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Copyright of Chinese Journal of Clinical Healthcare is the property of Chinese Journal of Clinical Healthcare and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objective To investigate and compare the efficacy of endoscopic-assisted hematoma evacuation and burr hole hematoma drainage combined with urokinase in the treatment of septated chronic subdural hematoma (CSDH). Methods A total of 38 patients with septated CSDH who were admitted in Peking Union Medical College Hospital and received endoscopic-assisted hematoma evacuation (endoscopy group, n = 19) or burr hole drainage combined with urokinase (burr hole group, n = 19) from January 2022 to December 2023. The change rate of the hematoma cavity was calculated, the modified Rankin Scale (mRS) and Barthel Index (BI) were used to evaluate neurological function prognosis and abilities of daily living. In addition, postoperative drainage time, postoperative hospitalization time, total hospitalization cost and incidence of cerebrospinal fluid leakage were recorded. Results The two treatment modalities did not show differences in the change rate of the hematoma cavity (t = 0.858, P = 0.396). Both endoscopy group (Z = -4.116, P = 0.000) and burr hole group (Z = -4.195, P = 0.000) had lower mRS scores on discharge than on admission, while the difference between the 2 groups on discharge was not significant (Z = -0.502, P = 0.616). The endoscopy group (Z = -1.557, P = 0.119) and burr hole group (Z = -0.091, P = 0.928) had no significant difference in BI scores on discharge versus on admission, and the difference in BI scores between the 2 groups on discharge was also not statistically significant (Z = -0.853, P = 0.394). Postoperative drainage time was longer in the endoscopy group (t = -2.488, P = 0.018), but postoperative hospitalization time was longer in the burr hole group (t = -3.894, P = 0.000). Total hospitalization cost in both 2 groups (t = 1.175, P = 0.248) and the incidence of cerebrospinal fluid leakage (Fisher's exact probability: P = 0.313) were not statistically significant. Conclusions Both neuroendoscopic surgery and burr hole drainage combined with urokinase treatment are safe and effective in treating septated CSDH. They can clear the hematoma and improve neurological function; neuroendoscopic surgery can faster improve the neurological function and shorter postoperative hospitalization time. [ABSTRACT FROM AUTHOR]

Patients with coronavirus disease-2019 (COVID-19) have an increased risk of thrombosis and acute respiratory distress syndrome (ARDS). Thrombosis is often attributed to increases in plasminogen activator inhibitor-1 (PAI-1) and a shut-down of fibrinolysis (blood clot dissolution). Decreased urokinase-type plasminogen activator (uPA), a protease necessary for cell-associated plasmin generation, and increased tissue-type plasminogen activator (tPA) and PAI-1 levels have been reported in COVID-19 patients. Because these factors can occur in free and complexed forms with differences in their biological functions, we examined the predictive impact of uPA, tPA, and PAI-1 in their free forms and complexes as a biomarker forCOVID-19 severity and the development of ARDS. In this retrospective study of 69 Japanese adults hospitalized with COVID-19 and 20 healthy donors, we found elevated free, non-complexed PAI-1 antigen, low circulating uPA, and uPA/ PAI-1 but not tPA/PAI-1 complex levels to be associatedwith COVID-19 severity and ARDS development. This biomarker profile was typical for patients in the complicated phase. Lack of PAI-1 activity in circulation despite free, noncomplexed PAI-1 protein and plasmin/a2anti-plasmin complex correlated with suPAR and sVCAM levels, markers indicating endothelial dysfunction. Furthermore, uPA/PAI-1 complex levels positively correlated with TNFa, a cytokine reported to trigger inflammatory cell death and tissue damage. Those levels also positively correlated with lymphopenia and the pro-inflammatory factors interleukin1b (IL1b), IL6, and C-reactive protein, markers associated with the anti-viral inflammatory response. These findings argue for using uPA and uPA/PAI-1 as novel biomarkers to detect patients at risk of developing severe COVID-19, including ARDS. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) is a serious health concern with high morbidity and high mortality worldwide. Recently, sexual dimorphism has become increasingly recognized as a factor influencing the severity of the disease. This study explores the gender-specific renoprotective pathways in αMUPA transgenic mice subjected to AKI. αMUPA transgenic male and female mice were subjected to ischemia–reperfusion (I/R)-AKI in the presence or absence of orchiectomy, oophorectomy, and L-NAME administration. Blood samples and kidneys were harvested 48 h following AKI for the biomarkers of kidney function, renal injury, inflammatory response and intracellular pathway sensing of or responding to AKI. Our findings show differing responses to AKI, where female αMUPA mice were remarkably protected against AKI as compared with males, as was evident by the lower SCr and BUN, normal renal histologically and attenuated expression of NGAL and KIM-1. Moreover, αMUPA females did not show a significant change in the renal inflammatory and fibrotic markers following AKI as compared with wild-type (WT) mice and αMUPA males. Interestingly, oophorectomized females eliminated the observed resistance to renal injury, highlighting the central protective role of estrogen. Correspondingly, orchiectomy in αMUPA males mitigated their sensitivity to renal damage, thereby emphasizing the devastating effects of testosterone. Additionally, treatment with L-NAME proved to have significant deleterious impacts on the renal protective mediators, thereby underscoring the involvement of eNOS. In conclusion, gender-specific differences in the response to AKI in αMUPA mice include multifaceted and keen interactions between the sex hormones and key biochemical mediators (such as estrogen, testosterone and eNOS). These novel findings shed light on the renoprotective pathways and mechanisms, which may pave the way for development of therapeutic interventions. [ABSTRACT FROM AUTHOR]

Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the antibody mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates shows that Ab binding causes uPAR internalization with a ∼20 min half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

