Activator uPA Research Articles

Bruton tyrosine kinase promotes wound healing after myocardial infarction by inhibiting the transcription of u-PA

BackgroundsBruton tyrosine kinase (BTK), which is highly expressed in immune cells, plays a critical role in regulating the function of macrophages. A growing body of evidence has demonstrated that the accumulation of macrophages in cardiac tissue after myocardial infarction (MI) significantly affects wound healing and ventricular remodeling during the early phase of repair after MI. However, the role of BTK in cardiac repair post-MI, especially in macrophage-mediated repair, remains unclear. MethodsMI was induced by permanent left anterior descending (LAD) artery ligation in wild-type (WT) mice and macrophage-specific BTK-knockout (BTKMAC-KO) mice. Expression of BTK and phosphorylated BTK were assessed by western blotting. Then, RNA sequencing and ChIP-qPCR assay were performed to explore potential BTK targets and transcriptional regulatory sites. ResultsBTK, which was mainly expressed in macrophages, was upregulated in mice after MI. Compared with WT mice, BTKMAC-KO mice had significantly greater mortality due to heart rupture, reduced wall thickness and severe impairment of left ventricular (LV) function after MI. In addition, increased matrix metalloproteinase-9 (MMP-9) expression and decreased α-SMA and collagen expression were observed in BTKMAC-KO mice after MI. Further experiments revealed that BTK deficiency in macrophages reduces the expression of VEGF and impairs angiogenesis after MI. By RNA sequencing, we found that Nf-kB family genes, as well as the urokinase-type plasminogen activator (uPA), were significantly upregulated in BTK-deficient macrophages. By ChIP-qPCR analysis, we confirmed that uPA was transcriptionally activated by the Nf-kB p65 subunit. Finally, the application of plasminogen activator inhibitor-1 (PAI-1), an uPA inhibitor, markedly protected against cardiac rupture, lowered the mortality rate, and improved cardiac function by increasing collagen deposition and promoting tissue healing in BTKMAC-KO mice after MI. ConclusionsThe present study identifies PAI-1 as a novel cardioprotective agent for cardiac repair post-MI that increases collagen deposition and promotes tissue healing. A therapeutic strategy targeting BTK may be a promising treatment for cardiac repair post-MI.

Activation of the Urokinase Plasminogen Activator/Urokinase Plasminogen Activator Receptor System in Periodontitis: A Case-Control Study.

The plasminogen activating (PA) system has a multitude of functions such as wound healing, proteolytic activity, collagen degradation and cell growth, and the role of the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system has been studied in many disease states. The aim of this study was to investigate salivary concentrations of uPA, uPAR and uPA activity in patients with periodontitis to identify biomarkers and novel pathogenic relationships. Saliva samples were obtained from 169 participants, comprising patients with periodontitis (n = 103) and periodontally healthy volunteers (n = 66) and analysed for uPA and uPAR with a multiplex protein assay using proximity extension analysis in a subset of samples, followed by validation with ELISA. The protease activity of salivary uPA was quantified using a fluorometric assay. Patients with periodontitis had a 4.0-fold higher (p < 0.001) salivary uPA and a 2.5-fold higher (p < 0.001) salivary uPAR concentration in comparison to periodontally healthy participants. The salivary uPA activity (median [IQR]) from patients with periodontitis (123.21 [188.29] U/mL) was 1.6-fold higher (p < 0.01) than the salivary uPA activity from periodontally healthy participants (76.83 [98.09] U/mL). Levels of uPA and uPAR were strongly correlated with periodontal indices, whereas only weak correlations were found with BMI and age. Activation of uPA/uPAR likely plays a role in the pathogenesis of periodontal diseases. uPA/uPAR may have potential utility as candidate salivary biomarkers in periodontal pathogenesis.

WITHDRAWN: THE ROLE OF THE PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI1) IN OARIAN CANCER: MECHANISMS AND THERAPEUTIC IMPLICATIONS

