# 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
The mean % of tissue area \(\pm\) SEM with collagen deposition is presented. *_p_ < 0.05, ***_p_ < 0.001, ***_p_ < 0.0001, 2-way ANOVA, [PERSON]’s test. The increased sensitivity of _Plaur-/-_ mice to bleomycin-induced fibrosis was further confirmed by measuring the mice's weight. The weight ratio at day 28 relative to day 0 was significantly decreased in _Plaur-/-_ mice after bleomycin (2-way ANOVA, [PERSON]'s test, \(p\) < 0.05, Figure 5B) as compared to the NaCl group. The worsened survival and considerable weight loss in _Plaur-/-_ mice suggested more severe lung damage and uncontrolled fibrosis upon bleomycin instillation. Further, longitudinal magnetic resonance imaging (MRI) scans were used to quantitatively assess the lung damage by analyzing the difference in the % of fibrotic tissue relative to the total area at day 28 and day 0. We found that 28 days after bleomycin administration, _Plaur-/-_ mice exhibited more profound lung damage than WT and _Plau-/-_ mice, with 48 \(\pm\) 6% of lung tissue being affected (2-way ANOVA, [PERSON]'s test, \(p\) < 0.001, \(p\) < 0.05, Figure 5C). In _Plau_ deficient mice, the area of lung damage also increased, but less prominently: 30 \(\pm\) 6% and 16 \(\pm\) 2% of lung tissue were injured in _Plau-/-_ and WT mice, respectively (2-way ANOVA, [PERSON]'s test, \(p\) < 0.05, Figure 5C). Figure 5D shows typical MRI images obtained 28 days after bleomycin administration to all three mice genotypes, with the affected lung tissue appearing light on the MRI images and the intact one dark. These data allowed us to propose a hypothesis that uPAR contributed more significantly to the pathogenesis of pulmonary fibrosis than uPA. Pulmonary fibrosis is characterized by the development of aberrant and disorganized collagen deposition in the lungs. Therefore, we evaluated the uPA and uPAR- mediated effects on collagen deposition upon lung injury. Mice were lethally anesthetized 7, 14, 21 and 28 days after bleomycin administration; lungs were isolated, perfused and placed in paraffin for further slicing with a thickness of 5 um. Tissue samples were stained using H&E, Mallory stain (to detect the total collagen), picrosirius red stain (to selectively detect type I and III collagen), and phosphotungscit acid-hematoxylin stain (PTAH, detection of fibrin) (Figure 5E). The % of the area containing type I and III collagen was estimated by analyzing the images of the picrosirius red stain. Collagen progressively accumulated after bleomycin-induced pulmonary fibrosis in all mice; collagen was intensively deposited in both _Plau-/-_ and _Plaur-/-_ deficient mice starting from day 7 and throughout the whole observation period (2-way ANOVA, [PERSON]'s test, \(p\) < 0.0001, Figure 5F). Thus, the absence of both uPA and uPAR led to augmented pulmonary fibrosis upon lung damage. However, uPAR deficiency exerted a more drastic effect on lung damage, weight loss and decreased survival, which confirmed its critical role in regulating fibrin deposition and cell functioning in pulmonary fibrosis. uPAR expression in WT mice resulted in no major changes throughout the observational period, serving as a valid control for further experiments (Figure 6A). The observed dramatic effect of uPAR knockout on pulmonary fibrosis can be explained by the receptor's role in regulating the extracellular presence and activity of its ligand uPA in the lungs. Although uPA activates fibrinolytic plasminogen, the absence of uPAR can significantly decrease uPA activity [21]. Moreover, it has been previously demonstrated that the high level of uPA exerts a prominent pro-fibrogenic effect in idiopathic pulmonary fibrosis that involves uPA-dependent IL-6 upregulation [17]. uPAR is implicated in uPA internalization and lysosomal degradation [38]; thereby, the lack of uPAR in the lung tissue of COVID-19 patients (Figures 1 and 3) can contribute to excessive uPA accumulation (Figure 4), aggravating pulmonary fibrosis.