Background: Cancer cells remodel their local physical environment through processes of matrix reorganisation, deposition, stiffening and degradation. Urokinase-type plasminogen activator (uPA), which is encoded by the PLAU gene, is an extracellular proteolytic enzyme known to be involved in cancer progression and tumour microenvironment (TME) remodelling. Perturbing uPA therefore has a strong potential as a mechano-based cancer therapy. This work is a bioengineering investigation to validate whether 1) uPA is involved in matrix degradation and 2) preventing matrix degradation by targeting uPA can reduce cancer cell invasion and metastasis. Methods: To this aim, we used an engineered 3D in vitro model, termed the tumouroid, that appropriately mimics the tumour’s native biophysical environment (3 kPa). A CRISPR-Cas9 mediated uPA knockout was performed to introduce a loss of function mutation in the gene coding sequence. Subsequently, to validate the translational potential of blocking uPA action, we tested a pharmacological inhibitor, UK-371,801. The changes in matrix stiffness were measured by atomic force microscopy (AFM). Invasion was quantified using images of the tumouroid, obtained after 21 days of culture. Results: We showed that uPA is highly expressed in invasive breast and colorectal cancers, and these invasive cancer cells locally degrade their TME. PLAU (uPA) gene knock-out (KO) completely stopped matrix remodelling and significantly reduced cancer invasion. Many invasive cancer gene markers were also downregulated in the PLAU KO tumouroids. Pharmacological inhibition of uPA showed similarly promising results, where matrix degradation was reduced and so was the cancer invasion. Conclusion: This work supports the role of uPA in matrix degradation. It demonstrates that the invasion of cancer cells was significantly reduced when enzymatic breakdown of the TME matrix was prevented. Collectively, this provides strong evidence of the effectiveness of targeting uPA as a mechano-based cancer therapy.

Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.

Acute kidney injury (AKI) is a serious health concern with high morbidity and high mortality worldwide. Recently, sexual dimorphism has become increasingly recognized as a factor influencing the severity of the disease. This study explores the gender-specific renoprotective pathways in αMUPA transgenic mice subjected to AKI. αMUPA transgenic male and female mice were subjected to ischemia–reperfusion (I/R)-AKI in the presence or absence of orchiectomy, oophorectomy, and L-NAME administration. Blood samples and kidneys were harvested 48 h following AKI for the biomarkers of kidney function, renal injury, inflammatory response and intracellular pathway sensing of or responding to AKI. Our findings show differing responses to AKI, where female αMUPA mice were remarkably protected against AKI as compared with males, as was evident by the lower SCr and BUN, normal renal histologically and attenuated expression of NGAL and KIM-1. Moreover, αMUPA females did not show a significant change in the renal inflammatory and fibrotic markers following AKI as compared with wild-type (WT) mice and αMUPA males. Interestingly, oophorectomized females eliminated the observed resistance to renal injury, highlighting the central protective role of estrogen. Correspondingly, orchiectomy in αMUPA males mitigated their sensitivity to renal damage, thereby emphasizing the devastating effects of testosterone. Additionally, treatment with L-NAME proved to have significant deleterious impacts on the renal protective mediators, thereby underscoring the involvement of eNOS. In conclusion, gender-specific differences in the response to AKI in αMUPA mice include multifaceted and keen interactions between the sex hormones and key biochemical mediators (such as estrogen, testosterone and eNOS). These novel findings shed light on the renoprotective pathways and mechanisms, which may pave the way for development of therapeutic interventions.

Since the proposition of the pro‐invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well‐characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein‐related peptidases, including KLK3 (prostate‐specific antigen, PSA), the most well‐known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration‐resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease‐activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens. [ABSTRACT FROM AUTHOR]

The major proteases that constitute the fibrinolysis system are tightly regulated. Protease inhibitors target plasmin, the protease responsible for fibrin degradation, and the proteases that convert plasminogen into plasmin, including tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). A second mechanism by which fibrinolysis is regulated involves exosite interactions, which localize plasminogen and its activators to fibrin, extracellular matrix (ECM) proteins, and cell surfaces. Once plasmin is generated in association with cell surfaces, it may cleave transmembrane proteins, activate growth factors, release growth factors from ECM proteins, remodel ECM, activate metalloproteases, and trigger cell-signaling by cleaving receptors in the Proteaseactivated Receptor (PAR) family. These processes are all implicated in cancer. It is thus not surprising that a family of structurally diverse but functionally similar cell-surface proteins, called Plasminogen Receptors (PlgRs), which increase the catalytic efficiency of plasminogen activation, have received attention for their possible function in cancer and as targets for anticancer drug development. In this review, we consider four previously described PlgRs, including: α-enolase, annexin-A2, Plg-RKT, and cytokeratin-8, in human cancer. To compare the PlgRs, we mined transcriptome profiling data from The Cancer Genome Atlas (TCGA) and searched for correlations between PlgR expression and patient survival. In glioma, the expression of specific PlgRs correlates with tumor grade. In a number of malignancies, including glioblastoma and liver cancer, increased expression of α-enolase or annexin-A2 is associated with an unfavorable prognosis. Whether these correlations reflect the function of PlgRs as receptors for plasminogen or other activities is discussed.

Understanding the coagulation process is critical to developing treatments for trauma and coagulopathies. Clinical studies on tranexamic acid (TXA) have resulted in mixed reports on its efficacy in improving outcomes in trauma patients. The largest study, CRASH-2, reported that TXA improved outcomes in patients who received treatment prior to 3 hours after the injury, but worsened outcomes in patients who received treatment after 3 hours. No consensus has been reached about the mechanism behind the duality of these results. In this paper we use a computational model for coagulation and fibrinolysis to propose that deficiencies or depletions of key anti-fibrinolytic proteins, specifically antiplasmin, a1-antitrypsin and a2-macroglobulin, can lead to worsened outcomes through urokinase-mediated hyperfibrinolysis.

BACKGROUND:Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway. METHODS:Primary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents. RESULTS:Astrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor. CONCLUSIONS:Plasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS.

