Urokinase System in Pathogenesis of Pulmonary Fibrosis: A Hidden Threat of COVID-19.

Pulmonary fibrosis is a common and threatening post-COVID-19 complication with poorly resolved molecular mechanisms and no established treatment. The plasminogen activator system, including urokinase (uPA) and urokinase receptor (uPAR), is involved in the pathogenesis of COVID-19 and contributes to the development of lung injury and post-COVID-19 pulmonary fibrosis, although their cellular and molecular underpinnings still remain obscure. The aim of the current study was to assess the role of uPA and uPAR in the pathogenesis of pulmonary fibrosis. We analyzed uPA and uPAR expression in human lung tissues from COVID-19 patients with pulmonary fibrosis using single-cell RNA-seq and immunohistochemistry. We modeled lung fibrosis in Plau-/- and Plaur-/- mice upon bleomycin instillation and explored the effect of uPAR downregulation in A549 and BEAS-2B lung epithelial cells. We found that uPAR expression drastically decreased in the epithelial airway basal cells and monocyte/macrophage cells, whereas uPA accumulation significantly increased in tissue samples of COVID-19 patients. Lung injury and fibrosis in Plaur-/- vs. WT mice upon bleomycin instillation revealed that uPAR deficiency resulted in pro-fibrogenic uPA accumulation, IL-6 and ACE2 upregulation in lung tissues and was associated with severe fibrosis, weight loss and poor survival. uPAR downregulation in A549 and BEAS-2B was linked to an increased N-cadherin expression, indicating the onset of epithelial-mesenchymal transition and potentially contributing to pulmonary fibrosis. Here for the first time, we demonstrate that plasminogen treatment reversed lung fibrosis in Plaur-/- mice: the intravenous injection of 1 mg of plasminogen on the 21st day of bleomycin-induced fibrosis resulted in a more than a two-fold decrease in the area of lung fibrosis as compared to non-treated mice as evaluated by the 42nd day. The expression and function of the plasminogen activator system are dysregulated upon COVID-19 infection, leading to excessive pulmonary fibrosis and worsening the prognosis. The potential of plasminogen as a life-saving treatment for non-resolving post-COVID-19 pulmonary fibrosis warrants further investigation.

T-Cadherin Deficiency Is Associated with Increased Blood Pressure after Physical Activity.

T-cadherin is a regulator of blood vessel remodeling and angiogenesis, involved in adiponectin-mediated protective effects in the cardiovascular system and in skeletal muscles. GWAS study has previously demonstrated a SNP in the Cdh13 gene to be associated with hypertension. However, the role of T-cadherin in regulating blood pressure has not been experimentally elucidated. Herein, we generated Cdh13∆Exon3 mice lacking exon 3 in the Cdh13 gene and described their phenotype. Cdh13∆Exon3 mice exhibited normal gross morphology, life expectancy, and breeding capacity. Meanwhile, their body weight was considerably lower than of WT mice. When running on a treadmill, the time spent running and the distance covered by Cdh13∆Exon3 mice was similar to that of WT. The resting blood pressure in Cdh13∆Exon3 mice was slightly higher than in WT, however, upon intensive physical training their systolic blood pressure was significantly elevated. While adiponectin content in the myocardium of Cdh13∆Exon3 and WT mice was within the same range, adiponectin plasma level was 4.37-fold higher in Cdh13∆Exon3 mice. Moreover, intensive physical training augmented the AMPK phosphorylation in the skeletal muscles and myocardium of Cdh13∆Exon3 mice as compared to WT. Our data highlight a critically important role of T-cadherin in regulation of blood pressure and stamina in mice, and may shed light on the pathogenesis of hypertension.

The Association of PLAUR Genotype and Soluble suPAR Serum Level with COVID-19-Related Lung Damage Severity.

Uncovering the risk factors for acute respiratory disease coronavirus 2019 (COVID-19) severity may help to provide a valuable tool for early patient stratification and proper treatment implementation, improving the patient outcome and lowering the burden on the healthcare system. Here we report the results of a single-center retrospective cohort study on 151 severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected symptomatic hospitalized adult patients. We assessed the association of several blood test measurements, soluble urokinase receptor (uPAR) serum level and specific single nucleotide polymorphisms of ACE (I/D), NOS3 (rs2070744, rs1799983), SERPINE1 (rs1799768), PLAU (rs2227564) and PLAUR (rs344781, rs2302524) genes, with the disease severity classified by the percentage of lung involvement on computerized tomography scans. Our findings reveal that the T/C genotype of PLAUR rs2302524 was independently associated with a less severe lung damage (odds ratio 0.258 [0.071-0.811]). Along with high C-reactive protein, fibrinogen and soluble uPAR serum levels turned out to be independently associated with more severe lung damage in COVID-19 patients. The identified factors may be further employed as predictors of a possibly severe COVID-19 clinical course.

