RESUMEN
Hemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8-/-mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8-/-/PN-1-/- mice displayed significantly reduced blood loss and bleeding time compared with F8-/-mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8-/-/PN-1-/- mice than in F8-/-mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8-/-/PN-1-/- and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia.
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Trastornos de la Coagulación Sanguínea Heredados/enzimología , Serpina E2/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/farmacología , Trastornos de la Coagulación Sanguínea Heredados/complicaciones , Hemorragia/etiología , Hemostasis/efectos de los fármacos , Humanos , Ratones , Ratones Noqueados , Activación Plaquetaria/efectos de los fármacosRESUMEN
OBJECTIVE: Plasminogen activator inhibitor-1 (PAI-1) is the key endogenous inhibitor of fibrinolysis, and enhances clot formation after injury. In traumatic brain injury, dysregulation of fibrinolysis may lead to sustained microthrombosis and accelerated lesion expansion. In the present study, we hypothesized that PAI-1 mediates post-traumatic malfunction of coagulation, with inhibition or genetic depletion of PAI-1 attenuating clot formation and lesion expansion after brain trauma. METHODS: We evaluated PAI-1 as a possible new target in a mouse controlled cortical impact (CCI) model of traumatic brain injury. We performed the pharmacological inhibition of PAI-1 with PAI-039 and stimulation by tranexamic acid, and we confirmed our results in PAI-1-deficient animals. RESULTS: PAI-1 mRNA was time-dependently upregulated, with a 305-fold peak 12 hours after CCI, which effectively counteracted the 2- to 3-fold increase in cerebral tissue-type/urokinase plasminogen activator expression. PAI-039 reduced brain lesion volume by 26% at 24 hours and 43% at 5 days after insult. This treatment also attenuated neuronal apoptosis and improved neurofunctional outcome. Moreover, intravital microscopy demonstrated reduced post-traumatic thrombus formation in the pericontusional cortical microvasculature. In PAI-1-deficient mice, the therapeutic effect of PAI-039 was absent. These mice also displayed 13% reduced brain damage compared with wild type. In contrast, inhibition of fibrinolysis with tranexamic acid increased lesion volume by 25% compared with vehicle. INTERPRETATION: This study identifies impaired fibrinolysis as a critical process in post-traumatic secondary brain damage and suggests that PAI-1 may be a central endogenous inhibitor of the fibrinolytic pathway, promoting a procoagulatory state and clot formation in the cerebral microvasculature. Ann Neurol 2019;85:667-680.
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Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Encéfalo/metabolismo , Encéfalo/patología , Fibrinólisis/fisiología , Serpina E2/metabolismo , Animales , Encéfalo/efectos de los fármacos , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Fibrinólisis/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Ácidos Indolacéticos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Serpina E2/antagonistas & inhibidoresRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) is increasingly recognized as a mediator in extracellular matrix (ECM) accumulation in diabetic nephropathy. Previous studies have implicated PAI-1 in adipose tissue (AT) expansion, while also contributing to insulin resistance. As inflammation is also known to occur in perirenal AT during obesity, we hypothesized that in a high-fat diet (HFD)-induced obese mouse model, PAI-1 contributes to macrophage-mediated inflammation and diabetic nephropathy. The HFD mice showed increased expression of PAI-1 in perirenal fat and also displayed increased fat weight and macrophage numbers. We found that the macrophage polarization, proinflammatory macrophage-M1-phenotype, including CD11c, IL-6, and monocyte chemoattractant protein-1, were increased by an HFD and decreased by either the genetic depletion of PAI-1 or treatment with the PAI-1 inhibitor, PAI-039. Similarly, an enhanced anti-inflammatory M2-phenotype, including CD206 and IL-10, was accompanied by either the genetic deletion of PAI-1 or PAI-039 treatment. Furthermore, the inhibition of PAI-1 reduced HFD-induced renal histological lesions and abated profibrotic/extracellular-matrix protein. Collectively, our findings provide support that PAI-1 contributes to the development of inflammation in perirenal fat and correlates with the development of diabetic nephropathy in HFD-induced obesity.