Urokinase‐type plasminogen activator receptor (uPAR) is a glycolipid‐anchored protein located on the cell surface that is implicated in the promotion of metastasis. New fluorescent probes for the detection of uPAR expression that feature a rapid "turn‐on" response are reported here. They consist of a donor‐π‐acceptor‐based fluorophore conjugated with a uPAR‐binding AE105 peptide. The resulting AE105‐coupled uPAR‐targeting probes are weakly emissive in aqueous buffer solutions; however, a fluorescence "turn‐on" signal is instantly triggered upon specific binding to uPAR (KD=63.2 nM for P1 and 49.5 nM for P2), which restricts the rotational deactivation of the fluorophore. Applications of the probes were demonstrated in the imaging of uPAR overexpressed on the membrane of cancer cell and in a cell‐based uPAR inhibitor assay. [ABSTRACT FROM AUTHOR]

The significance of plasminogen activation during the tympanic membrane (TM) healing is known mainly from studies performed on knock-out mice. In the previous study, we reported activation of genes coding proteins of plasminogen activation and inhibition system in rat's TM perforation healing. The aim of the present study was the evaluation of protein products expressed by these genes and their tissue distribution using Western blotting and immunofluorescent method, respectively, during 10-day observation period after injury. Otomicroscopical and histological evaluation were employed to assess the healing process. The expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) were significantly upregulated in the proliferation phase, with subsequent gradual attenuation during remodeling phase of healing process, when keratinocyte migration was weakening. The expression of plasminogen activator inhibitor type 1 (PAI-1) also showed the highest levels during the proliferation phase. The increase of tissue plasminogen activator (tPA) expression was observed during the whole observation period, with the highest activity during the remodeling phase. Immunofluorescence of these proteins was present mainly in migrating epithelium. Our study found that plasminogen activation (uPA, uPAR, tPA) and inhibitory (PAI-1) molecules form a well-structured regulatory system of the epithelial migration that is critical to the healing of TM after its perforation. [ABSTRACT FROM AUTHOR]

In the era of liquid biopsies, the reliable and cost-effective detection and screening of cancer biomarkers has become of fundamental importance, thus paving the way for the advancement of research in the field of point-of-care testing and the development of new methodologies and technologies. Indeed, the latter ones can help designing advanced diagnostic tools that can offer portability, ease of use with affordable production and operating costs. In this respect, impedance-based biosensing platforms might represent an attractive alternative. In this work, we describe a proof-of-concept study aimed at designing portable impedimetric biosensors for the monitoring of human urokinase-type plasminogen activator (h-uPA) cancer biomarker by employing small synthetic receptors. Aberrant levels of h-uPA were correlated with different types of cancers. Herein, we report the use of two bicyclic peptides (P2 and P3) which have been engineered to bind h-uPA with high affinity and exquisite specificity. The synthetic receptors were immobilized via biotin-streptavidin chemistry on the surface of commercial screen-printed electrodes. The impedimetric changes in the electrode/solution interface upon incubation of spiked h-uPA samples in the presence of a redox probe were followed via electrochemical impedance spectroscopy. The P3-based impedimetric assay showed the best outcomes in terms of dynamic range and linearity (0.01–1 μg mL−1) and sensitivity (LOD = 9 ng mL−1). To fully assess the performances of P3 over P2, and to compare the label-free architecture vs. labelled architecture, a voltammetric assay was also developed. [ABSTRACT FROM AUTHOR]

Current dosing of intrapleural fibrinolytic therapy (IPFT) in adults with complicated parapneumonic effusion (CPE) / empyema is empiric, as dose-escalation trials have not previously been conducted. We hypothesized that LTI-01 (scuPA), which is relatively resistant to PA inhibitor-1 (PAI-1), would be well-tolerated. This was an open-label, dose-escalation trial of LTI-01 IPFT at 50,000-800,000 IU daily for up to 3 days in adults with loculated CPE/empyema and failed pleural drainage. The primary objective was to evaluate safety and tolerability, and secondary objectives included assessments of processing and bioactivity of scuPA in blood and pleural fluid (PF), and early efficacy. LTI-01 was well tolerated with no bleeding, treatment-emergent adverse events or surgical referrals (n=14 subjects). uPA antigen increased in PFs at 3 hours after LTI-01 (p

Background: Achieving rapid and complete reperfusion is the ultimate purpose for ischemic stroke with large vessel occlusion (LVO). Although mechanical thrombectomy (MT) had been a proverbially important procedure, medium vessel occlusion (MeVO) with thrombus migration can sporadically occur after MT. Moreover, the safe and effective approach for such had been unknown. We reported thrombolysis with intraarterial urokinase for MeVO with thrombus migration after MT. Methods: We included 122 patients who were treated by MT with LVO stroke at our institution between April 2019 and March 2021. Of 26 patients (21.3%) who developed MeVO with thrombus migration after MT, 11 (9.0%) underwent additional MT (MT group) and 15 (12.3%) received intraarterial urokinase (UK group). The procedure time; angiographically modified Treatment in Cerebral Ischemia Scale (mTICI); functional independence, which was defined as modified Rankin Scale 0–2, on day 30 or upon discharge; and symptomatic and asymptomatic intracerebral hemorrhage (ICH) were compared between the UK and MT groups. Results: The procedure time, mTICI, and asymptomatic ICH did not significantly differ between the groups. In the UK group, 8 of 15 (53.3%) patients obtained functional independence, and the functional independence rate was significantly higher in the UK group than in the MT group (p < 0.05). Symptomatic ICH did not occur in the UK group, and its incidence was significantly smaller than that in the MT group (p < 0.05). Conclusion: The results of this study suggest that intraarterial urokinase for MeVO with thrombus migration after MT may safely improve angiographic reperfusion. [ABSTRACT FROM AUTHOR]

Background: Urokinase-type plasminogen activator (uPA) is a serine proteinase found in excitatory synapses located in the II/III and V cortical layers. The synaptic release of uPA promotes the formation of synaptic contacts and the repair of synapses damaged by various forms of injury, and its abundance is decreased in the synapse of Alzheimer's disease (AD) patients. Inactivation of the Wingless/Int1 (Wnt)-β-catenin pathway plays a central role in the pathogenesis of AD. Soluble amyloid-β (Aβ) prevents the phosphorylation of the low-density lipoprotein receptor-related protein-6 (LRP6), and the resultant inactivation of the Wnt-β-catenin pathway prompts the amyloidogenic processing of the amyloid-β protein precursor (AβPP) and causes synaptic loss.Objective: To study the role of neuronal uPA in the pathogenesis of AD.Methods: We used in vitro cultures of murine cerebral cortical neurons, a murine neuroblastoma cell line transfected with the APP-695 Swedish mutation (N2asw), and mice deficient on either plasminogen, or uPA, or its receptor (uPAR).Results: We show that uPA activates the Wnt-β-catenin pathway in cerebral cortical neurons by triggering the phosphorylation of LRP6 via a plasmin-independent mechanism that does not require binding of Wnt ligands (Wnts). Our data indicate that uPA-induced activation of the Wnt-β-catenin pathway protects the synapse from the harmful effects of soluble Aβ and prevents the amyloidogenic processing of AβPP by inhibiting the expression of β-secretase 1 (BACE1) and the ensuing generation of Aβ40 and Aβ42 peptides.Conclusion: uPA protects the synapse and antagonizes the inhibitory effect of soluble Aβ on the Wnt-β-catenin pathway by providing an alternative pathway for LRP6 phosphorylation and β-catenin stabilization. [ABSTRACT FROM AUTHOR]

