# Urokinase System in Pathogenesis of Pulmonary Fibrosis: A Hidden Threat of COVID-19
## 2 Results
### Plasminogen Administration Reversed Bleomycin-Induced Lung Fibrosis in Plaur-/- Mice  
As we have previously shown, in the absence of uPAR, inactive uPA accumulation could lead to accelerated lung fibrosis and lower survival in lung damage conditions. Apart from uPAR, uPA can be activated by plasmin in a powerful positive feedback loop [24; 25]. Thus, low levels of plasmin or its precursor plasminogen in COVID-19 patients [28; 44] may contribute to profibrogenic uPA accumulation and low-level fibrinolysis. We hypothesized that the external introduction of plasminogen in the absence of uPAR might disrupt this vicious circle and reverse lung fibrosis. In order to verify this hypothesis, we induced lung fibrosis via bleomycin instillation in _Plaur-/-_ mice on Day 0 and intravenously injected mouse plasminogen (1 mg) or isotonic NaCl (control) on Day 21 (Figure 9A). Lung fibrosis was monitored weekly by MRI for 3 weeks. On day 28, no differences were detected between mice assigned to different groups (Figure 9B). On Day 35, lung fibrosis had a tendency to decrease in mice treated with plasminogen (2-way ANOVA, [PERSON]'s test, \(p\) = 0.0765), while on Day 42, there was a dramatic statistically significant difference in the lung fibrosis: 23.6 \(\pm\) 6.4% of the fibrotic area in plasminogen-treated mice vs. 54.5 \(\pm\) 3.0% of the fibrotic area in control mice (2-way ANOVA, [PERSON]'s test, \(p\) < 0.001) (Figure 9B). Therefore, bleomycin-induced lung fibrosis progressively resolved in plasminogen-treated mice (Figure 9C). Figure S2 shows the individual % of lung fibrosis for each mouse enrolled in the experiment. Hence, the plasminogen treatment reverses lung fibrosis upon lung damage, which can serve as a promising therapeutic target for pulmonary fibrosis. Figure 8: uPAR expression affected the morphology of human lung epithelial cells and changed the expression of EMT-related markers: (**A**) The level of uPAR protein expression in the obtained  
A549-based stable cell lines. \(\beta\)-actin was used as a control of protein load. Densitometric analysis of uPAR band intensities normalized to \(\beta\)-actin band intensities is presented relative to A549 scrambled shRNA sample. (**B**) The level of uPAR protein expression in BEAS-2B-based stable cell lines. \(\beta\)-actin was used as a control of protein load. Densitometric analysis of uPAR band intensities normalized to \(\beta\)-actin is presented relative to A549 scrambled shRNA sample. (**C**) Representative images of A549 cells with downregulated uPAR expression (shPLAUR) and respective control (scrambled shRNA), scale bar--100 \(\upmu\)m (_left panel_). Results of cell area measurements of at least 100 cells for each cell type are presented (_right panel_). (**D**) Representative images of BEAS-2B cells with downregulated uPAR expression (shPLAUR) and respective control (scrambled shRNA), scale bar--100 \(\upmu\)m (_left panel_). Results of cell area measurements of at least 100 cells for each cell type are presented (_right panel_). Data are presented as the mean \(\pm\) SEM. ***** \(p\) < 0.0001, \(t\)-test. (**E**) A549 and (**F**) BEAS-2B cells with downregulated uPAR expression (shPLAUR) and respective control (scrambled shRNA) were cultured in the absence or presence of 20 nM of uPA, and 24 h later, a Western blot analysis of E-cadherin and N-cadherin EMT markers expression was performed. \(\beta\)-actin was used as a control of protein load. Reproducible results of three independent experiments are presented. Densitometric analysis of band intensities for each EMT marker is presented as the mean \(\pm\) SEM. * \(p\) < 0.05, ** \(p\) < 0.01, ** \(p\) < 0.001, ANOVA, [PERSON]'s post hoc test. **  
Figure 9: Treatment with plasminogen decreased bleomycin-induced lung fibrosis. (**A**) Overview of the experimental design. _Plaur-/-_ mice were subjected to intratracheal instillation with bleomycin (3 mg/kg)  
at day 0. On Day 21, mice were treated with 1 mg plasminogen (\(n=6\)) or isotonic NaCl (control, \(n=7\)) intravenously (i.v.).