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Tejido Adiposo/metabolismo , Nefropatías Diabéticas/genética , Riñón/metabolismo , Obesidad/metabolismo , Serpina E2/genética , Tejido Adiposo/inmunología , Animales , Nefropatías Diabéticas/inmunología , Dieta Alta en Grasa , Tasa de Filtración Glomerular , Ácidos Indolacéticos/farmacología , Inflamación , Riñón/efectos de los fármacos , Riñón/inmunología , Macrófagos/inmunología , Ratones , Ratones Noqueados , Serpina E2/antagonistas & inhibidores , Serpina E2/inmunologíaRESUMEN
This report describes the development of polyplexes based on CXCR4-inhibiting poly(ethylenimine) derivative (PEI-C) for pulmonary delivery of siRNA to silence plasminogen activator inhibitor-1 (siPAI-1) as a new combination treatment of pulmonary fibrosis (PF). Safety and delivery efficacy of the PEI-C/siPAI-1 polyplexes was investigated in vitro in primary lung fibroblasts isolated from mice with bleomycin-induced PF. Biodistribution analysis following intratracheal administration of fluorescently labeled polyplexes showed prolonged retention in the lungs. Treatment of mice with bleomycin-induced PF using the PEI-C/siPAI-1 polyplexes resulted in a significant down-regulation of the PAI-1 expression and decreased collagen deposition in the lung. The results of this study provide first evidence of the potential benefits of combined inhibition of CXCR4 and PAI-1 in the pulmonary treatment of PF.
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Sistemas de Liberación de Medicamentos , Polietileneimina/química , Polímeros/administración & dosificación , Fibrosis Pulmonar/prevención & control , ARN Interferente Pequeño/genética , Receptores CXCR4/antagonistas & inhibidores , Serpina E2/antagonistas & inhibidores , Animales , Antibióticos Antineoplásicos/toxicidad , Apoptosis , Bleomicina/toxicidad , Proliferación Celular , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patología , Silenciador del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Polímeros/química , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Receptores CXCR4/genética , Serpina E2/genéticaRESUMEN
To analyze the role of PAI-1 (plasminogen activator inhibitor 1) in endometriotic lesion growth, we studied the effect of PAI-1 inhibition by PAI-039 using a homologous mouse model of endometriosis that allows noninvasive monitoring. Endometrial tissue from donor mice was collected, labeled with mCherry adenovirus, and implanted into a subcutaneous pocket on the ventral abdomen of recipient mice. Seven days after transplantation, mice were randomly allocated in two groups and treated once daily for 2 weeks with either vehicle (control group) or PAI-1 inhibitor (PAI-039 group). Endometriotic lesion size generated in recipient mice was monitored by mCherry signal. Animals were euthanized 21 days after endometrial tissue implantation and endometriotic lesions were harvested for fibrin deposit and vascularization analyses. Collagen content was also examined to determine the overall effects of proteolysis on extracellular matrix degradation. We demonstrated that endometriotic lesions generated in recipient mice from both groups presented characteristics typical of human endometriotic lesions. We observed a significant decrease in fluorescence signal in endometriotic lesions from the PAI-039 group at the beginning of the treatment correlated with a decrease in endometriotic lesion size. PAI-1 inhibition significantly decreased lesion cell proliferation. In addition, endometriotic lesions from the PAI-1 inhibition group showed a decreased percentage of neovascularization as well as fibrin deposits. However, the density and distribution of collagen were not affected by PAI-039. Our results suggest that in vivo inhibition of PAI-1 by PAI-039 may be a useful strategy to reduce endometriotic lesion size by blocking angiogenesis.
