The urokinase-type plasminogen activator receptor (uPAR) is a unique protease binding receptor, now recognized as a key regulator of inflammation. Initially, uPA/uPAR was considered thrombolytic (clot-dissolving); however, recent studies have demonstrated its predominant immunomodulatory functions in inflammation and cancer. The uPA/uPAR complex has a multifaceted central role in both normal physiological and also pathological responses. uPAR is expressed as a glycophosphatidylinositol (GPI)-linked receptor interacting with vitronectin, integrins, G protein-coupled receptors, and growth factor receptors within a large lipid raft. Through protein-to-protein interactions, cell surface uPAR modulates intracellular signaling, altering cellular adhesion and migration. The uPA/uPAR also modifies extracellular activity, activating plasminogen to form plasmin, which breaks down fibrin, dissolving clots and activating matrix metalloproteinases that lyse connective tissue, allowing immune and cancer cell invasion and releasing growth factors. uPAR is now recognized as a biomarker for inflammatory diseases and cancer; uPAR and soluble uPAR fragments (suPAR) are increased in viral sepsis (COVID-19), inflammatory bowel disease, and metastasis. Here, we provide a comprehensive overview of the structure, function, and current studies examining uPAR and suPAR as diagnostic markers and therapeutic targets. Understanding uPAR is central to developing diagnostic markers and the ongoing development of antibody, small-molecule, nanogel, and virus-derived immune-modulating treatments that target uPAR.

The expression of human PLAUR gene, which encodes the urokinase plasminogen activator receptor (uPAR), is cell- and process-specific and elevated in inflammation, cancer and senescence. Its tight regulation is achieved by regulatory elements in the gene locus, such as the promoter and several enhancers. The promoter activity is not specific to a particular cell type and has been described earlier. The proximal enhancer is endothelial-specific and responsible for the PLAUR expression pattern in endothelial cells. In this study we described the enhancer activity and its cis-regulatory elements based on the published data. We showed a possible connection of the enhancer activity with known cellular phenotypes.

OBJECTIVE: Hepatic stellate cells (HSCs) are the key drivers of fibrosis in metabolic dysfunction-associated steatohepatitis (MASH), the fastest growing cause of hepatocellular carcinoma (HCC) worldwide. HSCs are heterogenous, and a senescent subset of HSCs is implicated in hepatic fibrosis and HCC. Administration of anti-uPAR (urokinase-type plasminogen activator receptor) CAR T cells has been shown to deplete senescent HSCs and attenuate fibrosis in murine models. However, the comprehensive features of senescent HSCs in MASH, as well as their cellular ontogeny have not been characterized; hence, we aimed to comprehensively characterize and define the origin of HSCs in human and murine MASH.
METHODS: To comprehensively characterize the phenotype and ontogeny of senescent HSCs in human and murine MASH, we integrated senescence-associated beta galactosidase activity with immunostaining, flow cytometry and single-nucleus RNA sequencing (snRNAseq). We integrated the immunohistochemical profile with a senescence score applied to snRNAseq data to characterize senescent HSCs and mapped the evolution of uPAR expression in MASH.
RESULTS: Using pseudotime trajectory analysis, we establish that senescent HSCs arise from activated HSCs. While uPAR is expressed in MASH, the magnitude and cell-specificity of its expression evolve with disease stage. In early disease, uPAR is more specific to activated and senescent HSCs, while it is also expressed by myeloid-lineage cells, including Trem2+ macrophages and myeloid-derived suppressor cells, in late disease. Furthermore, we identify novel surface proteins expressed on senescent HSCs in human and murine MASH that could be exploited as therapeutic targets.
CONCLUSIONS: These data define features of HSC senescence in human and murine MASH, establishing an important blueprint to target these cells as part of future antifibrotic therapies.
UNASSIGNED: Hepatic stellate cells (HSCs) are the primary drivers of scarring in chronic liver diseases. As injury develops, a subset of HSCs become senescent; these cells are non-proliferative and pro-inflammatory, thereby contributing to worsening liver injury. Here we show that senescent HSCs are expanded in MASH (metabolic dysfunction-associated steatohepatitis) in humans and mice, and we trace their cellular origin from the activated HSC subset. We further characterize expression of uPAR (urokinase plasminogen activated receptor), a protein that marks senescent HSCs, and report that uPAR is also expressed by activated HSCs in early injury, and in immune cells as liver injury advances. We have integrated high-resolution single-nucleus RNA sequencing with immunostaining and flow cytometry to identify five other novel proteins expressed by senescent HSCs, including mannose receptor CD206, which will facilitate future therapeutic development.