OC (OC) is one among most significantly fatal gynaecological cancers, with late-stage detection and an inadequate prognosis. Plasminogen Activator Inhibitor -1 (PAI1) gene anticipates negative outcomes in many different kinds of malignancies. Several research investigations are currently being done to examine the biological role of PAI1 in OC and the possible benefits of targeted pharmacotherapies. The PAI1 gene has been linked to the emergence and development of cancer in the ovary. PAI1, an inhibitor of serine protease, influences the fibrinolysis and extracellular matrix remodeling, both of which that are crucial for tumor expansion and metastatic growth. PAI1 levels have been discovered to be subsequently more elevated in malignant ovarian tissues than in usual ovarian tissue, demonstrating a potential connection among PAI1 overexpression and OC development. PAI1 promotes tumor cell proliferation, movement, and an invasion by influencing the urokinase-plasminogen activators (uPA) and through interactions with cell surface receptors. In addition, PAI1 gene contributes to angiogenesis and apoptotic cell death, which contribute to the more hostile phenotypes of OC. The prognostic and therapeutic consequences of focusing on PAI1 in OC are explored, demonstrating PAI1's potential to be a biomarker and emphasizing for novel treatment approaches. The PAI1 gene possesses several functions in OC, affecting tumor development, an invasion, and metastatic growth. Comprehending the complicated interactions and mechanisms that regulate PAI1 in OC may lead to more efficient evaluation and treatment strategies and ultimately enhance patient outcomes.

Causal correlations between inflammatory proteins and heart failure: A two-sample Mendelian randomization analysis.

Inflammation plays a critical role in both the development and progression of heart failure (HF), which is a leading cause of morbidity and mortality worldwide. However, the causality between specific inflammation-related proteins and HF risk remains unclear. This study aims to investigate the genetically supported causality between inflammatory proteins and HF using a two-sample Mendelian randomization (MR) analysis. We utilized genome-wide association study (GWAS) data of 91 inflammation-related proteins as exposures from the SCALLOP Consortium (14,824 participants), alongside outcome GWAS summary statistics from FinnGen (29,218 cases/381,838 controls) and HERMES (47,309 cases/930,014 controls) for HF, to conduct a two-sample MR analysis. For each inflammatory protein, instrumental variables (IVs) were chosen following the three foundational assumptions of the MR analysis, requiring a minimum of three qualifying single nucleotide polymorphisms (SNPs) each with a P<5e-8. Associations between inflammatory proteins and HF were assessed through inverse-variance weighted (IVW), MR-Egger regression, weighted median and weighted mode analysis. The reliability and validity of the results were evaluated by examining heterogeneity, horizontal pleiotropy, leave-one-out analysis, meta-analysis and reverse MR analysis. Heterogeneity refers to the variation in results across different genetic variants. Horizontal pleiotropy occurs when a genetic variant influences multiple traits through different biological pathways. Addressing both heterogeneity and horizontal pleiotropy is crucial for ensuring the reliability and interpretability of MR results. Our analysis identified associations between three inflammatory proteins and HF risk. Matrix metalloproteinase-1 (MMP-1) (OR, 1.09; 95% CI, 1.00-1.18; P=0.04) and TNF-beta (OR, 1.05; 95% CI, 1.01-1.09; P=0.01) were positively associated with HF risk in FinnGen. In contrast, urokinase-type plasminogen activator (uPA) was inversely associated with HF risk in both FinnGen (OR, 0.85; 95% CI, 0.78-0.92; P=3.27e-5) and HERMES (OR, 0.93; 95% CI, 0.87-0.99; P=0.03). No evidence of heterogeneity and horizontal pleiotropy was observed in the MR analysis, indicating the robustness of our findings. A meta-analysis further supported this association, indicating a reduced risk (OR, 0.89; 95% CI, 0.81-0.98; P=0.02). No reverse causality was found between HF and these three inflammatory proteins (P>0.05 for all). This study provides genetically supported evidence of the causal association of specific inflammatory proteins with HF risk. The positive association of MMP-1 and TNF-beta with HF suggests their roles in disease pathogenesis, whereas the inverse association of the uPA indicates its potential protective effect. Our findings highlight the potential of targeting specific inflammatory pathways as a therapeutic strategy for HF.

Mesenchymal stem/stromal cells alleviate early-stage pulmonary fibrosis in a uPAR-dependent manner.

