RESUMEN
Vascular injury models are indispensable for studying thrombotic processes in vivo. Amongst the available methods for inducing thrombosis, laser-induced endothelial injury (LIEI) has several unique advantages. However, a lack of methodological standardization and expensive instrumentation remain significant problems decreasing reproducibility and impeding the adoption of LIEI in the wider scientific community. In this, study, we developed a standardized protocol for scanning laser-induced endothelial injury (scanning-LIEI) of murine mesenteric veins using the intrinsic 405 nm laser of a conventional laser scanning confocal microscope. We show that our model produces thrombi with prominent core-shell architectures and minimal radiation-related fluorescence artefacts. In comparison with previous methods, the scanning-LIEI model exhibits reduced experimental variability, enabling the demonstration of dose-response effects for anti-thrombotic drugs using small animal cohorts. Scanning-LIEI using the intrinsic 405 nm laser of a confocal laser scanning microscope represents a new method to induce standardized vascular injury with improved reproducibility of thrombus formation. The reduced need for instrument customisation and user experience means that this model could be more readily adopted in the research community.
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Trombosis , Lesiones del Sistema Vascular , Animales , Microscopía Intravital , Rayos Láser , Ratones , Reproducibilidad de los ResultadosRESUMEN
Coronavirus disease 2019 (COVID-19) is associated with extreme inflammatory response, disordered hemostasis and high thrombotic risk. A high incidence of thromboembolic events has been reported despite thromboprophylaxis, raising the question of a more effective anticoagulation. First-line hemostasis tests such as activated partial thromboplastin time, prothrombin time, fibrinogen and D-dimers are proposed for assessing thrombotic risk and monitoring hemostasis, but are vulnerable to many drawbacks affecting their reliability and clinical relevance. Specialized hemostasis-related tests (soluble fibrin complexes, tests assessing fibrinolytic capacity, viscoelastic tests, thrombin generation) may have an interest to assess the thrombotic risk associated with COVID-19. Another challenge for the hemostasis laboratory is the monitoring of heparin treatment, especially unfractionated heparin in the setting of an extreme inflammatory response. This review aimed at evaluating the role of hemostasis tests in the management of COVID-19 and discussing their main limitations.
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Dexamethasone is frequently administered to surgical patients for anti-emetic prophylaxis. We have examined the immunomodulatory effects of a single bolus of dexamethasone on circulating peripheral blood mononuclear cells (PBMCs) in the same 10 healthy male volunteers, previously used in our investigation on early in vivo effects of a single anti-emetic dose of dexamethasone on innate immune cell gene expression and activation [1]. Blood samples were drawn at baseline, 2â¯h, 4â¯h and 24â¯h. Immune cell phenotypes were examined with flow cytometry. In this data article the expression strength of markers involved in immune activation and immunosuppression as well as maturation, migration, cell death and responsiveness to signalling on monocyte and cDC subsets, as well as NK cells, CD4+ and CD8+ T cells and regulatory T cells (Treg) are presented. These data improve our understanding of the immunomodulatory effects of the glucocorticoid dexamethasone in-vivo, which may be important for the optimisation of treatment regimens as well as the evaluation of new indications for glucocorticoid treatment.
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Portadores de Fármacos , Fibrinólisis , Fibrinolíticos/administración & dosificación , Trombosis Intracraneal/tratamiento farmacológico , Magnetismo/métodos , Accidente Cerebrovascular/tratamiento farmacológico , Terapia Trombolítica/métodos , Activador de Tejido Plasminógeno/administración & dosificación , Animales , Difusión de Innovaciones , Fibrinolíticos/efectos adversos , Humanos , Trombosis Intracraneal/sangre , Trombosis Intracraneal/diagnóstico , Trombosis Intracraneal/fisiopatología , Accidente Cerebrovascular/sangre , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/fisiopatología , Terapia Trombolítica/efectos adversos , Activador de Tejido Plasminógeno/efectos adversos , Resultado del TratamientoRESUMEN
We all know about classical fibrinolysis, how plasminogen activation by either tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) promotes fibrin breakdown, and how this process was harnessed for the therapeutic removal of blood clots. While this is still perfectly true and still applicable to thromboembolic conditions today, another dimension to this system came to light over two decades ago that implicated the plasminogen activating system in a context far removed from the dissolution of blood clots. This unsuspected area related to brain biology where t-PA was linked to a plethora of activities in the CNS, some of which do not necessarily require plasmin generation. Indeed, t-PA either directly or via plasmin, has been shown to not only have key roles in modulating astrocytes, neurons, microglia, and pericytes, but also to have profound effects in a number of CNS conditions, including ischaemic stroke, severe traumatic brain injury and also in neurodegenerative disorders. While compelling insights have been obtained from various animal models, the clinical relevance of aberrant expression of these components in the CNS, although strongly implied, are only just emerging. This review will cover these areas and will also discuss how the use of thrombolytic agents and anti-fibrinolytic drugs may potentially have impacts outside of their clinical intention, particularly in the CNS.