In the treatment of thrombosis, conventional nanocarriers inevitably have problems, such as adverse reactions and difficulties in synthesis. Inspired by the concept of 'medicine food homology,' we extracted and purified natural exosomes from mulberry leaves as carriers for the delivery of urokinase-type plasminogen activator (uPA) for targeted therapy. The obtained mulberry leaf exosomes (MLE) possessed a desirable hydrodynamic particle size (119.4 nm), a uniform size distribution (polydispersity index = 0.174), and a negative surface charge (-23.3 mv). Before loading with uPA, MLE were grafted with cyclic RGD (cRGD) to selectively bind activated platelets for thrombus targeting. The cytometry studies revealed that MLE@cRGD has a high thrombus targeting ability about 74.3 %. Animal tests demonstrated that the delivered uPA could dissolve clots almost completely in femoral vein thrombosis models. In addition, MLE could remodel venous microenvironments by effectively eliminating reactive oxygen species (ROS) and promoting the phenotypic transformation of macrophages from M1 to M2 for venous tissue repair., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Copyright of Chinese Journal of Clinical Healthcare is the property of Chinese Journal of Clinical Healthcare and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

June Yong Park,1,&ast; Yuseon Shin,1,&ast; Woong Roeck Won,1 Chaemin Lim,1,2 Jae Chang Kim,1 Kioh Kang,1 Patihul Husni,1 Eun Seong Lee,3 Yu Seok Youn,4 Kyung Taek Oh1,5 1Department of Global Innovative Drugs, The Graduate School of Chung-Ang University, Dongjak-gu, Seoul, 06974, Republic of Korea; 2Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA; 3Division of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do, 14662, Republic of Korea; 4School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, 16419, Republic of Korea; 5College of Pharmacy, Chung-Ang University, Dongjak-gu, Seoul, 06974, Republic of Korea&ast;These authors contributed equally to this workCorrespondence: Kyung Taek OhCollege of Pharmacy, Chung-Ang University, 221 Heukseok-dong, Dongjak-gu, Seoul, 06974, Republic of KoreaTel +82-2-824-5617Email kyungoh@cau.ac.krPurpose: An AE147 peptide-conjugated nanocarrier based on PEGylated liposomes was developed in order to target the metastatic tumors overexpressing urokinase-type plasminogen activator receptor (uPAR), which cancer progression via uPA signaling. Therefore, the AE147 peptide-conjugated nanocarrier system may hold the potential for active targeting of metastatic tumors.Methods: The AE147 peptide, an antagonist of uPAR, was conjugated to the PEGylated liposomes for targeting metastatic tumors overexpressing uPAR. Docetaxel (DTX), an anticancer drug, was incorporated into the nanocarriers. The structure of the AE147-conjugated nanocarrier, its physicochemical properties, and in vivo biodistribution were evaluated.Results: The DTX-loaded nanocarrier showed a spherical structure, a high drug-loading capacity, and a high colloidal stability. Drug carrying AE147 conjugates were actively taken up by the uPAR-overexpressing MDA-MB-231 cancer cells. In vivo animal imaging confirmed that the AE147-conjugated nanoparticles effectively accumulated at the sites of tumor metastasis.Conclusion: The AE147-nanocarrier showed potential for targeting metastatic tumor cells overexpressing uPAR and as a nanomedicine platform for theragnosis applications. These results suggest that this novel nano-platform will facilitate further advancements in cancer therapy.Keywords: tumor-targeting ligand, urokinase-type plasminogen activator, liposome, circulating tumor cell, metastatic tumor

Mutations in SLURP1, a secreted protein of keratinocytes, cause a palmoplantar keratoderma (PPK) known as mal de Meleda. Slurp1 deficiency in mice faithfully recapitulates the human disease, with increased keratinocyte proliferation and thickening of the epidermis on the volar surface of the paws. There has long been speculation that SLURP1 serves as a ligand for a receptor that regulates keratinocyte growth and differentiation. We were intrigued that mutations leading to increased signalling through the epidermal growth factor receptor (EGFR) cause PPK. Here, we sought to determine whether reducing EGFR signalling would ameliorate the PPK associated with SLURP1 deficiency. To address this issue, we bred Slurp1-deficient mice that were homozygous for a hypomorphic Egfr allele. The hypomorphic Egfr allele, which leads to reduced EGFR signalling in keratinocytes, did not ameliorate the PPK elicited by SLURP1 deficiency, suggesting that SLURP1 deficiency causes PPK independently (or downstream) from the EGFR pathway.

The effects of sex on the degree of liver damage and human cell engraftment were investigated in immunodeficient urokinase-type plasminogen activator-transgenic (uPA-NOG) mice. Liver damage, measured by serum alanine transaminase (ALT) levels, was compared in male and female uPA-NOG mice of different ages. Male mice had significantly higher ALT levels than females with a median of 334 versus 158 U/L in transgenic homozygous mice, respectively. Mice were transplanted with human adult hepatocytes or fetal liver cells and analyzed for any correlation of engraftment of hepatocytes, liver sinusoidal endothelial cells (LSECs), and hematopoietic cells with the degree of liver damage. Hepatocyte engraftment was measured by human albumin levels in the mouse serum. Higher ALT levels correlated with higher hepatocyte engraftment, resulting in albumin levels in male mice that were 9.6 times higher than in females. LSEC and hematopoietic cell engraftment were measured by flow cytometric analysis of the mouse liver and bone marrow. LSEC and hematopoietic engraftment did not differ between male and female transplant recipients. Thus, the sex of uPA-NOG mice affects the degree of liver damage, which is reflected in the levels of human hepatocyte engraftment. However, the high levels of LSEC engraftment observed in uPA-NOG mice are not further improved among male mice, suggesting that a lower threshold of liver damage is sufficient to enhance endothelial cell engraftment. Previously described sex differences in human hematopoietic stem cell engraftment in immunodeficient mice were not observed in this model.