Pulmonary fibrosis, a debilitating lung disorder characterised by excessive fibrous tissue accumulation in lung parenchyma, compromises respiratory function leading to a life-threatening respiratory failure. While its origins are multifaceted and poorly understood, the urokinase system, including urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plays a significant role in regulating fibrotic response, extracellular matrix remodelling, and tissue repair. Mesenchymal stem/stromal cells (MSCs) hold promise in regenerative medicine for treating pulmonary fibrosis. Our study aimed to investigate the potential of MSCs to inhibit pulmonary fibrosis as well as the contribution of uPAR expression to this effect. We found that intravenous MSC administration significantly reduced lung fibrosis in the bleomycin-induced pulmonary fibrosis model in mice as revealed by MRI and histological evaluations. Notably, administering the MSCs isolated from adipose tissue of uPAR knockout mice (Plaur-/- MSCs) attenuated lung fibrosis to a lesser extent as compared to WT MSCs. Collagen deposition, a hallmark of fibrosis, was markedly reduced in lungs treated with WT MSCs versus Plaur-/- MSCs. Along with that, endogenous uPA levels were affected differently; after Plaur-/- MSCs were administered, the uPA content was specifically decreased within the blood vessels. Our findings support the potential of MSC treatment in attenuating pulmonary fibrosis. We provide evidence that the observed anti-fibrotic effect depends on uPAR expression in MSCs, suggesting that uPAR might counteract the uPA accumulation in lungs.

Diastereoselective synthesis and biological evaluation of new fluorine-containing α-aminophosphonates as anticancer agents and scaffold to human urokinase plasminogen activator inhibitors

Phosphonate analogs of α-amino acids are increasingly valued for their significant potential in medicinal chemistry. Fluorine is a “magic” element that plays a huge role in modulating the properties of organic compounds. In this work, we combined the two pharmacophores in the synthesis of three series of new α-aminophosphonates. These compounds were obtained by diastereoselective hydrophosphonylation of imines prepared by an environmentally friendly mechanochemical approach.Results of computational SwissADME analysis suggested favorable drug-like properties of the α-aminophosphonates and indicated their potential for interaction with diverse biological targets including proteases, showing promising pharmacokinetic profiles compared to 5-fluoro-2'-deoxyuridine (FdU) used as a standard anticancer drug.Screening against ten cancer cell lines from seven types of cancer showed that five of the twenty compounds tested (1c, 2a, 2h, 3e, and 3f) exhibited superior activity against the HeLa cell line and lower cytotoxicity against normal MRC-5 cells than FdU. Compound 3e showed notable inhibitory effect on the MDA-MB-231 cell line, while 3a, 3h, and 3g demonstrated significant cytotoxic activity against U-87 MG and U-251 MG lines.Molecular docking highlighted the strong binding of compound 2a to the urokinase-type plasminogen activator (uPA) protein, with a binding affinity of −6.41 kcal/mol, suggesting the anti-metastatic potential of the compound. These findings enable to position the newly synthesized α-aminophosphonates as promising scaffolds for developing targeted anticancer therapies for metastatic cancers characterized by elevated uPA expression.

Circulating levels of neutrophil extracellular traps reflects local neutrophil activity but not fibrinolytic activity in coronary thrombi from patients with ST-elevation myocardial infarction

Abstract Introduction Neutrophil extracellular traps (NETs) are associated with impaired fibrinolysis in ischemic stroke, while NETs degradation enhances lysis of coronary thrombi in ex vivo experiments. Whether circulating NET markers are associated with local fibrinolytic activity in patients with ST-elevation myocardial infarction (STEMI) has not been reported. Purpose We investigated associations between circulating NET markers and gene expression of fibrinolytic markers in aspirated coronary thrombi from patients with STEMI. Furthermore, we explored whether local upregulation of NET formation by peptidylarginine deiminase 4 (PAD4) was reflected in the circulation. Methods Coronary thrombi from 35 STEMI patients undergoing primary percutaneous coronary intervention (PCI) were aspirated and promptly snap-frozen to -80°C. Peripheral blood samples were collected simultaneously. Median time from start of symptoms to PCI was 152 min. Thrombus gene expression of tissue Plasminogen Activator (tPA), urinary-type Plasminogen Activator (uPA), plasminogen-activation inhibitor type 1 (PAI-1) and PAD4 were quantified with RT-PCR. Gene expression of PAD4 was also measured in circulating leukocytes. Circulating NET markers were measured by a fluorescent nucleic acid stain using fluorometry (dsDNA), an in-house ELISA technique (MPO-DNA) and commercial ELISA (H3Cit). Correlations were tested using Spearman’s rho. Results There were no correlations between any of the circulating NET markers and gene expression of the fibrinolytic markers tPA, uPA and PAI-1 in the thrombus. Of the circulating NET markers, dsDNA correlated with thrombus gene expression of PAD4 (rs=0.491 p=0.006), whereas none of the NET markers correlated with gene expression of PAD4 in circulating leukocytes. Conclusion Circulating NET markers were not associated with gene expression of fibrinolytic activity in thrombi from patients with STEMI. Local NET formation in the thrombi, measured as PAD4 expression, was associated with the circulating NET marker dsDNA. These findings suggest that circulating NET markers have limited utility for assessing local fibrinolytic activity, but might reflect PAD4-dependent NET formation in coronary thrombi.