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Lesiones Traumáticas del Encéfalo/sangre , Encéfalo/metabolismo , Trastornos Cerebrovasculares/sangre , Fibrinólisis , Activador de Tejido Plasminógeno/sangre , Animales , Antifibrinolíticos/uso terapéutico , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/fisiopatología , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/fisiopatología , Trastornos Cerebrovasculares/tratamiento farmacológico , Trastornos Cerebrovasculares/fisiopatología , Fibrinolisina/metabolismo , Fibrinólisis/efectos de los fármacos , Fibrinolíticos/uso terapéutico , Humanos , Terapia Trombolítica , Activador de Tejido Plasminógeno/uso terapéuticoRESUMEN
The balance of inflammation and immunosuppression driven by changed ratios in diverse myeloid and T cell subsets, as well as their state of activation and ability to migrate to lymphoid compartments or inflammatory sites, has emerged as a highly active area of study across clinical trials of vaccines and therapies against cancer, trauma, as well as autoimmune and infectious diseases. There is a need for effective protocols which maximally use the possibilities offered by modern flow cytometers to characterize such immune cell changes in peripheral blood using small volumes of human blood. Additionally, longitudinal clinical studies often use cryopreserved samples, which can impact flow cytometric results. To efficiently gauge both the innate and the adaptive immune response, two novel 15-color antibody panels to identify key myeloid and T cell subsets and their functional potential were established. This approach was used to compare cellular immune profiles in fresh whole blood and in matched cryopreserved peripheral blood mononuclear cells (PBMCs). Cocktail I was designed to identify and characterize myeloid cell populations including dendritic cells (DCs), monocytic monocyte-derived suppressor cells (MO-MDSC), and monocytes, determining further core aspects of their state of maturity, T cell stimulatory (or inhibitory) potential, and migration capability. Cocktail II was used for phenotyping diverse T cells subsets, and their key migration and functional regulatory capabilities. The two 15-color antibody panels for the evaluation of both immune-stimulating and immunosuppressive processes presented herein allowed for efficient evaluation of the balance of immune activation versus immunosuppression across key blood cells, with good resolution for all 15 markers stained for in each panel. Gating strategies for the myeloid and T cells are presented to further support specific subset identification. This protocol was shown to be reproducible across donors and useful to study both RBC-lysed whole blood and cryopreserved PBMCs. © 2017 International Society for Advancement of Cytometry.