Mammalian target of rapamycin complex 2 (mTORC2) has been identified as a major regulator of bladder cancer cell migration and invasion. Upstream pathways that mediate mTORC2 activation remain poorly defined. Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored membrane protein and known activator of cell-signaling. We identified increased uPAR expression in 94% of invasive human bladder cancers and in 54-71% of non-invasive bladder cancers, depending on grade. Normal urothelium was uPAR-immunonegative. Analysis of publicly available datasets identified uPAR gene amplification or mRNA upregulation in a subset of bladder cancer patients with reduced overall survival. Using biochemical approaches, we showed that uPAR activates mTORC2 in bladder cancer cells. Highly invasive bladder cancer cell lines, including T24, J82 and UM-UC-3 cells, showed increased uPAR mRNA expression and protein levels compared with the less aggressive cell lines, UROtsa and RT4. uPAR gene-silencing significantly reduced phosphorylation of Serine-473 in Akt, an mTORC2 target. uPAR gene-silencing also reduced bladder cancer cell migration and Matrigel invasion. S473 phosphorylation was observed by immunohistochemistry in human bladder cancers only when the tumors expressed high levels of uPAR. S473 phosphorylation was not controlled by uPAR in bladder cancer cell lines that are PTEN-negative; however, this result probably did not reflect altered mTORC2 regulation. Instead, PTEN deficiency de-repressed alternative kinases that phosphorylate S473. Our results suggest that uPAR and mTORC2 are components of a single cell-signaling pathway. Targeting uPAR or mTORC2 may be beneficial in patients with bladder cancer.

The repair of injured tissue is a highly complex process that involves cell proliferation, differentiation, and migration. Cell migration requires the dismantling of intercellular contacts in the injured zone and their subsequent reconstitution in the wounded area. Urokinase-type plasminogen activator (uPA) is a serine proteinase found in multiple cell types including endothelial cells, smooth muscle cells, monocytes, and macrophages. A substantial body of experimental evidence with different cell types outside the central nervous system indicates that the binding of uPA to its receptor (uPAR) on the cell surface prompts cell migration by inducing plasmin-mediated degradation of the extracellular matrix. In contrast, although uPA and uPAR are abundantly found in astrocytes and uPA binding to uPAR triggers astrocytic activation, it is unknown if uPA also plays a role in astrocytic migration. Neuronal cadherin is a member of cell adhesion proteins pivotal for the formation of cell-cell contacts between astrocytes. More specifically, while the extracellular domain of neuronal cadherin interacts with the extracellular domain of neuronal cadherin in neighboring cells, its intracellular domain binds to β-catenin, which in turn links the complex to the actin cytoskeleton. Glycogen synthase kinase 3β is a serine-threonine kinase that prevents the cytoplasmic accumulation of β-catenin by inducing its phosphorylation at Ser33, Ser37, and Ser41, thus activating a sequence of events that lead to its proteasomal degradation. The data discussed in this perspective indicate that astrocytes release uPA following a mechanical injury, and that binding of this uPA to uPAR on the cell membrane induces the detachment of β-catenin from the intracellular domain of neuronal cadherin by triggering its extracellular signal-regulated kinase 1/2-mediated phosphorylation at Tyr650. Remarkably, this is followed by the cytoplasmic accumulation of β-catenin because uPA-induced extracellular signal-regulated kinase 1/2 activation also phosphorylates lipoprotein receptor-related protein 6 at Ser1490, which in turn, by recruiting glycogen synthase kinase 3β to its intracellular domain abrogates its effect on β-catenin. The cytoplasmic accumulation of β-catenin is followed by its nuclear translocation, where it induces the expression of uPAR, which is required for the migration of astrocytes from the injured edge into the wounded area.

In the era of liquid biopsies, the reliable and cost-effective detection and screening of cancer biomarkers has become of fundamental importance, thus paving the way for the advancement of research in the field of point-of-care testing and the development of new methodologies and technologies. Indeed, the latter ones can help designing advanced diagnostic tools that can offer portability, ease of use with affordable production and operating costs. In this respect, impedance-based biosensing platforms might represent an attractive alternative. In this work, we describe a proof-of-concept study aimed at designing portable impedimetric biosensors for the monitoring of human urokinase-type plasminogen activator (h-uPA) cancer biomarker by employing small synthetic receptors. Aberrant levels of h-uPA were correlated with different types of cancers. Herein, we report the use of two bicyclic peptides (P2 and P3) which have been engineered to bind h-uPA with high affinity and exquisite specificity. The synthetic receptors were immobilized via biotin-streptavidin chemistry on the surface of commercial screen-printed electrodes. The impedimetric changes in the electrode/solution interface upon incubation of spiked h-uPA samples in the presence of a redox probe were followed via electrochemical impedance spectroscopy. The P3-based impedimetric assay showed the best outcomes in terms of dynamic range and linearity (0.01–1 μg mL−1) and sensitivity (LOD = 9 ng mL−1). To fully assess the performances of P3 over P2, and to compare the label-free architecture vs. labelled architecture, a voltammetric assay was also developed.

Dementia is a clinical syndrome that affects approximately 47 million people worldwide and is characterized by progressive and irreversible decline of cognitive, behavioral and sesorimotor functions. Alzheimer’s disease (AD) accounts for approximately 60–80% of all cases of dementia, and neuropathologically is characterized by extracellular deposits of insoluble amyloid-β (Aβ) and intracellular aggregates of hyperphosphorylated tau. Significantly, although for a long time it was believed that the extracellular accumulation of Aβ was the culprit of the symptoms observed in these patients, more recent studies have shown that cognitive decline in people suffering this disease is associated with soluble Aβ-induced synaptic dysfunction instead of the formation of insoluble Aβ-containing extracellular plaques. These observations are translationally relevant because soluble Aβ-induced synaptic dysfunction is an early event in AD that precedes neuronal death, and thus is amenable to therapeutic interventions to prevent cognitive decline before the progression to irreversible brain damage. The plasminogen activating (PA) system is an enzymatic cascade that triggers the degradation of fibrin by catalyzing the conversion of plasminogen into plasmin via two serine proteinases: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Experimental evidence reported over the last three decades has shown that tPA and uPA play a role in the pathogenesis of AD. However, these studies have focused on the ability of these plasminogen activators to trigger plasmin-induced cleavage of insoluble Aβ-containing extracellular plaques. In contrast, recent evidence indicates that activity-dependent release of uPA from the presynaptic terminal of cerebral cortical neurons protects the synapse from the deleterious effects of soluble Aβ via a mechanism that does not require plasmin generation or the cleavage of Aβ fibrils. Below we discuss the role of the PA system in the pathogenesis of AD and the translational relevance of data published to this date.