Delivering urokinase-type plasminogen activator using mulberry leaf exosomes enables thrombolysis and remodeling of venous microenvironments

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.

Potential Nephroprotective Effect of uPA against Ischemia/Reperfusion-Induced Acute Kidney Injury in αMUPA Mice and HEK-293 Cells.

Background/Objectives: The incidence of acute kidney injury (AKI) has been steadily increasing. Despite its high prevalence, there is no pathogenetically rational therapy for AKI. This deficiency stems from the poor understanding of the pathogenesis of AKI. Renal ischemia/hypoxia is one of the leading causes of clinical AKI. This study investigates whether αMUPA mice, overexpressing the urokinase plasminogen activator (uPA) gene are protected against ischemic AKI, thus unraveling a potential renal damage treatment target. Methods: We utilized an in vivo model of I/R-induced AKI in αMUPA mice and in vitro experiments of uPA-treated HEK-293 cells. We evaluated renal injury markers, histological changes, mRNA expression of inflammatory, apoptotic, and autophagy markers, as compared with wild-type animals. Results: the αMUPA mice exhibited less renal injury post-AKI, as was evident by lower SCr, BUN, and renal NGAL and KIM-1 along attenuated adverse histological alterations. Notably, the αMUPA mice exhibited decreased levels pro-inflammatory, fibrotic, apoptotic, and autophagy markers like TGF-β, IL-6, STAT3, IKB, MAPK, Caspase-3, and LC3. By contrast, ACE-2, p-eNOS, and PGC1α were higher in the kidneys of the αMUPA mice. In vitro results of the uPA-treated HEK-293 cells mirrored the in vivo findings. Conclusions: These results indicate that uPA modulates key pathways involved in AKI, offering potential therapeutic targets for mitigating renal damage.

Association between circulating inflammatory proteins and benign prostatic disease: a Mendelian randomization study

Previous research has suggested that circulating inflammatory proteins are associated with benign prostatic disease (BPD). This Mendelian randomization (MR) study was conducted to further investigate the causal relationship between 91 inflammatory proteins and BPD. Genome-wide association study (GWAS) summarized data of benign prostatic hyperplasia (BPH) and prostatitis were obtained from the FinnGen Biobank. The latest study offered the GWAS data on 91 proteins related to inflammation. We performed a bidirectional MR to investigate the causal association between inflammatory proteins and BPD. The outcomes of the IVW method indicated that decreased levels of circulating interleukin-17 C (IL-17 C) (OR = 0.92, 95%CI = 0.85–0.99, p-value = 0.0344) were suggestively associated with a higher risk of BPH and elevated levels of interleukin-10 receptor subunit alpha (IL-10RA) (OR = 1.24, 95%CI = 1.05–1.47, p-value = 0.0132) and urokinase-type plasminogen activator (uPA) (OR = 1.13, 95%CI = 1.00–1.28, p-value = 0.0421) were suggestively related to a higher risk of prostatitis. Furthermore, reverse MR revealed that BPH may promote the expression of circulating factors, including natural killer cell receptor 2B4 (CD244) (OR = 1.07, 95%CI = 1.01–1.13, p-value = 0.0192), T-cell surface glycoprotein CD6 isoform (CD6) (OR = 1.07, 95%CI = 1.01–1.13, p-value = 0.0192), and leukemia inhibitory factor receptor (LIF-R) (OR = 1.07, 95%CI = 1.01–1.15, p-value = 0.0163). Moreover, the results of sensitivity analyses indicate that heterogeneity and horizontal pleiotropy are unlikely to distort the findings. The results of this study indicate a potential association between circulating inflammatory proteins and BPD, which may become new diagnostic indicators or drug targets for clinical application in the prevention and treatment of BPD. However, further investigation is required.

