# Urokinase System in Pathogenesis of Pulmonary Fibrosis: A Hidden Threat of COVID-19
## 2 Results
### uPA and uPAR Were Differentially Expressed in Lung Tissue of COVID-19 Patients
Normalized expression levels of _PLAU_ (**B**) and _PLAU_ (**C**) in different cell types of mesenchymal cells; statistics were calculated only for the cell types with the median normalized expression value \(>\) 0. *** _Padistated_ < 0.0001 as assessed by FDR. IPF/other PF, idiopathic pulmonary fibrosis and other pulmonary fibrosis; umap, Uniform Manifold Approximation and Projection. patients, while in MoM1 and MoM3 cells, its expression was slightly elevated. Overall, _PLAU_ expression was low in all monocytes/macrophages in control and COVID-19 samples, but in PF, several AM2, MoM1 and MoM3 cells expressed _PLAU_ (Figure 3B). Therefore, the scRNA-seq data presented here demonstrated that COVID-19 and pulmonary fibrosis displayed remarkable similarities in _PLAU_/_PLAU_ gene expression across cell lineages. _PLAU_ expression was significantly decreased in airway basal cells and in lung monocytes/macrophages, the latter being the main source of _PLAU_ expression. Along with the generally decreased _PLAU_ expression in airway basal cells, specific monocytes/macrophages and fibroblasts from PF patients exhibited an upregulated _PLAU_ expression. Next, we analyzed the uPA and uPAR presence in the peripheral and hilar regions of lung tissue from COVID-19 patients and the control by immunohistochemistry (Figure 4). In line with the previous data, histopathological analysis of lung autopsy samples revealed diffuse alveolar damage and features of organizing pneumonia (Figure 4A) [37]. uPA and  
Figure 3: _PLAU_ and _PLAU_ expression in the immune cells of control, COVID-19 and PF lung samples: (**A**) UMAP embedding of jointly analyzed single-cell transcriptomes annotated by cell type (color) and disease status. Lung tissue transcriptomes of patients with COVID-19-associated pneumonia, pulmonary fibrosis (PF) and control lung samples from healthy donors, reported in [PERSON] et al. and [PERSON] et al. (GSE158127, GSE1535893 [35; 36]), were analyzed. Normalized expression levels of _PLAU_ (**B**) and _PLAU_ (**C**) in different types of immune cells; statistics were calculated only for cells with the median normalized expression value > 0. ns—non-significant, **\(p_{altistical}\)** < 0.001, ***\(p_{altistical}\)** < 0.0001 as assessed by FDR. AM, alveolar macrophages; IPF/other PF, idiopathic pulmonary fibrosis and other pulmonary fibrosis; Mo, monocyte-derived macrophages; umap, Uniform Manifold Approximation and Projection. uPAR mild immunoreactivity was detected in the alveolar epithelium of healthy control lung tissue (Figure 4A), which was consistent with scRNA-seq data that showed generally low levels of _PLAU_ and _PLAUR_ expression in epithelial and mesenchymal cells. The lung tissue from COVID-19 patients (\(n=4\)) was intensively stained for uPA, which was significantly higher than in the control (Mann-Whitney test, * \(p<0.05\), Figure 4A,B). uPAR staining was also detected in COVID-19 patients with no significant difference from the control (Figure 4A,C). Thus, uPA was excessively accumulated in the lesioned lung regions of COVID-19 patients, consistent with its reported contribution to pulmonary fibrosis [17]. However, according to scRNA-seq data, _PLAU_ expression increased only in certain cell types (KRT17+ KRT5-) in COVID-19 patients; uPA protein accumulation may be linked to abnormal internalization and lysosomal degradation, directed by uPAR [38]. The significant decrease in _PLAUR_ mRNA expression in basal epithelial cells and lung monocytes/macrophages (Figure 1) and its relative decrease at the background of the significantly increased amount of its ligand (Figure 4) might also contribute to the pathogenesis of pulmonary fibrosis in COVID-19.