# Urokinase System in Pathogenesis of Pulmonary Fibrosis: A Hidden Threat of COVID-19
## 3 Discussion
These data allowed us to draw the following conclusion: uPAR downregulation, complemented by inactive uPA accumulation, led to augmented pulmonary fibrosis upon lung damage. Furthermore, we confirmed that the worsened pulmonary fibrosis in _Plaur-/-_ mice was accompanied by IL-6 upregulation (Figure 6C), which was in agreement with the previously established link between uPA and IL-6 in lung fibrosis [17]. IL-6, in turn, is known to upregulate ACE2, the \"SARS-CoV-2 receptor\" [39], and, indeed, ACE2 was upregulated in _Plaur-_/- mice lungs after bleomycin-induced lung injury (Figure 6D,E). Our findings indicate that uPAR downregulation and the increased uPA levels in lung tissue of COVID-19 patients may substantially aggravate pulmonary fibrosis and lead to IL-6 and ACE2 upregulation. High plasma levels of IL-6 are strongly associated with lower survival in COVID-19 patients [63; 64; 65]. ACE2 enhances the interaction of SARS-CoV-2 with epithelial cells, exacerbating the disease and COVID-19 prognosis at large [66; 67; 68]. As reported earlier, both uPAR and IL-6 regulate EMT progression [41; 42; 43], a process that actively contributes to pulmonary fibrosis upon lung injury [40]. Here we demonstrate that significantly more severe lung damage in _Plaur-_/- mice can also result from EMT induction: the expression of \(\alpha\)-smooth muscle actin (_Acta_2), a mesenchymal marker, was significantly increased in _Plaur-_/- mice compared to WT mice (Figure 7C). Modeling uPAR downregulation in lung epithelial cell lines A549 and BEAS-2B showed similar results: uPAR downregulation increased N-cadherin expression (a mesenchymal marker) (Figure 8E,F), indicating the EMT induction. We also observed characteristic morphologic changes in lung epithelial cells with downregulated uPAR, which exhibited a more rounded and spread morphology (Figure 8C,D), in accordance with the previous published data on neuroblastoma, melanoma and colon cancer cells [43; 69; 70]. Our current results suggest that uPAR is a factor that sustains the epithelial phenotype in lung epithelial cells, and the absence of uPAR in lung tissue may additionally stimulate EMT induced by pulmonary fibrosis, worsening lung damage and overall outcome. Moreover, our data correlate well with the previous report on the role of uPAR in alveolar type II epithelial cells EMT, which showed that upon exposure to bleomycin, uPAR-deficient alveolar type II lung epithelial cells undergo increased EMT [50]. Coming full circle, we have previously shown that uPAR downregulation induces uPA- and IL-6-dependent EMT in Neuro2a cells [50]. Apart from uPAR, uPA can be activated by plasmin in a powerful positive feedback loop [24; 25]; hence, excessive lung fibrosis, associated with low uPAR levels and the accumulation of inactive uPA, can be resolved by plasmin or its precursor plasminogen. For the first time, we demonstrated that the introduction of the external plasminogen could indeed disrupt this vicious circle and reverse lung fibrosis in the absence of uPAR: the intravenous injection of 1 mg of plasminogen on the 21 st day of bleomycin-induced fibrosis in _Plaur-_/- mice resulted in a 2-fold reduction in the fibrosis area in the lungs as compared to the non-treated mice by day 42 (Figure 9). Although initially, plasminogen is not efficiently activated in the presence of inactive uPA, the reaction can still be initiated by the low intrinsic catalytic activity of pro-uPA. Following the lag-phase, plasmin can be exponentially generated through the feedback loop when pro-uPA is activated by the already generated plasmin [26]. The described mechanism can provide a rationale for the effect of plasminogen treatment on pulmonary fibrosis resolution being detected three weeks after its injection (Figure 9). Low blood levels of plasmin and its precursor plasminogen correlate with a high risk for mortality and are associated with IL-6 upregulation in COVID-19 patients [27; 28; 44], reflecting its potential contribution to profibrogenic uPA accumulation and low-level fibrinolysis.