Abstract: The aim of this study was to analyze and compare the immunohistochemical expression of plasminogen activator system (PAS) proteins (uPA, uPAR, and PAI-1) in ameloblastomas (AMBs), odontogenic keratocysts (OKCs), and dental follicles (DFs) representing normal odontogenic tissue, as well as to investigate possible correlations between these proteins. Twenty AMBs, 20 OKCs, and 10 DFs were selected for immunohistochemical analysis. In each case, the immunoexpression of uPA, uPAR, and PAI-1 was evaluated semiquantitatively based on the percentage of positivity in odontogenic epithelial and connective tissue cells. The epithelial immunoexpression of uPA was significantly lower in AMBs when compared to OKCs (p = 0.001) and DFs (p = 0.029). Significantly higher epithelial immunostaining for uPAR was observed in AMBs when compared to OKCs (p < 0.001). There were no significant differences in the epithelial immunoexpression of PAI-1 between AMBs and OKCs (p = 1.000). The correlations found for the expression of the studied proteins were not statistically significant (p > 0.05). However, the epithelial and connective tissue expressions of uPAR have a strong positive and statistically significant correlation in AMBs. The present results suggest that uPA is involved in the pathogenesis of OKCs and that uPAR may participate in tumorigenesis in AMBs. The high percentage of PAI-1-positive cells suggests a possible role for this protein in the development of AMBs and OKCs. Furthermore, the studied proteins do not seem to act synergistically in AMBs, OKCs, and DFs.

BackgroundKnown risk factors for intracerebral hemorrhage (ICH) include age, hypertension, smoking, alcohol intake, and anticoagulant use. Some previous reports have indicated that hemostatic factors measured many years before the onset of ICH might predict the later occurrence of ICH. The objective of this analysis was to test whether selected hemostatic factors measured years before the onset of ICH could identify patients at higher risk for future ICH.MethodsWe performed a nested case-control study within the Women's Health Initiative (WHI) cohort. Postmenopausal women aged 50-79 years (mean 68) at baseline (1993-1998) were enrolled at 40 Clinical Centers in the United States and followed for adjudicated ICH for a mean of 11.4 years. ICH cases (N = 75) and controls (N = 75) were matched on age, ethnicity, blood pressure, anticoagulant use, and treated hypertension. Stored blood samples from the baseline WHI examination were tested for von Willebrand factor (vWF), a disintegrin-like and metalloprotease domain with thrombospondin type-1 motif, number 13 (ADAMTS13), tissue plasminogen activator (t-PA), and urokinase plasminogen activator (u-PA). Platelet count, white blood cell count, and hemoglobin concentration were also measured.ResultsMean baseline levels of vWF (1.03 and .95 U/mL), ADAMTS13 (1.0 and 1.1 µg/mL), vWF : ADAMTS13 ratio (.99 and .92), t-PA (14.75 and 14.80 IU/mL), and u-PA (.09 and .10 IU/mL) were not significantly different by case-control status. Significant differences were also not identified for platelet count, hemoglobin, white blood count, or reported alcohol use.ConclusionNone of the 4 baseline hemostatic factors nor the platelet count was predictive of future ICH risk in this long-term study of older postmenopausal women.

SLURP1, a member of the lymphocyte antigen 6 protein family, is secreted by suprabasal keratinocytes. Mutations in SLURP1 cause a palmoplantar keratoderma (PPK) known as mal de Meleda. SLURP2, another secreted lymphocyte antigen 6 protein, is encoded by a gene located ?20 kb downstream from SLURP1. SLURP2 is produced by suprabasal keratinocytes. To investigate the importance of SLURP2, we first examined Slurp2 knockout mice in which exon 2-3 sequences had been replaced with lacZ and neo cassettes. Slurp2(-/-) mice exhibited hyperkeratosis on the volar surface of the paws (i.e., palmoplantar keratoderma), increased keratinocyte proliferation, and an accumulation of lipid droplets in the stratum corneum. They also exhibited reduced body weight and hind limb clasping. These phenotypes are similar to those of Slurp1(-/-) mice. To solidify a link between Slurp2 deficiency and palmoplantar keratoderma and to be confident that the disease phenotypes in Slurp2(-/-) mice were not secondary to the effects of the lacZ and neo cassettes on Slurp1 expression, we created a new line of Slurp2 knockout mice (Slurp2X(-/-)) in which Slurp2 was inactivated with a simple nonsense mutation. Slurp2X(-/-) mice exhibited the same disease phenotypes. Thus, Slurp2 deficiency and Slurp1 deficiencies cause the same disease phenotypes.