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Endometriosis/metabolismo , Endometrio/metabolismo , Neovascularización Patológica/metabolismo , Serpina E2/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Endometrio/efectos de los fármacos , Femenino , Ácidos Indolacéticos/farmacología , Ratones , Serpina E2/antagonistas & inhibidoresAsunto(s)
Fibrinólisis/efectos de los fármacos , Polisacáridos/farmacología , Serpina E2/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Activador de Tejido Plasminógeno/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos BALB C , Serpina E2/metabolismo , Trombosis/sangre , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/farmacologíaRESUMEN
Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of tissue-type and urokinase-type plasminogen activators (t/uPA) and plays an important role in fibrinolysis. Inhibition of PAI-1 activity prevents thrombosis and accelerates fibrinolysis, indicating that PAI-1 inhibitors may be used as effective antithrombotic agents. We previously designed a PAI-1 inhibitor (PAItrap) which is a variant of inactivated urokinase protease domain. In the present study, we fused PAItrap with human serum albumin (HSA) to develop a long-acting PAI-1 inhibitor. Unfortunately, the fusion protein PAItrap-HSA lost some potency compared to PAItrap (33 nM vs 10 nM). Guided by computational method, we carried out further optimisation to enhance inhibitory potency for PAI-1. The new PAItrap, denominated PAItrap(H37R)-HSA, which was the H37R variant of PAItrap fused to HSA, gave a six-fold improvement of IC50 (5 nM) for human active PAI-1 compared to PAItrap-HSA, and showed much longer plasma half-life (200-fold) compared to PAItrap. We further demonstrated that the PAItrap(H37R)-HSA inhibited exogenous or endogenous PAI-1 to promote fibrinolysis in fibrin-clot lysis assay. PAItrap(H37R)-HSA inhibits murine PAI-1 with IC50 value of 12 nM, allowing the inhibitor to be evaluated in murine models. Using an intravital microscopy, we demonstrated that PAItrap(H37R)-HSA blocks thrombus formation and platelet accumulation in vivo in a laser-induced vascular injury mouse model. Additionally, mouse tail bleeding assay showed that PAItrap(H37R)-HSA did not affect the global haemostasis. These results suggest that PAItrap(H37R)-HSA have the potential benefit to prevent thrombosis and accelerates fibrinolysis.
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Fibrinolíticos/farmacología , Inhibidor 1 de Activador Plasminogénico/farmacología , Trombosis/prevención & control , Animales , Tiempo de Sangría , Modelos Animales de Enfermedad , Diseño de Fármacos , Fibrinólisis/efectos de los fármacos , Fibrinolíticos/sangre , Fibrinolíticos/química , Semivida , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/farmacología , Inhibidor 1 de Activador Plasminogénico/sangre , Inhibidor 1 de Activador Plasminogénico/química , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacología , Inhibidores de Serina Proteinasa/sangre , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Serpina E2/antagonistas & inhibidores , Activador de Plasminógeno de Tipo Uroquinasa/farmacologíaRESUMEN
Adhesive small bowel obstruction remains a common problem for surgeons. After surgery, platelet aggregation contributes to coagulation cascade and fibrin clot formation. With clotting, fibrin degradation is simultaneously enhanced, driven by tissue plasminogen activator-mediated cleavage of plasminogen to form plasmin. The aim of this study was to investigate the cellular events and proteolytic responses that surround plasminogen activator inhibitor (PAI-1; Serpine1) inhibition of postoperative adhesion. Peritoneal adhesion was induced by gauze deposition in the abdominal cavity in C57BL/6 mice and those that were deficient in fibrinolytic factors, such as Plat-/- and Serpine1-/- In addition, C57BL/6 mice were treated with the novel PAI-1 inhibitor, TM5275. Some animals were treated with clodronate to deplete macrophages. Epidermal growth factor (EGF) experiments were performed to understand the role of macrophages and how EGF contributes to adhesion. In the early phase of adhesive small bowel obstruction, increased PAI-1 activity was observed in the peritoneal cavity. Genetic and pharmacologic PAI-1 inhibition prevented progression of adhesion and increased circulating plasmin. Whereas Serpine1-/- mice showed intra-abdominal bleeding, mice that were treated with TM5275 did not. Mechanistically, PAI-1, in combination with tissue plasminogen activator, served as a chemoattractant for macrophages that, in turn, secreted EGF and up-regulated the receptor, HER1, on peritoneal mesothelial cells, which led to PAI-1 secretion, further fueling the vicious cycle of impaired fibrinolysis at the adhesive site. Controlled inhibition of PAI-1 not only enhanced activation of the fibrinolytic system, but also prevented recruitment of EGF-secreting macrophages. Pharmacologic PAI-1 inhibition ameliorated adhesion formation in a macrophage-dependent manner.-Honjo, K., Munakata, S., Tashiro, Y., Salama, Y., Shimazu, H., Eiamboonsert, S., Dhahri, D., Ichimura, A., Dan, T., Miyata, T., Takeda, K., Sakamoto, K., Hattori, K., Heissig, B. Plasminogen activator inhibitor-1 regulates macrophage-dependent postoperative adhesion by enhancing EGF-HER1 signaling in mice.
