Fig 1: Possible pathological pathways driven by PM2.5-induced PAI-1 and the beneficial effect of PAI-1 inhibitor TM5614.Air pollution stressors increase the levels of proinflammatory and prothrombotic mediators/regulators that cause cellular and vascular dysfunction and contribute to cardiopulmonary vascular pathologies. Neutralization of PM2.5-induced PAI-1 with TM5614 reduces PM2.5-induced cardiopulmonary vascular pathologies. In the depicted model, the individual step and feed-back loop are supported by the present study and or previous published works by other investigators. 1. Air-pollutant: PM2.5 increases the level of PAI-1 (present study and Upadhyay et al., 2010; Budinger et al., 2011); 2. PAI-1 increases inflammation and apoptosis (This study and Kubala et al., 2018); 3. Inflammation increases ROS (Mittal et al., 2014); 4. Inflammation induces thrombin (Margetic S. 2012, Foley and Conway, 2016); 5. Thombin induces the levels of ROS (Carrim et al., 2015); 6. Thrombin increases inflammation/apoptosis (Lopez et al., 2007; Chen and Dorling, 2009; Danckwardt et al., 2013; Foley and Conway, 2016); 7. Thrombin increases PAI-1 level (Hsieh et al., 2019); 8. Thrombin induces cellular and vascular abnormalities (present study and Rabiet et al., 1994); 9. Increased cellular dysfunction, elevation of thrombin-induced fibrinogen to fibrin deposition leads to cardiopulmonary pathologies, hypertension, thrombosis (present study and Savoia et al., 2011), 10. The results of the present biochemical, histological, immunohistological and cellular studies provide evidence on the pivotal role of air-pollutant PM2.5-induced PAI-1 in cardiopulmonary vascular pathologies, and the efficacy of a novel PAI-1 inhibitor TM5614 in improving air pollutant-induced cardiopulmonary vascular pathologies. Red upward arrow indicates induction of pathological factors by air-pollutants. Red downward arrow indicate downregulation of anti-pathological factor. Blue downward arrow indicates amelioration of pathological events by PAI-1 inhibitor TM5614. Blue upward arrow indicates upregulation of anti-pathological factor.
Fig 2: Effect of PAI-1 inhibitor TM5614 on PM2.5-induced cellular apoptosis and inflammation in hearts.Hearts collected from 4 groups of mice (n = 4–6) were processed for immunohistochemistry using an anti-cleaved caspase 3 antibody (A) and anti-Mac3 antibody (C). Day 1–6: TM5614 (10 mg/kg/day); Day 7: PM2.5 (200 µg/mouse) or PBS instillation. After 72 h, hearts were collected and processed for immunohistochemistry. Representative images are reduced form of original 40X images. The levels of cleaved caspase 3 and Mac3 in several fields of each heart section were determined by ImageJ. Quantitative data are shown in (B) for cleaved caspase 3 and (D) for Mac3. Data are presented as Mean (blue bar) ± SEM (red bar). Gr1: PBS; Gr2: PM2.5; Gr3: TM5614; Gr4: TM5614 + PM2.5. Arrows indicate the presence of cleaved caspase 3 (A) and Mac3 (C) positive cells.
Fig 3: Expression and activity of thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) in vein endothelial cells and a rat model of deep venous thrombosis. (A and B) TAFI (A) and PAI-1 (B) protein expression levels after treatment with rivaroxaban in vein endothelial cells. (C and D) Activity of TAFI (C) and PAI-1 (D) was detected in vein endothelial cells after treatment with rivaroxaban or PBS. (E and F) Plasma concentration levels of TAFI (E) and PAI-1 (F) were decreased in serum in rivaroxaban-treated rat. (G and H) Activity of TAFI (G) and PAI-1 (H) was also decreased in rivaroxaban-treated rat. (I and J) Activity of fibrinolysis (I) and plasma concentration of fibrinolysis (J) in a rat model of deep venous thrombosis. All data are represented as means ± SEM of triplicate samples. One-way ANOVA revealed a significant effect. *P<0.05 and **P<0.01 vs. the control.
Fig 4: PHZ-OH inhibits coagulation and attenuates organ injury in mice challenged with LPS.WT or Casp11-/- mice were primed with LPS (0.4 mg/kg) for 7 h prior to an intervention of PHZ-OH (5 mg/kg) and/or a challenge of LPS (4 mg/kg for 6 h for SD-IVM images or 10 mg/kg for 12 h for coagulation markers). A Representative SD-IVM images showing the circulating blood (Red) and the occlusion (as indicated by arrows) of the liver microvasculature that was clued by the signal of circulating blood (Red) and the autofluorescence of hepatocytes (Green); B Percent occluded region of the microvasculature related to the imaging field; Plasma concentrations of PAI-1 and TAT (C), and Fibrinogen and D-dimer (D); E Fibrin concentrations in the lung and the liver; F IHC staining showing the fibrin deposition in the lung (Dark brown, as indicated by arrows). Data are mean ± SEM of three mice for (B) and six mice for (C–E) in one experiment. *P < 0.05. ns, not significant. Scale bar = 50 µm.
Fig 5: Effect of PAI-1 inhibitor TM5614 on PM2.5-induced inflammation in lungs.Lungs collected from 4 groups of mice (n = 4–6) were processed for immunohistochemistry using anti-pSTAT3 antibody (A,B) and antiMac3 antibody (C,D). Day 1–6: TM5614 (10 mg/kg/day); Day 7: PM2.5 (200 µg/mouse) or PBS instillation; Day 10: Lungs were collected and processed for immunohistochemistry. Representative images of pSTAT3 stained lung sections (A) and Mac3 stained lung sections (C) are shown. Images are reduced form of original 20X images. The levels of nuclear pSTAT3 in several fields of each lung section were determined by ImageJ software followed by statistical analysis. Quantitative data are shown in (B) for pSTAT3 and (D) for Mac3. Data presented as Mean (blue bar) ± SEM (red bar). Gr1: PBS; Gr2: PM2.5; Gr3: TM5614; Gr4: TM5614 + PM2.5. Arrows indicate the presence of pSTAT3 (A) and Mac3 (B) positive cells.
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