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The aim of this study was to analyze and compare the immunohistochemical expression of plasminogen activator system (PAS) proteins (uPA, uPAR, and PAI-1) in ameloblastomas (AMBs), odontogenic keratocysts (OKCs), and dental follicles (DFs) representing normal odontogenic tissue, as well as to investigate possible correlations between these proteins. Twenty AMBs, 20 OKCs, and 10 DFs were selected for immunohistochemical analysis. In each case, the immunoexpression of uPA, uPAR, and PAI-1 was evaluated semiquantitatively based on the percentage of positivity in odontogenic epithelial and connective tissue cells. The epithelial immunoexpression of uPA was significantly lower in AMBs when compared to OKCs (p = 0.001) and DFs (p = 0.029). Significantly higher epithelial immunostaining for uPAR was observed in AMBs when compared to OKCs (p < 0.001). There were no significant differences in the epithelial immunoexpression of PAI-1 between AMBs and OKCs (p = 1.000). The correlations found for the expression of the studied proteins were not statistically significant (p > 0.05). However, the epithelial and connective tissue expressions of uPAR have a strong positive and statistically significant correlation in AMBs. The present results suggest that uPA is involved in the pathogenesis of OKCs and that uPAR may participate in tumorigenesis in AMBs. The high percentage of PAI-1-positive cells suggests a possible role for this protein in the development of AMBs and OKCs. Furthermore, the studied proteins do not seem to act synergistically in AMBs, OKCs, and DFs. [ABSTRACT FROM AUTHOR]

BACKGROUND: Oral squamous cell carcinoma (OSCC) usually originates from oral potentially malignant disorders (OPMD), such as oral leukoplakia (OLK) and oral lichen planus (OLP). Identifying biomarkers for the early diagnosis and evaluation of malignant transformation in OPMD could improve the survival rate of OSCC patients. OBJECTIVE: The present study aimed to screen for potential salivary biomarkers for evaluating the malignant transformation of OPMD. METHODS: Salivary proteases from OLK and OSCC patients or healthy donors and proteases in cultural medium from DOK and Cal-27 cells were detected with a human protease array kit. The concentrations of the salivary Kallikrein 5 (KLK5) and urokinase-type plasminogen activator (uPA) proteases were measured by ELISA. Receiver operating characteristics (ROC) to determine the potential value of these proteases in clinical diagnosis were calculated using SPSS software. Immunohistochemistry was used to detect the KLK5 and uPA expression in the oral organizations. RESULTS: The salivary protease spectrum was different among patients with OLK and OSCC and healthy donors. KLK5 and uPA levels in saliva tended to increase as the disease progressed (healthy < OPMD [OLK and OLP] < OSCC). ROC curves showed the optimum diagnostic cutoffs for KLK5 as a biomarker for OLK, OLP, and OSCC were 5.97, 6.03, and 9.45 pg/mL, respectively, while the cutoffs for uPA were 17.19, 17.26, and 20.96 pg/mL. Their combined analysis showed a higher sensitivity for the differential diagnosis of disease. Furthermore, higher levels of KLK5 and uPA were observed in OSCC tissues than in OLK and OLP. CONCLUSIONS: Salivary KLK5 and uPA are potential biomarkers for evaluating OLK and OLP malignant transformation and early diagnosis of OSCC. [ABSTRACT FROM AUTHOR]

Study Question: Does fibrin promote trophoblast growth in human and mouse blastocysts during early embryo implantation?Summary Answer: Mouse blastocysts were unaffected by fibrin; however, human blastocysts were significantly suppressed by fibrin in trophoblast growth and then switched to growth promotion through increased fibrinolysis with urokinase-type plasminogen activator (uPA) activity.What Is Known Already: Fibrin(ogen) plays an important role in various physiological processes and is also critical for maintaining feto-maternal attachment during pregnancy. The addition of fibrin to embryo transfer media has been used to increase implantation rates in human ART; however, its mechanism of action' in vitro has not yet been characterized.Study Design, Size, Duration: Vitrified mouse and human blastocysts were warmed and individually cultured in vitro for up to 120 and 168 h, respectively, on a fibrin substrate. Blastocysts were cultured at 37°C in 6% CO2, 5% O2 and 89% N2. Blastocyst development and related fibrinolytic factors were analyzed.Participants/materials, Setting, Methods: ICR strain mouse embryos were purchased from a commercial supplier. Human blastocysts were donated with informed consent from two fertility centers. Mouse and human blastocysts cultured on fibrin-coated plates were compared to those on non-coated and collagen-coated plates in vitro. Trophoblast growth and fibrin degradation were assessed based on the cell area and fibrin-free area, respectively. Fibrinolytic factors were detected in supernatants using plasminogen-casein zymography. The fibrinolytic activity of blastocysts was investigated using a selective uPA inhibitor, exogenous uPA, plasminogen activator inhibitor-1 (PAI-1) inhibitor and fibrin degradation products (FDPs). Fibrinolysis-related mRNA expression level was detected using quantitative real-time PCR.Main Results and the Role Of Chance: Fibrin did not affect the developmental speed or morphology of mouse blastocysts, and a large fibrin-degrading region was observed in the attachment stage. In contrast, fibrin significantly suppressed the outgrowth of trophoblasts in human blastocysts, and trophoblasts grew after the appearance of small fibrin-degrading regions. uPA was identified as a fibrinolytic factor in the conditioned medium, and uPA activity was significantly weaker in human blastocysts than in mouse blastocysts. The inhibition of uPA significantly reduced the outgrowth of trophoblasts in mouse and human blastocysts. Human blastocysts expressed PLAU (uPA), PLAUR (uPA receptor), SERPINE1 (PAI-1) and SERPINB2 (PAI-2), whereas mouse blastocysts were limited to Plau, Plaur and Serpine1. In a subsequent experiment on human blastocysts, the addition of exogenous uPA and the PAI-1 inhibitor promoted trophoblast growth in the presence of fibrin, as did the addition of FDPs.Limitations, Reasons For Caution: This model excludes maternal factors and may not be fully reproduced in vivo. Donated human embryos are surplus embryos that may inherently exhibit reduced embryonic development. In addition, donated ART-derived embryos may exhibit weak uPA activity, because women with sufficient uPA-active embryos may not originally require ART. The present study used orthodox culture methods, and results may change with the application of recently developed protocols for culture blastocysts beyond the implantation stage.Wider Implications Of the Findings: The present results suggest that the distinct features of trophoblast outgrowth in human blastocysts observed in the presence of fibrin are regulated by a phenotypic conversion induced by contact with fibrin and FDPs. Mouse embryos did not exhibit the human phenomenon, indicating that the present results may be limited to humans.Study Funding/competing Interest(s): The present study was supported by the Department of Obstetrics and Gynecology at the Hamamatsu University School of Medicine and Kishokai Medical Corporation. None of the authors have any conflicts of interest to declare.Trial Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Abstract Background Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway. Methods Primary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents. Results Astrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor. Conclusions Plasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS.