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Receptores ErbB/metabolismo , Macrófagos/fisiología , Piperazinas/uso terapéutico , Serpina E2/antagonistas & inhibidores , Adherencias Tisulares/patología , para-Aminobenzoatos/uso terapéutico , Animales , Antígeno CD11b , Ensayos de Migración Celular , Movimiento Celular/efectos de los fármacos , Cetuximab/farmacología , Factor de Crecimiento Epidérmico , Receptores ErbB/genética , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos C57BL , Complicaciones Posoperatorias/prevención & control , Células RAW 264.7 , Serpina E2/genética , Serpina E2/metabolismo , Transducción de Señal , Adherencias Tisulares/metabolismo , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismoRESUMEN
Doxorubicin, an anthracycline antibiotic, is a commonly used anticancer drug. In spite of its widespread usage, its therapeutic effect is limited by its cardiotoxicity. On the cellular level, Doxorubicin-induced cardiotoxicity manifests as stress induced premature senescence. Previously, we demonstrated that plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of serine proteases, is an important biomarker and regulator of cellular senescence and aging. Here, we tested the hypothesis that pharmacological inhibition of cellular PAI-1 protects against stress- and aging-induced cellular senescence and delineated the molecular basis of protective action of PAI-1 inhibition. Results show that TM5441, a potent small molecule inhibitor of PAI-1, effectively prevents Doxorubicin-induced senescence in cardiomyocytes, fibroblasts and endothelial cells. TM5441 exerts its inhibitory effect on Doxorubicin-induced cellular senescence by decreasing reactive oxygen species generation, induction of antioxidants like catalase and suppression of stress-induced senescence cadre p53, p21, p16, PAI-1 and IGFBP3. Importantly, TM5441 also reduces replicative senescence of fibroblasts. Together these results for the first time demonstrate the efficacy of PAI-1 inhibitor in prevention of Doxorubicin-induced and replicative senescence in normal cells. Thus PAI-1 inhibitor may form an important adjuvant component of chemotherapy regimens, limiting not only Doxorubicin-induced cardiac senescence but also ameliorating the prothrombotic profile.
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Cardiotoxicidad/etiología , Cardiotoxicidad/prevención & control , Doxorrubicina/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Piperazinas/farmacología , Serpina E2/antagonistas & inhibidores , para-Aminobenzoatos/farmacología , Animales , Antibióticos Antineoplásicos/toxicidad , Cardiotoxicidad/metabolismo , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , RatasRESUMEN
The extracellular serine protease inhibitor serpinE2 is overexpressed in breast cancer and has been shown to foster metastatic spread. Here, we investigated the hypothesis that serpinE2 creates tumor-promoting conditions in the tumor microenvironment (TME) by affecting extracellular matrix remodeling. Using two different breast cancer models, we show that blocking serpinE2, either by knock-down (KD) in tumor cells or in response to a serpinE2 binding antibody, decreases metastatic dissemination from primary tumors to the lungs. We demonstrate that in response to serpinE2 KD or antibody treatment there are dramatic changes in the TME. Multiphoton intravital imaging revealed deposition of a dense extracellular collagen I matrix encapsulating serpinE2 KD or antibody-treated tumors. This is accompanied by a reduction in the population of tumor-promoting macrophages, as well as a decrease in chemokine ligand 2, which is known to affect macrophage abundance and polarization. In addition, TIMP-1 secretion is increased, which may directly inhibit matrix metalloproteases critical for collagen degradation in the tumor. In summary, our findings suggest that serpinE2 is required in the extracellular milieu of tumors where it acts in multiple ways to regulate tumor matrix deposition, thereby controlling tumor cell dissemination.
