, Yujing Lin, Monica P. Goldklang, Richard H. Simon, Daniel A. Lawrence, and Thomas H. Sisson
Blood
Volume 118(8):
August 25, 2011
©2011 by American Society of Hematology

2. PAI-1 protein concentrations in bleomycin-injured WT mice.
PAI-1 protein concentrations in bleomycin-injured WT mice. WT mice were given intratracheal bleomycin (1.15 μg/kg in 50 μL of sterile PBS) on day 0. On day 9 or day 19, animals were killed and plasma and bronchoalveolar lavage (BAL) fluid were collected. The concentrations of total and active PAI-1 were measured with a bead-based ELISA. (A) Total PAI-1 concentration in plasma. (B) Active PAI-1 concentration in plasma. (C) Total PAI-1 concentration in BAL fluid. (D) Active PAI-1 concentration in BAL fluid. Results are reported as the mean concentration in pg/mL ± SEM. N = 6-8 mice per time point.
Anthony J. Courey et al. Blood 2011;118:
©2011 by American Society of Hematology

3. Day 9 mutant PAI-1 protein concentrations in bleomycin-injured PAI-1−/− mice.
Day 9 mutant PAI-1 protein concentrations in bleomycin-injured PAI-1−/− mice. PAI-1−/− mice were given intratracheal bleomycin (1.15 μg/kg in 50 μL of sterile PBS) on day 0. On day 8, subgroups of mice were treated with either intraperitoneal injections of PBS or 100 μg/injection every 12 hours of PAI-1(V+P+), PAI-1(V+P-) or PAI-1(V-P+). On day 9, 1 hour after the last injection, plasma and BAL fluid were collected from each animal and the concentrations of total and active PAI-1 were measured with a bead-based ELISA. (A) Total PAI-1 concentration in plasma. (B) Active PAI-1 concentration in plasma. (C) Total PAI-1 concentration in BAL fluid. (D) Active PAI-1 concentration in BAL fluid. Results are reported as the mean concentration in ng/mL ± SEM. N = 5 mice per group. Groups are compared with a 1-way ANOVA with Newman-Kuels posthoc multiple comparison test.
Anthony J. Courey et al. Blood 2011;118:
©2011 by American Society of Hematology

4. Day 9 bronchoalveolar lavage fluid fibrinolytic activity.
Day 9 bronchoalveolar lavage fluid fibrinolytic activity. PAI-1−/− and WT mice were treated with intratracheal bleomycin (1.15 μg/kg in 50 μL of sterile PBS) on day 0. On day 8, subgroups of PAI-1−/− animals were treated with either intraperitoneal injections of PBS or 100 μg/injection every 12 hours of PAI-1(V+P+), PAI-1(V+P-) or PAI-1(V-P+). WT mice received the same dosing regimen of intraperitoneal PBS. On day 9, 1 hour after the last injection, BAL fluid was collected from each animal and the amount of fibrinolytic activity was measured using a clot lysis assay. Results are reported as a scatter plot of t1/2, which represents the time in minutes required for 50% of the clot to be degraded (as assessed by absorbance at 405nm). N = mice. Groups are statistically compared with a Kruskal-Wallis test with post hoc group comparison using the Dunn Multiple Comparison Test.
Anthony J. Courey et al. Blood 2011;118:
©2011 by American Society of Hematology

5. Lung hydroxyproline concentration after bleomycin injury.
Lung hydroxyproline concentration after bleomycin injury. PAI-1−/− and WT mice were treated with intratracheal bleomycin (1.15 μg/kg in 50 μL of sterile PBS) on day 0. PAI-1(V+P+), PAI-1(V+P-), and PAI-1(V-P+) were administered to subgroups of injured PAI-1−/− animals at 100 μg/injection every 12 hours beginning on day 8 and continuing through day 19. Control groups in each of the 3 studies included bleomycin-injured PAI-1−/− mice that received intraperitoneal PBS, bleomycin-injured WT mice that received intraperitoneal PBS, and untreated WT mice. On day 19, lung hydroxyproline levels were measured as an indicator of collagen content. Hydroxyproline results from the 3 studies were pooled and are normalized to the extent of fibrosis measured in the bleomycin treated WT group which is set at a 100% increase in lung collagen above baseline ± SEM. N = mice per group in each study. Groups are compared with a 1-way ANOVA with Newman-Kuels posthoc multiple comparison test.
Anthony J. Courey et al. Blood 2011;118:
©2011 by American Society of Hematology