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Citometría de Flujo/métodos , Inmunofenotipificación/métodos , Células Mieloides/citología , Subgrupos de Linfocitos T/citología , Criopreservación , Humanos , Inmunidad Innata , Leucocitos Mononucleares/citologíaRESUMEN
Essentials Stimulating endogenous fibrinolysis could be a novel antithrombotic strategy. The effect of valproic acid on endothelial tissue plasminogen activator in mice was investigated. Valproic acid increased tissue plasminogen activator expression in vascular endothelium. Valproic acid reduced fibrin deposition and thrombus formation after vascular injury. SUMMARY: Background The endogenous fibrinolytic system has rarely been considered as a target to prevent thrombotic disease. Tissue-type plasminogen activator (t-PA) production is potently increased by histone deacetylase (HDAC) inhibitors in endothelial cells in vitro, but whether this translates into increased vascular t-PA production and an enhanced fibrinolytic capacity in vivo is unknown. Objectives To determine whether the HDAC inhibitor valproic acid (VPA) stimulates production of t-PA in the vasculature of mice, and whether VPA pretreatment affects fibrin deposition and clot formation after mechanical vessel injury. Methods Mice were injected with VPA twice daily for up to 5 days. t-PA mRNA, and antigen expression in the mouse aorta and the circulating levels of t-PA were determined. Fibrin and thrombus dynamics after mechanical vessel injury were monitored with intravital confocal microscopy. Potential effects of VPA on platelets and coagulation were investigated. Results and Conclusions We found that VPA treatment increased vascular t-PA production in vivo and, importantly, that VPA administration was associated with reduced fibrin accumulation and smaller thrombi in response to vascular injury, but still was not associated with an increased risk of bleeding. Furthermore, we observed that higher concentrations of VPA were required to stimulate t-PA production in the brain than in the vasculature. Thus, this study shows that VPA can be dosed to selectively manipulate the fibrinolytic system in the vascular compartment and reduce thrombus formation in vivo.
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Endotelio Vascular/metabolismo , Trombosis/tratamiento farmacológico , Activador de Tejido Plasminógeno/metabolismo , Ácido Valproico/farmacología , Animales , Aorta/metabolismo , Coagulación Sanguínea , Plaquetas/metabolismo , Inhibidores Enzimáticos/farmacología , Fibrinólisis , Hemorragia , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Pruebas de Función Plaquetaria , ARN Mensajero/metabolismoRESUMEN
UNLABELLED: Essentials C57BL/6J-tissue plasminogen activator (tPA)-deficient mice are widely used to study tPA function. Congenic C57BL/6J-tPA-deficient mice harbor large 129-derived chromosomal segments. The 129-derived chromosomal segments contain gene mutations that may confound data interpretation. Passenger mutation-free isogenic tPA-deficient mice were generated for study of tPA function. SUMMARY: Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density single nucleotide polymorphism (SNP) analysis, bioinformatics and genome editing were used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat(-/-) mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel 'passenger mutation'-free isogenic C57BL/6J-Plat(-/-) and FVB/NJ-Plat(-/-) mouse strains by introducing an 11 bp deletion into the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes.
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Mutación , Activador de Tejido Plasminógeno/genética , Alelos , Animales , Encéfalo/metabolismo , Cromosomas/ultraestructura , Biología Computacional , Cruzamientos Genéticos , Células Madre Embrionarias/citología , Exones , Femenino , Fibrinólisis , Edición Génica , Regulación de la Expresión Génica , Marcación de Gen , Genotipo , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Polimorfismo de Nucleótido Simple , Señales de Clasificación de Proteína , Serina Proteasas/metabolismo , Dedos de ZincRESUMEN
Traumatic brain injury (TBI) represents a significant global health burden and causes long-lasting neuromotor deficits, particularly in individuals who sustain severe TBI. A better understanding of gait impairment after experimental TBI will provide valuable information for the recovery and rehabilitation of TBI survivors. Here we utilised the DigiGait system to perform kinematic gait analysis in mice subjected to brain injury induced by the controlled cortical impact (CCI) TBI model. Naïve mice, non-craniotomised and craniotomised mice were included as controls. The temporal and spatial profile of gait was mapped from 3h to 1-week post-TBI. Remarkably, there was a noticeable alteration in some aspects of gait in craniotomised sham mice from their pre-surgery baseline at various time-points over the testing period. This was not observed in naïve mice or non-craniotomised sham controls over the same time period. This finding indicates that the craniotomy procedure alone effects gait. When craniotomised mice were subjected to TBI, additional deleterious effects on gait function were observed, including forelimb stance and swing duration as well as left hindlimb swing and stride duration and frequency. Hence, mice subjected to CCI-induced TBI develop clear alterations in gait but part of this is attributable to the effect of craniotomy alone. This study also highlights the need to include both non-craniotomised and craniotomised sham mice as controls when undertaking the CCI-induced model of TBI, particularly when early time points are being evaluated.