Shilajit (Mumio) Elicits Apoptosis and Suppresses Cell Migration in Oral Cancer Cells Through Targeting Urokinase-type Plasminogen Activator and Its Receptor and Chemokine Signaling Pathways

Background Shilajit a natural phytomineral has a proven record of treating many human body ailments. Purpose This study aimed to explore the influence of Shilajit on cell proliferation and apoptosis induction in oral cancer cells (OCC) and to comprehend the molecular mechanism associated with OCC migration. Materials and Methods Human gingival fibroblast (hGF) and OCC (KB-1 (KERATIN-forming tumor cell line HeLa)) were exposed to Shilajit solution dilutions. Cell growth and apoptosis were measured by MTT and Annexin-V tests (control/test group). Cellular morphology using an inverted microscope, cellular apoptosis using acridine orange/ethidium bromide dual staining, reactive oxygen species production analysis using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and gene protein expression using real-time polymerase chain reaction test were used to measure study outcomes. The data enumerated were the average of three trial experiments. Categorical and numerical data were expressed as frequency distribution and means, respectively. Differences in groups (control/test; zero/24 h/48 h) were determined using Student’s t-test and one way analysis of variance, with probability ‘ p’ value considered significant at ≤0.05. Results The viability of OCC exhibited a concentration and time-dependent response to Shilajit. Notably, Shilajit demonstrated selectivity against cancer cells. Through an examination of the Annexin-V apoptosis assay, it was observed that Shilajit induces apoptosis by upregulating the proapoptotic gene expression ( p ≤ 0.05) and downregulating antiapoptotic proteins ( p ≤ 0.05). Furthermore, the impact of Shilajit on cell migration decreased significantly when compared to control cells through modulating the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) and chemokines gene expression. Conclusion Shilajit exhibited greater cytotoxicity, decreased OCC proliferation and migration, and initiated OCC apoptosis as equivalent to normal cells. These promising outcomes indicate that Shilajit holds potential as a robust, promising option for oral cancer treatment, underscoring the need for further research in this domain.

Inherited Disorders of the Fibrinolytic Pathway: Pathogenic Phenotypes and Diagnostic Considerations of Extremely Rare Disorders

AbstractFibrinolysis is initiated by the activation of plasminogen to plasmin via tissue-plasminogen activator (tPA) and urokinase-plasminogen activator (uPA); plasmin then converts fibrin to fibrin degradation products (FDPs). The antifibrinolytics counterbalancing this system include plasminogen activator inhibitor-1 (PAI-1), which inhibits tPA and uPA, α-2 antiplasmin (α2AP), which inhibits plasmin, and thrombin activatable fibrinolysis inhibitor, which inhibits the conversion of fibrin to FDP. Inherited disorders of the fibrinolytic pathway are rare and primarily have hemorrhagic phenotypes in humans: PAI-1 deficiency, α2AP deficiency, and Quebec platelet disorder. Patients with these disorders are usually treated for bleeds or receive prophylaxis to prevent bleeds in the surgical setting, with pharmacological antifibrinolytics such as aminocaproic acid and tranexamic acid. Disorders of the fibrinolytic pathway with fibrin deposition are extremely rare, mostly noted in patients with plasminogen deficiency, who have more recently benefited from advances in human plasma-derived plasminogen concentrates administered intravenously or locally. These disorders can be very difficult to diagnose using conventional or even specialized coagulation testing, as testing can be nonspecific or have low sensitivity. Testing of the corresponding protein's activity and antigen (where applicable) can be obtained in specialized centres, and routine laboratory measures are not diagnostic. Genetic testing of the pathogenic mutations is recommended in patients with a high suspicion of an inherited disorder of the fibrinolytic pathway.

Regulation of macrophage fibrinolysis during venous thrombus resolution

BackgroundVenous thromboembolism (VTE), which includes pulmonary embolism (PE) and deep vein thrombosis (DVT), is a serious cardiovascular disease with significant mortality and morbidity. Clinically, patients with faster resolution of a venous thrombi have improved prognosis. Urokinase-plasminogen activator (uPA), produced by macrophages, is a key mediator of fibrinolysis required for resolving venous thrombi and restoring vascular integrity. The major macrophage protein, plasminogen activator inhibitor type-2 (PAI-2), was originally identified as an inhibitor of uPA and is implicated in the modulation of pathways affecting fibrinolytic uPA activity, however its direct role in blocking uPA-mediated clot lysis is not known. ObjectiveTo determine the contribution of macrophage PAI-2 in inhibiting uPA-mediated fibrinolysis during resolution of DVT. MethodsUsing a murine model of venous thrombosis and resolution, we determined histological changes and molecular features of fibrin degradation in venous thrombi from WT mice and mice genetically deficient in PAI-2 and PAI-1, and determined the fibrinolytic activities of macrophages from these genotypes ex vivo. ResultsAcceleration of venous thrombus resolution by PAI-2−/− mice increases fibrin degradation in venous thrombi showing a pattern similar to genetic deficiency of PAI-1, the major attenuator of fibrinolysis. PAI-2 deficiency was not associated with increased macrophage infiltration into thrombi or changes in macrophage PAI-1 expression. uPA-initiated fibrinolysis by macrophages in vitro could be accelerated by PAI-1 deficiency, but not PAI-2 deficiency. ConclusionPAI-2 has an alternate anti-fibrinolytic activity that is macrophage uPA independent, where PAI-1 is the dominant uPA inhibitor during DVT resolution.

