# Urokinase System in Pathogenesis of Pulmonary Fibrosis: A Hidden Threat of COVID-19
## 2 Results
### Beomycin-Induced Pulmonary Fibrosis Was More Severe in Plaur-/-Mice
This assumption was further confirmed by uPA expression analysis in _Plaur-/-_ mice 7 days after bleomycin instillation: the absence of uPAR resulted in a profound accumulation of uPA in lung tissue following the bleomycin-induced damage as compared to WT mice (Figure 6B). Moreover, _IL6_ mRNA expression was significantly increased in _Plaur-/-_ mice 14 days after bleomycin instillation (_p_ < 0.05, 2-way ANOVA, [PERSON]'s test, Figure 6C), confirming uPA and IL-6 in lung fibrosis to be intertwined and closely linked. In turn, IL-6 is known to upregulate ACE2, a.k.a \"SARS-CoV-2 receptor\" [39]; thus, we also analyzed ACE2 expression in mice lungs after bleomycin-induced pulmonary fibrosis. We found that ACE2 was upregulated in _Plaur-/-_ mice at both: mRNA (_p_ < 0.01, 2-way ANOVA, [PERSON]'s test, Figure 6D) and protein levels (Figure 6E), correlating well with higher IL-6 expression in this group (Figure 6C). Figure 6: uPAR deficiency led to uPA accumulation and increased IL-6 and ACE2 expression in lung tissue following bleomycin-induced lung damage: (**A**) Representative sections of lung tissue from wild-type (WT) mice 7, 14 or 28 days after instillation with bleomycin, stained for uPAR. uPAR was revealed with DAB chromogen substrate (brown). The scale bar in images is 100 \(\upmu\)m. (**B**) Representative  
sections of lung tissue from wild-type (WT) and _Plaur_-deficient (_Plaur_-/-) mice 7 days after instillation with bleomycin, stained for uPA. uPA was revealed with DAB chromogen substrate (brown), and the slides were counterstaineded with hematoxylin (blue). The scale bar is 100 \(\upmu\)m. (**C**) _IL6_ mRNA expression analyzed by RT-qPCR. The mRNA level was normalized to _Rpl13a_ expression as a housekeeping gene; the normalization was carried out assuming the mean level of transcript in WT cells as 1. Data are presented as the mean \(\pm\) SEM. * \(p\) < 0.05, 2-way ANOVA, Holm-Sidak's test. (**D**) Representative sections of lung tissue from wild-type (WT) and _Plaur_-deficient (_Plaur_-/-) mice 28 days after instillation with bleomycin, stained for ACE2. ACE2 was revealed with DAB chromogen substrate (brown), and the slides were counterstaineded with hematoxylin (blue). The scale bar is 100 \(\upmu\)m. (**E**) _Ac2_ mRNA expression as analyzed by RT-qPCR. The mRNA level was normalized to _Rpl13a_ expression as a housekeeping gene; the normalization was carried out assuming the mean level of transcript in WT cells as 1. Data are presented as the mean \(\pm\) SEM. **_p_ < 0.01, 2-way ANOVA, [PERSON]'s test. EMT of alveolar epithelial cells is acknowledged to largely contribute to pulmonary fibrosis upon lung injury [40]. Since both uPAR and IL-6 considerably stimulate the EMT progression [41; 42; 43], we assumed that EMT induction in _Plaur_-/- mice could give a reason for more severe lung damage. We next addressed how the expression of EMT-inducing transcription factors, _Snai1_, _Snai2_, _Twist1_, _Twist2_, _Zeb1_ and _Zeb2_, changed in the lungs from WT and _Plaur_-/- mice at Day 7, 14, 21 and 28 after bleomycin-induced pulmonary fibrosis. The expression of _Twist1_ and _Twist2_ progressively increased on Day 21 and Day 28 after bleomycin instillation (Figure 7A,B), indicating their substantial contribution to EMT in our model. We found a tendency towards their higher expression in _Plaur_-/- mice as compared to WT control, especially for _Twist1_ at Day 28 (_p_ > 0.05, 2-way ANOVA, [PERSON]'s test), though statistically non-significant. Except for a moderate increase in _Snai1_ expression at Day 21 in WT mice (Figure S1A), we found no increase in the expression of the other transcription factors after Day 7 of bleomycin-induced lung injury in WT and _Plaur_-/- mice throughout the whole experiment (Figure S1B-D). Along with this, no changes were detected in _Cdh1_ (encodes for E-cadherin, an epithelial marker) and _Cdh1_2 expression (encodes for N-cadherin, a mesenchymal marker) (Figure S1E,F).