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Lesiones Encefálicas/fisiopatología , Marcha , Trastornos del Movimiento/diagnóstico , Trastornos del Movimiento/fisiopatología , Animales , Fenómenos Biomecánicos , Lesiones Encefálicas/complicaciones , Craneotomía , Modelos Animales de Enfermedad , Miembro Anterior/fisiopatología , Marcha/fisiología , Miembro Posterior/fisiopatología , Masculino , Ratones Endogámicos C57BL , Trastornos del Movimiento/etiologíaRESUMEN
The timely removal of blood clots and fibrin deposits is essential in the regulation of haemostasis. This is achieved by the fibrinolytic system, an enzymatic process that regulates the activation of plasminogen into its proteolytic form, plasmin. This is a self-regulated event as the very presence of fibrin initiates plasminogen activation on the fibrin surface due to the presentation of exposed C-terminal lysine residues in fibrin that allow plasminogen to position itself via its lysine binding sites and to be more efficiently cleaved by tissue-type plasminogen activator (t-PA). Hence fibrin, the ultimate substrate of plasmin during fibrinolysis, is indeed an essential cofactor in the cascade. What has now come to light is that the fibrinolytic system is not solely designed to eliminate fibrin. Indeed, it is a broad acting system that processes a variety of proteins, including many in the brain where there is no fibrin. So what drives t-PA-mediated plasminogen activation when fibrin is not available? This review will describe the broadening role of the fibrinolytic system highlighting the importance of fibrin and other key proteins as facilitators during t-PA-mediated plasminogen activation.
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Coagulación Sanguínea/fisiología , Fibrina/metabolismo , Fibrinólisis/fisiología , Fibrinolíticos/administración & dosificación , Modelos Cardiovasculares , Activador de Tejido Plasminógeno/metabolismo , Animales , Fibrinólisis/efectos de los fármacos , HumanosRESUMEN
Platelet activation is a frontline response to injury, not only essential for clot formation but also important for tissue repair. Indeed, the reparative influence of platelets has long been exploited therapeutically where application of platelet concentrates expedites wound recovery. Despite this, the mechanisms of platelet-triggered cytoprotection are poorly understood. Here, we show that activated platelets accumulate in the brain to exceptionally high levels following injury and release factors that potently protect neurons from apoptosis. Kinomic microarray and subsequent kinase inhibitor studies showed that platelet-based neuroprotection relies upon paracrine activation of the epidermal growth factor receptor (EGFR) and downstream DNA-dependent protein kinase (DNA-PK). This same anti-apoptotic cascade stimulated by activated platelets also provided chemo-resistance to several cancer cell types. Surprisingly, deep proteomic profiling of the platelet releasate failed to identify any known EGFR ligand, indicating that activated platelets release an atypical activator of the EGFR. This study is the first to formally associate platelet activation to EGFR/DNA-PK--an endogenous cytoprotective cascade.
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Apoptosis , Plaquetas/enzimología , Lesiones Encefálicas/enzimología , Proteína Quinasa Activada por ADN/metabolismo , Receptores ErbB/metabolismo , Neuronas/citología , Adolescente , Adulto , Anciano , Animales , Plaquetas/metabolismo , Encéfalo/citología , Encéfalo/enzimología , Lesiones Encefálicas/genética , Lesiones Encefálicas/fisiopatología , Línea Celular Tumoral , Células Cultivadas , Proteína Quinasa Activada por ADN/genética , Receptores ErbB/genética , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Activación Plaquetaria , Adulto JovenRESUMEN
We have identified a single-nucleotide polymorphism (SNP) in the t-PA enhancer (-7351C>T), which is associated with endothelial t-PA release in vivo. In vitro studies demonstrated that this SNP is functional at the level of transcription. In the brain, t-PA has been implicated in both physiologic and pathophysiologic processes. The aim of the present study was to examine the effect of the t-PA -7351C>T SNP on t-PA gene expression in human brain tissue. Allelic mRNA expression was measured in heterozygous post-mortem brain tissues using quantitative TaqMan genotyping assay. Protein-DNA interactions were assessed using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Significantly higher levels of t-PA mRNA were generated from chromosomes that harboured the wild-type -7351C allele, as compared to those generated from the mutant T allele (for the hippocampus, C to T allelic ratio of ~1.3, p=0.010, n=12; and for the cortex, C to T allelic ratio of ~1.2, p=0.017, n=12). EMSA showed reduced neuronal and astrocytic nuclear protein binding affinity to the T allele, and identified Sp1 and Sp3 as the major transcription factors that bound to the -7351 site. ChIP analyses confirmed that Sp1 recognises this site in intact cells. In conclusion, the t-PA -7351C>T SNP affects t-PA gene expression in human brain tissue. This finding might have clinical implications for neurological conditions associated with enhanced t-PA levels, such as in the acute phase of cerebral ischaemia, and also for stroke recovery.