MTA2 knockdown suppresses human osteosarcoma metastasis by inhibiting uPA expression.

The relationship between metastasis-associated protein 2 (MTA2) overexpression and tumor growth and metastasis has been extensively studied in a variety of tumor cells but not in human osteosarcoma cells. This study aims to elucidate the clinical significance, underlying molecular mechanisms, and biological functions of MTA2 in human osteosarcoma in vitro and in vivo. Our results show that MTA2 was elevated in osteosarcoma cell lines and osteosarcoma tissues and was associated with tumor stage and overall survival of osteosarcoma patients. Knockdown of MTA2 inhibited osteosarcoma cell migration and invasion by reducing the expression of urokinase-type plasminogen activator (uPA). Bioinformatic analysis demonstrated that high levels of uPA in human osteosarcoma tissues correlated positively with MTA2 expression. Furthermore, treatment with recombinant human uPA (Rh-uPA) caused significant restoration of OS cell migration and invasion in MTA2 knockdown osteosarcoma cells. We found that ERK1/2 depletion increased the expression of uPA, facilitating osteosarcoma cell migration and invasion. Finally, MTA2 depletion significantly reduced tumor metastasis and the formation of lung nodules in vivo. Overall, our study suggests that MTA2 knockdown suppresses osteosarcoma cell metastasis by decreasing uPA expression via ERK signaling. This finding provides new insight into potential treatment strategies against osteosarcoma metastasis by targeting MTA2.

A new insight on alleviating the inhibitory effect of aflatoxin B1 on muscle development in grass carp (Ctenopharyngodon idella): The effect of 4-Methylesculetin in vivo and in vitro

Aflatoxin B1 (AFB1), an important fungal toxin, exists mainly in plant feed ingredients and animals consuming feed contaminated with AFB1 will have reduced growth and impaired health condition mainly due to oxidative stress and reduced immunity. Our previous study found that AFB1 caused oxidative damage and inhibited muscle development of zebrafish. 4-Methylesculetin (4-ME), a coumarin derivative, is now used in biochemistry and medicine widely because of its antioxidant function. Whether 4-ME could alleviate the inhibition of muscle development in grass carp induced by AFB1 has not been reported. In this experiment, 720 healthy grass carp (11.40 ± 0.01 g) were randomly divided into 4 groups with 3 replicates of 60 fish each, including control group, AFB1 group (60 μg/kg diet AFB1), 4-ME group (10 mg/kg diet 4-ME), and AFB1+4-ME group (60 μg/kg diet AFB1 + 10 mg/kg 4-ME diet), for a 60-d growth experiment. In vitro, we also set up 4 treatment groups for grass carp primary myoblast, including control group, AFB1 group (15 μmol/L AFB1), 4-ME group (0.5 μmol/L 4-ME) and AFB1+4-ME group (15 μmol/L AFB1+0.5 μmol/L 4-ME). The results showed that dietary AFB1 decreased growth performance of grass carp, damaged the ultrastructure and induced oxidative damage in grass carp muscle, and significantly decreased the mRNA and protein expression levels of myogenin (MyoG), myogenic differentiation (MyoD), myosin heavy chain (MYHC), as well as the protein expression levels of laminin β1, fibronectin and collagen Ⅰ (P < 0.05), significantly activated the protein expression levels of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and phosphorylate-38 mitogen-activated protein kinase (p38 MAPK) both in grass carp muscle and grass carp primary myoblast (P < 0.05). Supplementation of AFB1 with 4-ME significantly improved the growth performance inhibition and alleviated the muscle fiber development inhibition and extracellular matrix (ECM) degradation in grass carp induced by AFB1 (P < 0.05). The present results revealed that supplementation of AFB1 contaminated feed with 4-ME reduced the inhibition of growth and muscle development by alleviating AFB1-induced ECM degradation in grass carp, which might be related to the p38 MAPK/uPA/MMP/ECM pathway. The results implied that 4-ME could be used as a valuable mycotoxin scavenger in animal feed.