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Neoplasias de la Mama/metabolismo , Movimiento Celular , Matriz Extracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Macrófagos/metabolismo , Serpina E2/metabolismo , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiocina CCL2/metabolismo , Colágeno Tipo I/metabolismo , Matriz Extracelular/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Macrófagos/efectos de los fármacos , Macrófagos/patología , Ratones Endogámicos BALB C , Ratones SCID , Invasividad Neoplásica , Fenotipo , Interferencia de ARN , Serpina E2/antagonistas & inhibidores , Serpina E2/genética , Transducción de Señal , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Transfección , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND AND PURPOSE: Obesity is one of the most prevalent chronic diseases worldwide, and dysregulated adipocyte function plays an important role in obesity-associated metabolic disorder. The level of plasma plasminogen activator inhibitor-1 (PAI-1) is increased in obese subjects, and PAI-1 null mice show improved insulin sensitivity when subjected to high-fat and high-sucrose diet-induced metabolic stress, suggesting that a best-in-class PAI-1 inhibitor may become a novel therapeutic agent for obesity-associated metabolic syndrome. TM5441 is a novel orally active PAI-1 inhibitor that does not cause bleeding episodes. Hence, in the present study we examined the preventive effect of TM5441 on high-fat diet (HFD)-induced adipocyte dysfunction. EXPERIMENTAL APPROACH: Ten-week-old C57BL/6J mice were fed a normal diet (18% of total calories from fat) or HFD (60% of total calories from fat) for 10 weeks, and TM5441 (20 mg·kg(-1) oral gavage) was administered daily with the initiation of HFD. KEY RESULTS: TM5441 prevented HFD-induced body weight gain and systemic insulin resistance. TM5441 normalized HFD-induced dysregulated JNK and Akt phosphorylation, suggesting that it prevents the insulin resistance of adipocytes. TM5441 also attenuated the macrophage infiltration and increased expression of pro-inflammatory cytokines, such as inducible nitric oxide synthase, induced by the HFD. In addition, TM5441 prevented the HFD-induced down-regulation of genes involved in mitochondrial biogenesis and function, suggesting that it may prevent adipocyte inflammation and dysregulation by maintaining mitochondrial fitness. CONCLUSION AND IMPLICATIONS: Our data suggest that TM5441 may become a novel therapeutic agent for obesity and obesity-related metabolic disorders.
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Adipocitos/efectos de los fármacos , Dieta Alta en Grasa/efectos adversos , Obesidad/tratamiento farmacológico , Obesidad/prevención & control , Piperazinas/farmacología , Serpina E2/antagonistas & inhibidores , para-Aminobenzoatos/farmacología , Adipocitos/citología , Adipocitos/patología , Animales , Relación Dosis-Respuesta a Droga , Ratones , Ratones Endogámicos C57BL , NG-Nitroarginina Metil Éster , Obesidad/patología , Piperazinas/química , Serpina E2/metabolismo , Relación Estructura-Actividad , para-Aminobenzoatos/químicaRESUMEN
The post translational modification of lysine acetylation is a key mechanism that regulates chromatin structure. Epigenetic readers, such as the BET domains, are responsible for reading histone lysine acetylation which is a hallmark of open chromatin structure, further providing a scaffold that can be accessed by RNA polymerases as well as transcription factors. Recently, several reports have assessed and highlighted the roles of epigenetic readers in various cellular contexts. However, little is known about their role in the regulation of inflammatory genes, which is critical in exquisitely tuning inflammatory responses to a variety of immune stimuli. In this study, we investigated the role of epigenetic readers BRD2 and BRD4 in the lipopolysaccharide (LPS)-induced immune responses in mouse primary astrocytes. Inflammatory stimulation by LPS showed that the levels of Brd2 mRNA and protein were increased, while Brd4 mRNA levels did not change. Knocking down of Brd2 mRNA using specific small interfering RNA (siRNA) in cultured mouse primary astrocytes inhibited LPS-induced mRNA expression and secretion of plasminogen activator inhibitor-1 (PAI-1). However, no other pro-inflammatory cytokines, such as Il-6, Il-1ß and Tnf-α, were affected. Indeed, treatment with bromodomain-containing protein inhibitor, JQ1, blocked Pai-1 mRNA expression through the inhibition of direct BRD2 protein-binding and active histone modification on Pai-1 promoter. Taken together, our data suggest that BRD2 is involved in the modulation of neuroinflammatory responses through PAI-1 and via the regulation of epigenetic reader BET protein, further providing a potential novel therapeutic strategy in neuroinflammatory diseases.