MicroRNAs in Cancer: From Gene Expression Regulation to the Metastatic Niche Reprogramming.

By 2003, the Human Genome project had been completed; however, it turned out that 97% of genome sequences did not encode proteins. The explanation came later when it was found the untranslated DNA contain sequences for short microRNAs (miRNAs) and long noncoding RNAs that did not produce any mRNAs or tRNAs, but instead were involved in the regulation of gene expression. Initially identified in the cytoplasm, miRNAs have been found in all cell compartments, where their functions are not limited to the degradation of target mRNAs. miRNAs that are secreted into the extracellular space as components of exosomes or as complexes with proteins, participate in morphogenesis, regeneration, oncogenesis, metastasis, and chemoresistance of tumor cells. miRNAs play a dual role in oncogenesis: on one hand, they act as oncogene suppressors; on the other hand, they function as oncogenes themselves and inactivate oncosuppressors, stimulate tumor neoangiogenesis, and mediate immunosuppressive processes in the tumors, The review presents current concepts of the miRNA biogenesis and their functions in the cytoplasm and nucleus with special focus on the noncanonical mechanisms of gene regulation by miRNAs and involvement of miRNAs in oncogenesis, as well as the authors' opinion on the role of miRNAs in metastasis and formation of the premetastatic niche.

Early Induction of Neurotrophin Receptor and miRNA Genes in Mouse Brain after Pentilenetetrazole-Induced Neuronal Activity.

Neurotrophin receptors regulate neuronal survival and network formation, as well as synaptic plasticity in the brain via interaction with their ligands. Here, we examined early changes in the expression of neurotrophin receptor genes Ntk1 (TrkA), Ntrk2 (TrkB), Ntrk3 (TrkC), Ngfr (p75NTR) and miRNAs that target theses gens in the mouse brain after induction of seizure activity by pentylenetetrazol. We found that expression of Ntrk3 and Ngfr was upregulated in the cortex and the hippocampus 1-3 hours after the seizures, while Ntrk2 expression increased after 3-6 hours in the anterior cortex and after 1 and 6 hours in the hippocampus. At the same time, the ratio of Bcl-2/Bax signaling proteins increased in the anterior and posterior cortex, but not in the hippocampus, suggesting the activation of anti-apoptotic signaling. Expression of miRNA-9 and miRNA-29a, which were predicted to target Ntrk3, was upregulated in the hippocampus 3 hours after pentylenetetrazol injection. Therefore, early cellular response to seizures in the brain includes induction of the Ntrk2, Ntrk3, Ngfr, miRNA-9, and miRNA-29a expression, as well as activation of Bcl-2 and Bax signaling pathways, which may characterize them as important mediators of neuronal adaptation and survival upon induction of the generalized brain activity.

Downregulation of uPAR promotes urokinase translocation into the nucleus and epithelial to mesenchymal transition in neuroblastoma.

The urokinase system is involved in a variety of physiological processes, such as fibrinolysis, matrix remodeling, wound healing, and regeneration. Upon binding to its cognate receptor urokinase-type plasminogen activator receptor (uPAR), urokinase-type plasminogen activator (uPA) catalyzes the conversion of plasminogen to plasmin and the activation of matrix metalloproteases. Apart from this, uPA-uPAR interaction can lead to the activation of transcription factors, mitogen-activated protein kinase signaling pathways and RTK cascades. Elevated expression of uPA and uPAR is markedly associated with cancer progression and metastasis and correlates with a poor prognosis in clinics. Targeting the urokinase system has proved to be effective in experimental models in vitro and in vivo, however, in clinics the inhibition of the uPA/uPAR system has fallen short of expectations, suggesting that the question of the functional relevance of uPA/uPAR system is far from being moot. Recently, using CRISPR/Cas9 technology, we have shown that uPAR knockout decreases the proliferation of neuroblastoma Neuro2a cells in vitro. In the present study we demonstrate that uPAR expression is essential for maintaining the epithelial phenotype in Neuro2a cells and that uPAR silencing promotes epithelial-mesenchymal transition (EMT) and increased cell migration. Accordingly, uPAR knockout results in the downregulation of epithelial markers (E-cadherin, occludin, and claudin-5) and in the increase of mesenchymal markers (N-cadherin, α-smooth muscle actin, and interleukin-6). In search of the molecular mechanism underlying these changes, we identified uPA as a key component. Two key insights emerged as a result of this work: in the absence of uPAR, uPA is translocated into the nucleus where it is presumably involved in the activation of transcription factors (nuclear factor κB and Snail) resulting in EMT. In uPAR-expressing cells, uPAR functions as a uPA "trap" that binds uPA on the cell surface and promotes controlled uPA internalization and degradation in lysosomes.