Anti-Cancer Potential of Isoflavone-Enriched Fraction from Traditional Thai Fermented Soybean against Hela Cervical Cancer Cells.

Cervical cancer is a leading cause of gynecological malignancies and cancer-related deaths among women worldwide. This study investigates the anti-cancer activity of Thua Nao, a Thai fermented soybean, against HeLa cervical carcinoma cells, and explores its underlying mechanisms. Our findings reveal that the ethyl acetate fraction of Thua Nao (TN-EA) exhibits strong anti-cancer potential against HeLa cells. High-performance liquid chromatography (HPLC) analysis identified genistein and daidzein as the major isoflavones in TN-EA responsible for its anti-cancer activity. TN-EA and genistein reduced cell proliferation and induced G2/M phase arrest, while daidzein induced G1 arrest. These responses were associated with the downregulation of cell cycle regulators, including Cyclin B1, cycle 25C (Cdc25C), and phosphorylated cyclin-dependent kinase 1 (CDK-1), and the upregulation of the cell cycle inhibitor p21. Moreover, TN-EA and its active isoflavones promoted apoptosis in HeLa cells through the intrinsic pathway, evidenced by increased levels of cleaved Poly (ADP-ribose) polymerase (PARP) and caspase-3, loss of mitochondrial membrane potential, and the downregulation of anti-apoptotic proteins B-cell leukemia/lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL), cellular inhibitor of apoptosis proteins 1 (cIAP), and survivin. Additionally, TN-EA and its active isoflavones effectively reduced cell invasion and migration by downregulating extracellular matrix degradation enzymes, including Membrane type 1-matrix metalloproteinase (MT1-MMP), urokinase-type plasminogen activator (uPA), and urokinase-type plasminogen activator receptor (uPAR), and reduced the levels of the mesenchymal marker N-cadherin. At the molecular level, TN-EA suppressed STAT3 activation via the regulation of JNK and Erk1/2 signaling pathways, leading to reduced proliferation and invasion of HeLa cells.

Transcriptional Induction of SERPINE1 and Fibrinolysis Inhibition as Predominant Effects of Glucocorticoids on the Cancer Coagulome.

How tumors regulate the genes of the coagulome is crucial for cancer-associated thrombosis and the occurrence of venous thromboembolic complications in patients with cancer. We have previously reported potent yet complex effects of glucocorticoids (GC) on the expression of three genes that play a key role in the regulation of thrombin/plasmin activation (F3, PLAU, and SERPINE1). This study aimed to extend the investigation of GC effects to the whole tumor coagulome and assess the resulting impact on the ability of cancer cells to activate thrombin and plasmin. Cancer RNA expression data were retrieved from various sources. Additionally, oral squamous cell carcinoma (OSCC) cells exposed to GC in vitro were analyzed using QPCR, enzymatic assays measuring thrombin and urokinase-type Plasminogen Activator (uPA) activity, and D-dimer concentrations. Our findings highlight the potent and specific stimulatory effect of GC on SERPINE1 expression across different types of cancer. Consistently, GC were found to inhibit uPA proteolytic activity and reduce the concentrations of D-dimers in OSCC in vitro. Fibrinolysis inhibition is a key consequence of cancer cell exposure to GC, possibly leading to the stabilization of the fibrin clot in cancer.

Drug delivery under cover of erythrocytes extends drug half-life: A thrombolytic targeting therapy utilizing microenvironment-responsive artificial polysaccharide microvesicles