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Astrocitos/metabolismo , Proteínas Cromosómicas no Histona/genética , Epigénesis Genética/genética , Lipopolisacáridos/farmacología , Serpina E2/biosíntesis , Serpina E2/genética , Animales , Astrocitos/efectos de los fármacos , Azepinas/farmacología , Citocinas/biosíntesis , Citocinas/genética , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Ratones , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Cultivo Primario de Células , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño , Serpina E2/antagonistas & inhibidores , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transfección , Triazoles/farmacologíaRESUMEN
Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor beta 1 (TGF-ß1), plays a role in inducing apoptosis, modulates fibrosis, and ECM accumulation. Plasminogen activator inhibitor 1 (PAI-1) plays an important role in the development hepatic fibrosis. The overexpression of PAI-1 induces ECM accumulation, the main hallmark of chronic liver diseases. Death of hepatocytes is a characteristic feature of chronic liver disease due to various causes. The TGF-ß1-mediated apoptotic pathway is regarded as a promising therapeutic target in hepatic fibrosis. Gamma-linolenic acid (GLA) is of special interest as it possesses anti-fibrosis, anti-inflammatory, and anti-cancer properties. However, the precise mechanism for GLA in chronic liver disease is not still clear. The aim of the present study was to determine whether GLA prevents hepatic PAI-1 expression and apoptosis through the inhibition of TGF-ß1-mediated molecular mediators. GLA attenuated TGF-ß1-stimulated PAI-1 expression, and inhibited PAI-1 promoter activity in AML12 cells. This effect was mediated by Smad3/4, the p38 pathways. We also found that GLA suppressed TGF-ß1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase 1 cleavage. GLA ameliorates the pro-fibrotic and pro-apoptotic effects of TGF-ß1 in hepatocytes, suggesting GLA exerts a protective effect on hepatocytes and has a therapeutic potential for the treatment of chronic liver disease.
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Antiinflamatorios no Esteroideos/farmacología , Hepatocitos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Serpina E2/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Ácido gammalinolénico/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular , Fibrosis , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Hepatocitos/patología , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Inflamación/prevención & control , Ratones , Mitocondrias/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Serpina E2/genética , Serpina E2/metabolismo , Transducción de Señal , Proteína smad3/genética , Proteína smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
PURPOSE: Altered expression of serine protease inhibitor peptidase inhibitor clade E member 2 (SERPINE2) associates with human cancer development and progression; thus, this study investigated SERPINE2 expression in gastric cancer tissues for association with clinicopathological and survival data from the patients and then investigated the role of SERPINE2 in gastric cancer cells in vitro. METHODS: The levels of SERPINE2 mRNA in 243 gastric cancer tissues and paired non-cancerous mucosa were determined using quantitative PCR. Inhibition of SERPINE2 expression by small interfering RNA (siRNA) was detected by Western blotting. tetrazolium, soft agar, and transwell assays were performed to evaluate the proliferation, anchorage-independent growth, and motility of gastric cancer SGC7901 cells transfected with SERPINE2 siRNA. RESULTS: Compared with the normal mucosa, SERPINE2 mRNA was increased in gastric cancer tissues and cells. Analysis of the 243 matched specimens showed that high SERPINE2 levels were associated with lymph node metastasis, distant metastasis, and clinical stage. Patients with high SERPINE2 mRNA levels had poorer survival compared with patients with low SERPINE2 mRNA levels. In vitro, SERPINE2 inhibited anchorage-independent growth, migration, and invasion of SGC7901 cells, but not proliferation. CONCLUSIONS: Our findings indicate that upregulated SERPINE2 may contribute to the aggressive phenotype of gastric cancer and suggest that SERPINE2 can be used as a novel prognostic factor and anticancer target in patients with gastric cancer.