Background: Polytrauma is one of the leading mortality factors in younger patients, and in particular, the presence of cardiac damage correlates with a poor prognosis. Currently, troponin T is the gold standard, although troponin is limited as a biomarker. Therefore, there is a need for new biomarkers of cardiac damage early after trauma. Methods: Polytraumatized patients (ISS ≥ 16) were divided into two groups: those with cardiac damage (troponin T > 50 pg/mL, n = 37) and those without cardiac damage (troponin T < 12 pg/mL, n = 32) on admission to the hospital. Patients\' plasma was collected in the emergency room 24 h after trauma, and plasma from healthy volunteers (n = 10) was sampled. The plasma was analyzed for the expression of HFABP, GDF-15 and uPAR proteins, as well as miR-21, miR-29, miR-34, miR-122, miR-125b, miR-133, miR-194, miR-204, and miR-155. Results were correlated with patients\' outcomes. Results: HFABP, uPAR, and GDF-15 were increased in polytraumatized patients with cardiac damage (p < 0.001) with a need for catecholamines. HFABP was increased in non-survivors. Analysis of systemic miRNA concentrations showed a significant increase in miR-133 (p < 0.01) and miR-21 (p < 0.05) in patients with cardiac damage. Conclusion: All tested plasma proteins, miR-133, and miR-21 were found to reflect the cardiac damage in polytrauma patients. GDF-15 and HFABP were shown to strongly correlate with patients\' outcomes.

Urokinase plasminogen activator receptor (uPAR) encoded by the PLAUR gene is known as a clinical marker for cell invasiveness in glioblastoma multiforme (GBM). It is additionally implicated in various processes, including angiogenesis and inflammation within the tumor microenvironment. However, there has not been a comprehensive study that depicts the overall functions and molecular cooperators of PLAUR with respect to intra-tumoral subtypes of GBM. Using single-cell RNA sequencing data from 37 GBM patients, we identified PLAUR as a marker gene for two distinct subtypes in GBM. One subtype is featured by inflammatory activities and the other subtype is marked by ECM remodeling processes. Using the whole-transcriptome data from single cells, we are able to uncover the molecular cooperators of PLAUR for both subtypes without presuming biological pathways. Two protein networks comprise the molecular context of PLAUR, with each of the two subtypes characterized by a different dominant network. We concluded that targeting PLAUR directly influences the mechanisms represented by these two protein networks, regardless of the subtype of the targeted cell.

Cancer remains one of the most pressing challenges to global healthcare, exerting a significant impact on patient life expectancy. Cancer metastasis is a critical determinant of the lethality and treatment resistance of cancer. The urokinase-type plasminogen activator receptor (uPAR) shows great potential as a target for anticancer and antimetastatic therapies. In this work, we aimed to identify potential uPAR inhibitors by structural dynamics-based virtual screenings against a natural product library on four representative apo-uPAR structural models recently derived from long-timescale molecular dynamics (MD) simulations. Fifteen potential inhibitors (NP1-NP15) were initially identified through molecular docking, consensus scoring, and visual inspection. Subsequently, we employed MD-based molecular mechanics-generalized Born surface area (MM-GBSA) calculations to evaluate their binding affinities to uPAR. Structural dynamics analyses further indicated that all of the top 6 compounds exhibited stable binding to uPAR and interacted with the critical residues in the binding interface between uPAR and its endogenous ligand uPA, suggesting their potential as uPAR inhibitors by interrupting the uPAR-uPA interaction. We finally predicted the ADMET properties of these compounds. The natural products NP5, NP12, and NP14 with better binding affinities to uPAR than the uPAR inhibitors previously discovered by us were proven to be potentially orally active in humans. This work offers potential uPAR inhibitors that may contribute to the development of novel effective anticancer and antimetastatic therapeutics.Communicated by Ramaswamy H. Sarma.