Urokinase receptor and tissue plasminogen activator as immediate-early genes in pentylenetetrazole-induced seizures in the mouse brain.

Epileptogenesis progressively leads to the rearrangement of normal neuronal networks into more excitable ones and can be viewed as a form of neuroplasticity, the molecular mechanisms of which still remain obscure. Here, we studied pentylenetetrazole seizure-induced regulation of genes for plasminogen activator system in the mouse brain. We found that expression of tissue plasminogen activator (tPA) and urokinase receptor (uPAR) mRNA was strongly increased in the mouse cerebral cortex, hippocampus, striatum and amygdala as early as 3 hr after pentylenetetrazole seizures. Such early activity-induced expression of uPAR in the central nervous system has not been demonstrated before. uPAR mRNA accumulation was followed by elevation of uPAR protein, indicating a complete transcription-translation process. Both tPA gene induction and uPAR gene induction were independent of the protein synthesis, suggesting that they are regulated by neural activity as immediate-early genes. In contrast to tPA and uPAR genes, the expression of which returned to the basal level 6 hr following seizures, urokinase and plasminogen activator inhibitor-1 gene expression showed a delayed activation only at 3 days after seizures. In conclusion, our results suggest an important sensitivity of the brain plasminogen activator system to seizure activity which raises the question of its role in activity-dependent neural tissue remodeling in pathological and normal conditions.

Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis.

T-cadherin is a unique member of the cadherin superfamily of adhesion molecules. In contrast to "classical" cadherins, T-cadherin lacks transmembrane and cytoplasmic domains and is anchored to the cell membrane via a glycosilphosphoinositol moiety. T-cadherin is predominantly expressed in cardiovascular system. Clinical and biochemical studies evidence that expression of T-cadherin increases in post-angioplasty restenosis and atherosclerotic lesions-conditions associated with endothelial dysfunction and pathological expression of adhesion molecules. Here, we provide data suggesting a new signaling mechanism by which T-cadherin regulates endothelial permeability. T-cadherin overexpression leads to VE-cadherin phosphorylation on Y731 (ß-catenin-binding site), VE-cadherin clathrin-dependent endocytosis and its degradation in lysosomes. Moreover, T-cadherin overexpression results in activation of Rho GTPases signaling and actin stress fiber formation. Thus, T-cadherin up-regulation is involved in degradation of a key endothelial adhesion molecule, VE-cadherin, resulting in the disruption of endothelial barrier function. Our results point to the role of T-cadherin in regulation of endothelial permeability and its possible engagement in endothelial dysfunction.

Urokinase Receptor uPAR Downregulation in Neuroblastoma Leads to Dormancy, Chemoresistance and Metastasis.

uPAR is a membrane receptor that binds extracellular protease urokinase, contributes to matrix remodeling and plays a crucial role in cellular adhesion, proliferation, survival, and migration. uPAR overexpression in tumor cells promotes mitogenesis, opening a prospective avenue for targeted therapy. However, uPAR targeting in cancer has potential risks. We have recently shown that uPAR downregulation in neuroblastoma promotes epithelial-mesenchymal transition (EMT), potentially associated with metastasis and chemoresistance. We used data mining to evaluate the role of uPAR expression in primary and relapsed human neuroblastomas. To model the decreased uPAR expression, we targeted uPAR using CRISPR/Cas9 and shRNA in neuroblastoma Neuro2a cells and evaluated their chemosensitivity in vitro as well as tumor growth and metastasis in vivo. We demonstrate that the initially high PLAUR expression predicts poor survival in human neuroblastoma. However, relapsed neuroblastomas have a significantly decreased PLAUR expression. uPAR targeting in neuroblastoma Neuro2a cells leads to p38 activation and an increased p21 expression (suggesting a dormant phenotype). The dormancy in neuroblastoma cells can be triggered by the disruption of uPAR-integrin interaction. uPAR-deficient cells are less sensitive to cisplatin and doxorubicin treatment and exhibit lower p53 activation. Finally, low uPAR-expressing Neuro2a cells formed smaller primary tumors, but more frequent metastasis in mice. To the best of our knowledge, this is the first study revealing the pathological role of dormant uPAR-deficient cancer cells having a chemoresistant and motile phenotype.

Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene (Plaur) and Its Molecular Target Mef2d.

Urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored receptor of urokinase (uPA), which is involved in brain development, nerve regeneration, wound healing and tissue remodeling. We have recently shown that Plaur, which encodes uPAR, is an early response gene in murine brain. Assumingly, diverse functions of Plaur might be attributed to hypothetical, unidentified microRNAs encoded within introns of the Plaur gene. Using a bioinformatic approach we identified novel small RNAs within the Plaur gene and named them Plaur-miR1-3p and Plaur-miR1-5p. We confirmed Plaur-dependent expression of Plaur-miR1-3p and Plaur-miR1-5p in the mouse brain and mouse neuroblastoma Neuro2a cells. Utilizing an in silico MR-microT algorithm in DianaTools we selected two target genes - Mef2d and Emx2 with the highest binding scores to small RNAs selected from identified Plaur-Pre-miR1. Furthermore, sequencing of mouse brain samples for Plaur-miR1-5p target genes revealed two more genes-Nrip3 and Snrnp200. The expression of Emx2, Mef2d, and Snrnp200 in the mouse brain and Mef2d and Snrnp200 in Neuro2a cells correlated with expression of Plaur and small RNAs-Plaur-miR1-3p and Plaur-miR1-5p. Finally, we demonstrated elevated MEF2D protein expression in the mouse brain after Plaur induction and displayed activating effects of Plaur-miR1-5p on Mef2d expression in Neuro2a cells using Luciferase reporter assay. In conclusion, we have identified Plaur-miR1-3p and Plaur-miR1-5p as novel small RNAs encoded in the Plaur gene. This finding expands the current understanding of Plaur function in brain development and functioning.

CRISPR/Cas9 nickase mediated targeting of urokinase receptor gene inhibits neuroblastoma cell proliferation.

Neuroblastoma is a tumor arising from pluripotent sympathoadrenal precursor cells of neural cell origin. Neuroblastoma is one of the most aggressive childhood tumors with highly invasive and metastatic potential. The increased expression of urokinase and its receptor is often associated with a negative prognosis in neuroblastoma patients. We have shown that targeting of the Plaur gene in mouse neuroblastoma Neuro 2A cells by CRISPR/Cas9n results in ~60% decrease in cell proliferation (p<0.05), reduction in the number of Ki-67 positive cells, caspase 3 activation and PARP-1 cleavage. Knockout of uPAR leads to downregulation of mRNA encoding full-length TrkC receptor, which is involved in p38MAPK and Akt signalling pathways. This finding provides a rationale to study a role of uPAR in neuroblastoma progression, since uPAR could be considered a potential therapeutic target in neuroblastoma treatment.

T-Cadherin Expression in Melanoma Cells Stimulates Stromal Cell Recruitment and Invasion by Regulating the Expression of Chemokines, Integrins and Adhesion Molecules.

T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored member of the cadherin superfamily involved in the guidance of migrating cells. We have previously shown that in vivo T-cadherin overexpression leads to increased melanoma primary tumor growth due to the recruitment of mesenchymal stromal cells as well as the enhanced metastasis. Since tumor progression is highly dependent upon cell migration and invasion, the aim of the present study was to elucidate the mechanisms of T-cadherin participation in these processes. Herein we show that T-cadherin expression results in the increased invasive potential due to the upregulated expression of pro-oncogenic integrins, chemokines, adhesion molecules and extracellular matrix components. The detected increase in chemokine expression could be responsible for the stromal cell recruitment. At the same time our previous data demonstrated that T-cadherin expression inhibited neoangiogenesis in the primary tumors. We demonstrate molecules and reduction in pro-angiogenic factors. Thus, T-cadherin plays a dual role in melanoma growth and progression: T-cadherin expression results in anti-angiogenic effects in melanoma, however, this also stimulates transcription of genes responsible for migration and invasion of melanoma cells.