The development of thrombolytic drug carriers capable of thrombus-targeting, prolonged circulation time, intelligent responsive release, and the ability to inhibit thrombotic recurrences remains a promising but significant challenge. To tackle this, an artificial polysaccharide microvesicle drug delivery system (uPA-CS/HS@RGD-ODE) was constructed. It is composed of cationic chitosan and anionic heparin assembled in a layer by layer structure, followed by surface modification using RGD peptide and 2-(N-oxide-N,N-diethylamino) ethylmethacrylate (ODE) before encapsulation of urokinase-type plasminogen activator (uPA). The effect of chitosan on the basic performances of uPA-CS/HS@RGD-ODE was estimated. The in vitro results suggest the uPA carrier, CS/HS@RGD-ODE, displayed outstanding targeting specific to activated platelets (61 %) and microenvironment-responsiveness at pH 6.5, facilitating thrombus-targeting and a controlled drug release, respectively. Most importantly, in vivo experiment suggests ODE from uPA-CS/HS@RGD-ODE substantially extends the half-life of uPA (120 min), as uPA-CS/HS@RGD-ODE can adhere onto erythrocytes and deliver uPA under cover of erythrocytes enabling a prolonged circulation time in the bloodstream. Further tail vein and abdominal aorta thrombosis models confirmed uPA-CS/HS@RGD-ODE exhibited superior targeting and thrombolysis capabilities compared to systemic administration of free uPA. To the knowledge of authors, this may be the first study to develop new drug carriers for delivery of thrombolytic drugs under the cover of erythrocytes for extended drug half-lives.

Protease-activated CendR peptides targeting tenascin-C: mitigating off-target tissue accumulation.

To achieve precision and selectivity, anticancer compounds and nanoparticles (NPs) can be targeted with affinity ligands that engage with malignancy-associated molecules in the blood vessels. While tumor-penetrating C-end Rule (CendR) peptides hold promise for precision tumor delivery, C-terminally exposed CendR peptides can accumulate undesirably in non-malignant tissues expressing neuropilin-1 (NRP-1), such as the lungs. One example of such promiscuous peptides is PL3 (sequence: AGRGRLVR), a peptide that engages with NRP-1 through its C-terminal CendR element, RLVR.Here, we report thedevelopment of PL3 derivatives that bind to NRP-1 only after proteolytic processing by urokinase-type plasminogen activator (uPA), while maintaining binding to the other receptor of the peptide, the C-domain of tenascin-C (TNC-C). Through a rational design approach and screening of a uPA-treated peptide-phage library (PL3 peptide followed by four random amino acids) on the recombinant NRP-1, derivatives of the PL3 peptide capable of binding to NRP-1 only post-uPA processing were successfully identified. In vitro cleavage, binding, and internalization assays, along with in vivo biodistribution studies in orthotopic glioblastoma-bearing mice, confirmed the efficacy of two novel peptides, PL3uCendR (AGRGRLVR↓SAGGSVA) and SKLG (AGRGRLVR↓SKLG), which exhibit uPA-dependent binding to NRP-1, reducing off-target binding to healthy NRP-1-expressing tissues. Our study not only unveils novel uPA-dependent TNC-C targeting CendR peptides but also introduces a broader paradigm and establishes a technology for screening proteolytically activated tumor-penetrating peptides.

Dissecting the mediating role of cytokines in the interaction between immune traits and sepsis: insights from comprehensive mendelian randomization.

Sepsis is a life-threatening organ dysfunction resulting from a dysregulated host response to infection, yet the potential causal relationship between the immunophenotype and sepsis remains unclear. Genetic variants associated with the immunophenotype served as instrumental variables (IVs) in Mendelian randomization (MR) to elucidate the causal impact of the immunophenotype on three sepsis outcomes. Additionally, a two-step MR analysis was conducted to identify significant potential mediators between the immunophenotype and three sepsis outcomes. Our MR analysis demonstrated a significant association between the immunophenotype and sepsis outcome, with 36, 36, and 45 the immunophenotype associated with the susceptibility, severity, and mortality of sepsis, respectively. Specifically, our analysis highlighted the CD14+ CD16+ monocyte phenotype as a significant factor across all three sepsis outcomes, with odds ratios (ORs) and corresponding confidence intervals (CIs) indicating its impact on sepsis (OR = 1.047, CI: 1.001-1.096), sepsis in Critical Care Units (OR = 1.139, CI: 1.014-1.279), and sepsis-related 28-day mortality (OR = 1.218, CI: 1.104-1.334). Mediation analyses identified seven cytokines as significant mediators among 91 potential cytokines, including interleukin-5 (IL-5), S100A12, TNF-related apoptosis-inducing ligand (TRAIL), T-cell surface glycoprotein CD6 isoform, cystatin D, interleukin-18 (IL-18), and urokinase-type plasminogen activator (uPA). Furthermore, reverse MR analysis revealed no causal effect of sepsis outcomes on the immunophenotype. Our MR study suggests that the immunophenotype is significantly associated with the susceptibility, severity, and mortality of patient with sepsis, providing, for the first time, robust evidence of significant associations between immune traits and their potential risks. This information is invaluable for clinicians and patients in making informed decisions and merits further attention.