One of the largest challenges to the implementation of cardiac cell therapy is identifying selective reparative targets to enhance stem/progenitor cell therapeutic efficacy. In this work, we hypothesized that such a target could be an urokinase-type plasminogen activator receptor (uPAR)-a glycosyl-phosphatidyl-inositol-anchored membrane protein, interacting with urokinase. uPAR is able to form complexes with various transmembrane proteins such as integrins, activating intracellular signaling pathway and thus regulating multiple cell functions. We focused on studying the CD117+ population of cardiac mesenchymal progenitor cells (MPCs), expressing uPAR on their surface. It was found that the number of CD117+ MPCs in the heart of the uPAR-/- mice is lower, as well as their ability to proliferate in vitro compared with cells from wild-type animals. Knockdown of uPAR in CD117+ MPCs of wild-type animals was accompanied by a decrease in survival rate and Akt signaling pathway activity and by an increase in the level of caspase activity in these cells. That suggests the role of uPAR in supporting cell survival. After intramyocardial transplantation of uPAR(-) MPCs, reduced cell retention and angiogenesis stimulation were observed in mice with myocardial infarction model compared to uPAR(+) cells transplantation. Taken together, the present results appear to prove a novel mechanism of uPAR action in maintaining the survival and angiogenic properties of CD117+ MPCs. These results emphasize the importance of the uPAR as a potential pharmacological target for the regulation of reparative properties of myocardial mesenchymal progenitor cells.

Human cytochrome P450 1B1 (CYP1B1) catalyzes estrogen metabolism to produce metabolites that promote the progression of breast cancer. Since the invasive properties of cancer cells cause cancer relapse, which dramatically reduces patient survival, we investigated the new pro-invasive mechanism involving CYP1B1 in breast cancer. Exploring clinical data from invasive breast cancer patients revealed that CYP1B1 exhibits a potential correlation with urokinase-type plasminogen activator receptor (uPAR). Interestingly, uPAR mRNA expression was elevated in invasive breast cancer patients carrying TP53 genes with driver mutations, and our results showed that CYP1B1 activates the uPAR pathway following regulation of p53 according to its mutant status. CYP1B1 suppressed wild-type (WT) p53 whereas it induced the oncogenic gain-of-function mutant p53R280K, not only via transcriptional regulation but also the protein stabilization and activation following phosphorylation on Ser15 residue of p53R280K. Intriguingly, results from CYP1B1 polymorphic gene study and 4-hydroxyestradiol (4-OHE2) treatment showed that CYP1B1 regulates p53s and uPAR through its enzymatic activity. Furthermore, effects of DMBA and TMS on uPAR expression disappeared in HCT116p53-/- cells, indicating that p53 is critical for uPAR induction by CYP1B1. Collectively, our results demonstrate that CYP1B1 may reduce the relapse-free survival rate of breast cancer patients by inducing invasive traits in cancer cells via p53 regulation based on the mutation status of TP53 genes and further activation of the uPAR pathway. The elucidation of the previously unknown molecular mechanism of CYP1B1 may provide evidence for the development of effective anti-cancer therapeutic strategies that target the progression of cancer invasion.

Distant metastasis is the primary reason for treatment failure in patients with nasopharyngeal carcinoma (NPC). In this study, we investigated the effect of ulinastatin (UTI) on NPC metastasis and its underlying mechanism. Highly-metastatic NPC cell lines S18 and 58F were treated with UTI and the effect on cell proliferation, migration, and invasion were determined by MTS and Transwell assays. S18 cells with luciferase-expressing (S18-1C3) were injected into the left hind footpad of nude mice to establish a model of spontaneous metastasis from the footpad to popliteal lymph node (LN). The luciferase messenger RNA (mRNA) was measured by quantitative polymerase chain reaction (qPCR), and the metastasis inhibition rate was calculated. Key molecular members of the UTI-related uPA, uPAR, and JAT/STAT3 signaling pathways were detected by qPCR and immunoblotting. UTI suppressed the migration and infiltration of S18 and 5-8F cells and suppressed the metastasis of S18 cells in vivo without affecting cell proliferation. uPAR expression decreased from 24 to 48 h after UTI treatment. The antimetastatic effect of UTI is partly due to the suppression of uPA and uPAR. UTI partially suppresses NPC metastasis by downregulating the expression of uPA and uPAR.

SARS-CoV-2 acute respiratory distress syndrome (ARDS) induces uncontrolled lung inflammation and coagulopathy with high mortality. Anti-viral drugs and monoclonal antibodies reduce early COVID-19 severity, but treatments for late-stage immuno-thrombotic syndromes and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases. The myxoma virus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated thrombotic, thrombolytic, and complement proteases as a self-defense strategy to combat clearance. Serp-1 is effective in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 as a therapy for immuno-coagulopathic complications during ARDS. Treatment with PEGSerp-1 in two mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved outcomes. PEGSerp-1 significantly reduced M1 macrophages in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR), thrombotic proteases, and complement membrane attack complex (MAC). Sequential changes in gene expression for uPAR and serpins (complement and plasminogen inhibitors) were observed. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for severe viral ARDS, immuno-coagulopathic responses, and Long COVID.