Purpose: We sought to validate the association of plasma levels of urokinase-type plasminogen activator (uPA), its soluble receptor (SuPAR) and its inhibitor (PAIone) with oncologic outcomes in a large cohort of patients treated with radical cystectomy (RC) for urothelial carcinoma of the bladder (UCB). Materials and Methods: We collected preoperative blood samples from 1,036 consecutive patients treated with RC for UCB. Plasma specimens were assessed for levels of uPA, SuPAR and PAI-one. Retrospective logistic and Cox regression analyses were performed to assess their correlation with clinical outcomes. The additional clinical net benefit provided by the biomarkers was evaluated using decision curve analysis. Results: Preoperative plasma uPA, SuPAR and PAI-one levels were significantly elevated in patients harboring adverse pathological features. Higher levels of all biomarkers were independently associated with an increased risk of lymph node metastasis; uPA levels were also independently associated with ≥pT3 disease. Preoperative uPA and SuPAR were independently associated with recurrencefree and cancer-specific survival. The addition of these biomarkers to standard pre-treatment and post-treatment models improved the discriminatory power for prediction of lymph node metastasis, ≥pT3 disease, and recurrence-free and cancer-specific survival by a prognostically significant margin. Conclusions: We confirmed that elevated preoperative plasma levels of uPA, SuPAR and PAI-one are associated with features of aggressive disease and worse survival outcomes in patients treated with RC for UCB. These biomarkers hold potential in identifying patients who are likely to benefit from intensified/multimodal therapy. They also demonstrated the ability to improve the discriminatory power of predictive/prognostic models, thus refining personalized clinical decision-making. [ABSTRACT FROM AUTHOR]

Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are serine proteases and major activators of fibrinolysis in mammalian systems. Because fibrinolysis is an essential component of the response to tissue injury, diverse cells, including cells that participate in the response to injury, have evolved receptor systems to detect tPA and uPA and initiate appropriate cell-signaling responses. Formation of functional receptor systems for the plasminogen activators requires assembly of diverse plasma membrane proteins, including but not limited to: the urokinase receptor (uPAR); integrins; N-formyl peptide receptor-2 (FPR2), receptor tyrosine kinases (RTKs), the N-methyl-D-aspartate receptor (NMDA-R), and low-density lipoprotein receptor-related protein-1 (LRP1). The cell-signaling responses elicited by tPA and uPA impact diverse aspects of cell physiology. This review describes rapidly evolving knowledge regarding the structure and function of plasminogen activator receptor assemblies. How these receptor assemblies regulate innate immunity and inflammation is then considered. [ABSTRACT FROM AUTHOR]

Urokinase-type plasminogen activator (uPA) is a serine proteinase that catalyzes the generation of plasmin on the cell surface and activates cell signaling pathways that promote remodeling and repair. Neuronal cadherin (NCAD) is a transmembrane protein that in the mature brain mediates the formation of synaptic contacts in the II/III and V cortical layers. Our studies show that uPA is preferentially found in the II/III and V cortical laminae of the gyrencephalic cortex of the non-human primate. Furthermore, we found that in murine cerebral cortical neurons and induced pluripotent stem cell (iPSC)-derived neurons prepared from healthy human donors, most of this uPA is associated with pre-synaptic vesicles. Our in vivo experiments revealed that in both, the gyrencephalic cortex of the non-human primate and the lissecephalic murine brain, cerebral ischemia decreases the number of intact synaptic contacts and the expression of uPA and NCAD in a band of tissue surrounding the necrotic core. Additionally, our in vitro data show that uPA induces the synthesis of NCAD in cerebral cortical neurons, and in line with these observations, intravenous treatment with recombinant uPA three hours after the onset of cerebral ischemia induces NCAD-mediated repair of synaptic contacts in the area surrounding the necrotic core. [ABSTRACT FROM AUTHOR]

5-Benzylglycinyl-amiloride (UCD38B) is the parent molecule of a class of anticancer small molecules that kill proliferative and nonproliferative high-grade glioma cells by programmed necrosis. UCD38B intracellularly triggers endocytosis, causing 40-50% of endosomes containing proteins of the urokinase plasminogen activator system (uPAS) to relocate to perinuclear mitochondrial regions. Endosomal "mis-trafficking" caused by UCD38B in human glioma cells corresponds to mitochondrial depolarization with the release and nuclear translocation of apoptotis-inducing factor (AIF) followed by irreversible caspase-independent cell demise. High-content quantification of immunocytochemical colocalization studies identified that UCD38B treatment increased endocytosis of the urokinase plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) into the early and late endosomes by 4- to 5-fold prior to AIF nuclear translocation and subsequent glioma demise. PAI-1 was found to comparably relocate with a subset of early and late endosomes in four different human glioma cell lines after UCD38B treatment, followed by caspase-independent, nonapoptotic cell death. Following UCD38B treatment, the receptor guidance protein LRP-1, which is required for endosomal recycling of the uPA receptor to the plasmalemma, remained abnormally associated with PAI-1 in early and late endosomes. The resultant aberrant endosomal recycling increased the total cellular content of the uPA-PAI-1 protein complex. Reversible inhibition of cellular endocytosis demonstrated that UCD38B bypasses the plasmalemmal uPAS complex and directly acts intracellularly to alter uPAS endocytotic trafficking. UCD38B represents a class of small molecules whose anticancer cytotoxicity is a consequence of causing the mis-trafficking of early and late endosomes containing uPAS cargo and leading to AIF-mediated necrotic cell death.

The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU). In the full-length immunoglobulin form, U33 IgG labeled with near-infrared fluorophores or radionuclides allowed us to noninvasively detect active uPA in prostate cancer xenograft models using optical and single-photon emission computed tomography imaging modalities. U33 IgG labeled with (111)In had a remarkable tumor uptake of 43.2% injected dose per gram (%ID/g) 72 hours after tail vein injection of the radiolabeled probe in subcutaneous xenografts. In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissemination prostate cancer model that recapitulated metastatic human cancer. The favorable imaging properties were the direct result of U33 IgG internalization through an uPA receptor-mediated mechanism in which U33 mimicked the function of the endogenous inhibitor of uPA to gain entry into the cancer cell. Overall, our imaging probe targets a prostate cancer-associated protease, through a unique mechanism, allowing for the noninvasive preclinical imaging of prostate cancer lesions.

Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.