6. Lung histology after bleomycin injury.
Lung histology after bleomycin injury. PAI-1−/− and WT mice were treated with intratracheal bleomycin (1.15 μg/kg in 50 μL of sterile PBS) on day 0. Subgroups of PAI-1−/− mice were administered PAI-1(V+P+), PAI-1(V+P-), and PAI-1(V-P+) at 100 μg/injection every 12 hours beginning on day 8 and continuing through day 19. For comparison, groups of bleomycin-injured PAI-1−/− mice and WT mice received twice-daily intraperitoneal injections of PBS. On day 19, the left lung was fixed, and 5 micron sections were stained using Masson's trichrome (A) or immunostained with antibodies to α-SMA (B) and F4/80 (C). Images of representative sections from 2 mice (Masson trichrome) or a single mouse (immunostaining) in each treatment group are shown. Micrographs for panels A and B were obtained with a Nikon Eclipse E400 microscope (Melville, NY) and a Nikon 10×/0.25 lens (A) and a Nikon 40×/0.65 lens (B) and captured with a Diagnostic Instruments camera and SPOT Basic Version acquisition software. Micrographs for panel C were obtained with a Nikon Eclipse 80i microscope and a Nikon 20× lens and captured with a Nikon DS-Qi1Mc camera and Nikon NIS-Elements BR 3.10 software.
Anthony J. Courey et al. Blood 2011;118:
©2011 by American Society of Hematology

7. Day 21 mutant PAI-1 protein concentrations in bleomycin-injured transgenic mice.
Day 21 mutant PAI-1 protein concentrations in bleomycin-injured transgenic mice. PAI-1−/− mice that transgenically express PAI-1(V+P+), PAI-1(V+P-) or PAI-1(V-P+) off of the preproendothelin promoter were treated with intratracheal bleomycin (1.15μg/kg in 50 μL of sterile PBS) on day 0. Groups of WT and PAI-1−/− mice were also treated with bleomycin. A control group of WT mice was left uninjured. On day 21, bronchoalveolar lavage fluid (BAL) was collected from each animal and the concentrations of total (A) and active (B) PAI-1 were measured with a bead-based ELISA. Results are reported as the mean concentration in pg/mL ± SEM. N = 6-8 mice per time point. Groups are compared with a 1-way ANOVA with Newman-Kuels posthoc multiple comparison test.
Anthony J. Courey et al. Blood 2011;118:
©2011 by American Society of Hematology

8. Day 21 lung hydroxyproline concentrations in bleomycin-injured transgenic mice.
Day 21 lung hydroxyproline concentrations in bleomycin-injured transgenic mice. PAI-1−/− mice that transgenically express PAI-1(V+P+), PAI-1(V+P-) or PAI-1(V-P+) off of the preproendothelin promoter were treated with intratracheal bleomycin (1.15 μg/kg in 50 μL of sterile PBS) on day 0. Groups of WT and PAI-1−/− mice were also treated with bleomycin. A control group of WT mice was left uninjured and was used to establish a baseline level of lung collagen. On day 21, lungs were harvested from each animal and the concentration of hydroxyproline was measured. Hydroxyproline results from the 2 separate studies were pooled and are normalized to the extent of fibrosis measured in the bleomycin treated WT which is set at a 100% increase in lung collagen above baseline ± SEM. N = 6-8 mice per time point. Groups are compared with a 1-way ANOVA with Newman-Kuels posthoc multiple comparison test.
Anthony J. Courey et al. Blood 2011;118:
©2011 by American Society of Hematology