Asunto(s)
Serpina E2/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Anciano , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Pronóstico , ARN Interferente Pequeño/metabolismo , Serpina E2/antagonistas & inhibidores , Serpina E2/genética , Regulación hacia ArribaRESUMEN
Intracerebral-intraventricular hemorrhages (ICH/IVH) in very preterm neonates are responsible for high mortality and subsequent disabilities. In humans, tissue plasminogen activator (t-PA) initiates fibrinolysis and activates endoluminal-endothelial receptors; dysfunction of the t-PA inhibitor (PAI-1) results in recurrent hemorrhages. We used PAI-1 knockout (PAI-1) mice to examine the role of t-PA in age-dependent intracranial hemorrhages as a possible model of preterm ICH/IVH. Intracortical injection of 2 µL of phosphate-buffered saline produced a small traumatic injury and a high rate of hemorrhage in PAI-1 pups at postnatal day 3 (P3) or P5, whereas it had no effect in wild-type neonates. This resulted in white matter and cortical lesions, ventricle enlargement, hyperlocomotion, and altered cortical levels of serotonin and dopamine in the adult PAI mice. N-methyl-D-aspartate receptor blockers, plasmin- and matrix metalloproteinases inhibitors reduced hemorrhage and tissue lesions. In contrast to P3 to P5, no significant hemorrhages were induced in P10 PAI-1 pups and there were no behavioral or neurochemical alterations in adulthood. These data suggest that microvascular immaturity up to P5 in mice is a determinant factor required for t-PA-dependent vascular rupture. Neonatal PAI-1 mice could be a useful ICH/IVH model for studying the ontogenic window of vascular immaturity and vascular protection against later neurodisabilities.
Asunto(s)
Envejecimiento/fisiología , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/fisiopatología , Serpina E2/deficiencia , Factores de Edad , Ácido Aminocaproico/administración & dosificación , Animales , Animales Recién Nacidos , Aprotinina/administración & dosificación , Hemorragia Cerebral/genética , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Fenotipo , Serpina E2/antagonistas & inhibidores , Serpina E2/fisiologíaRESUMEN
The prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT) depends on the rapid recovery and sustained life-long hematopoiesis. The activation of the fibrinolytic pathway promotes hematopoietic regeneration; however, the role of plasminogen activator inhibitor-1 (PAI-1), a negative regulator of the fibrinolytic pathway, has not yet been elucidated. We herein demonstrate that bone marrow (BM) stromal cells, especially osteoblasts, produce PAI-1 in response to myeloablation, which negatively regulates the hematopoietic regeneration in the BM microenvironment. Total body irradiation in mice dramatically increased the local expression levels of fibrinolytic factors, including tissue-type plasminogen activator (tPA), plasmin, and PAI-1. Genetic disruption of the PAI-1 gene, or pharmacological inhibition of PAI-1 activity, significantly improved the myeloablation-related mortality and promoted rapid hematopoietic recovery after HSCT through the induction of hematopoiesis-promoting factors. The ability of a PAI-1 inhibitor to enhance hematopoietic regeneration was abolished when tPA-deficient mice were used as recipients, thus indicating that PAI-1 represses tPA-dependent hematopoietic regeneration. The PAI-1 inhibitor not only accelerated the expansion of the donor HSCs during the early-stage of regeneration, but also supported long-term hematopoiesis. Our results indicate that the inhibition of PAI-1 activity could be a therapeutic approach to facilitate the rapid recovery and sustained hematopoiesis after HSCT.
Asunto(s)
Médula Ósea/metabolismo , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Regeneración , Serpina E2/antagonistas & inhibidores , Nicho de Células Madre , Aloinjertos , Animales , Humanos , Ratones , Serpina E2/metabolismo , Células del Estroma/metabolismoRESUMEN
Protease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with ß-strand 3A and the loop connecting α-helix A with ß-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1's protease inhibitory activity.