# 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  
Having identified that uPA and uPAR were implicated in pulmonary fibrosis in COVID-19 and PF patients, we revealed that their expression was significantly decreased in epithelial basal cells. Next, to scrutinize the sequelae of uPA and uPAR downregulation, we used the model of pulmonary fibrosis in _Plau-_ and _Plaur-deficient mice. Figure 4: uPA and uPAR immunoreactive cells were detected in lung tissue of COVID-19 patients. (**A**) Representative sections of parenchymal tissue from COVID-19 patients and control healthy donors. uPA and uPAR were stained and revealed with ImmPACT Vector Red Substrate (uPA, pink) and DAB HRP Substrate (uPAR, brown). Scale bars—100 μm (upper panels) and 300 μm (lower panels). (**B**) uPA and (**C**) uPAR staining scores in the images of lung tissue sections of COVID-19 patients and control healthy donors. Images were analyzed using the Positive Pixel Count v9 algorithm of ImageScope (Aperio), which counts pixels of predetermined color (pink for uPA and brown for uPAR, positive pixels) and pixels related to other colors (negative pixels). A staining score was calculated as the number of positive pixels/(number of positive + negative pixels). Data are presented as median and interquartile range. * \(p<0.05\), Mann-Whitney test. We used the bleomycin-induced lung injury model, which mimics the major features of pulmonary fibrosis in humans, in order to study the mechanisms of pulmonary fibrosis in wild-type (WT), _Plau_-deficient (_Plau-/-_) and _Plaur_-deficient (_Plaur-_/-) mice. Isotonic NaCl instillation was used as a control. The mice were monitored regularly for a 28-day period. Both WT and _Plau-/-_ mice survived bleomycin-induced lung injury well; however, _Plaur-/-_ mice demonstrated a significant mortality rate, with the overall survival being 36% at day 28 (log-rank test, \(p\) < 0.001, Figure 5A). Zero mortality rate was observed in the NaCl control group for all genotypes. Figure 5: Differential progress of pulmonary fibrosis between wild-type (WT) and _Plau_-deficient (_Plau-_/-) or _Plaur_-deficient (_Plaur-_/-) mice after instillation with bleomycin. Bleomycin (3 mg/kg) was administered into WT, _Plau-_/- and _Plaur-_/- mice by intratracheal instillation at day 0. Instillation of NaCl solution was used as a control. (**A**) [PERSON]–Meier survival curves, % \(\pm\) SE, for WT, _Plau-_/- and _Plaur-_/- mice following intratracheal instillation of bleomycin. ***_p_ < 0.001, log-rank test. (**B**) Mice were weighed before bleomycin or NaCl administration (day 0) and 28 days after. Body weight at day 28 divided by body weight day 0 (weight ratio) individual values, mean \(\pm\) SEM are presented. *_p_ < 0.05, 2-way ANOVA, [PERSON]–[PERSON]’s test. (**C**) The % of lung tissue with fibrosis at day 0 and day 28 of bleomycin intratracheal instillation in WT, _Plau-_/- and _Plaur-_/- mice. *_p_ < 0.05, **_p_ < 0.01, ***_p_ < 0.001, 2-way ANOVA, [PERSON]–[PERSON]’s test. (**D**) Typical MRI images obtained 28 days after bleomycin administration to WT, _Plau-_/- and _Plaur-_/- mice, the affected lung areas on the MRI images appear light, and the intact lungs appear dark. Asterisk indicates the area of severe lung damage. (**E**) Representative micrographs of 5-\(\upmu\) paraffin lung tissue sections stained with hematoxylin and eosin (H&E), Mallory stain (detection of total collagen), picrosirius red stain (selective detection of type I and III collagen) and phosphotungstic acid-hematoxylin stain (PTAH, detection of fibrin). Histological sections (HS) of lung tissues of WT, _Plau-_/- and _Plaur-_/- mice 28 days after bleomycin administration are presented. B, bronchus or bronchiole; F, fibrosis; V, vessel. The arrow points to collagen fibers in the pulmonary interstitium in the area of fibrosis. Scale bar—300 \(\upmu\)m. (**F**) The analysis of collagen deposition in WT, _Plau-_/- and _Plaur-_/- mice 7, 14, 21 and 28 days after bleomycin administration by picrosirius red stain.