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Astrocitos/metabolismo , Isquemia Encefálica/metabolismo , Neuronas/metabolismo , Accidente Cerebrovascular/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Astrocitos/patología , Encéfalo/patología , Isquemia Encefálica/genética , Análisis Mutacional de ADN , Elementos de Facilitación Genéticos/genética , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Neuronas/patología , Polimorfismo de Nucleótido Simple , Unión Proteica/genética , Accidente Cerebrovascular/genética , Activador de Tejido Plasminógeno/genéticaAsunto(s)
Contaminación de Medicamentos , Fibrinolisina/farmacología , Neuronas/efectos de los fármacos , Activador de Tejido Plasminógeno/farmacología , Animales , Células Cultivadas , Diálisis , Fibrinolisina/aislamiento & purificación , Ratones , Peso Molecular , Activador de Tejido Plasminógeno/aislamiento & purificaciónRESUMEN
The maintenance of a given physiological process demands a coordinated and spatially regulated pattern of gene regulation. This applies to genes encoding components of enzyme cascades, including those of the plasminogen activating system. This family of proteases is vital to fibrinolysis and dysregulation of the expression pattern of one or more of these proteins in response to inflammatory events can impact on hemostasis. Gene regulation occurs on many levels, and it is apparent that the genes encoding the plasminogen activator (fibrinolytic) proteins are subject to both direct transcriptional control and significant post-transcriptional mechanisms. It is now clear that perturbation of these genes at either of these levels can dramatically alter expression levels and have a direct impact on the host's response to a variety of physiological and pharmacological challenges. Inflammatory processes are well known to impact on the fibrinolytic system and to promote thrombosis, cancer and diabetes. This review discusses how inflammatory and other signals affect the transcriptional and post-transcriptional expression patterns of this system, and how this modulates fibrinolysis in vivo.
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Fibrinólisis , Inflamación/metabolismo , Activadores Plasminogénicos/metabolismo , Coagulación Sanguínea , Citocinas/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamación/fisiopatología , Procesamiento Postranscripcional del ARN , Transcripción GenéticaAsunto(s)
Células Endoteliales/metabolismo , Regulación de la Expresión Génica/genética , Polimorfismo de Nucleótido Simple , Activador de Tejido Plasminógeno/genética , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Genotipo , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/agonistas , Activador de Tejido Plasminógeno/metabolismo , Tretinoina/agonistas , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Increased plasminogen activator inhibitor type 1 (PAI-1) levels are observed in endothelial cells stimulated by tumour necrosis factor alpha (TNFalpha). Thiazolidinediones (TZDs) may inhibit elevated endothelial cell PAI-1 accounting, in part, for the putative atheroprotective effects of TZDs. In an endothelial cell line, Rosiglitazone (RG) and Pioglitazone (PG) inhibited induction of PAI-1 by TNFalpha. The specific peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor, SR-202, failed to modulate this effect. RG also inhibited the effect of TNFalpha on a reporter gene construct harbouring the proximal PAI-1 promoter and PAI-1 mRNA in cells co-transfected with a dominant-negative PPARgamma construct. RG and PG attenuated TNFalpha-mediated induction of trans-acting factor(s) Nur77/Nurr1 and binding of nuclear proteins (NP) to the cis-acting element (NBRE). SR-202 failed to modulate these effects. The observations suggest TZDs inhibit TNFalpha-mediated PAI-1 induction independent of inducible PPARgamma activation and this may involve in the modulation of Nur77/Nurr1 expression and NP binding to the PAI-1 NBRE.
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Células Endoteliales/metabolismo , PPAR gamma/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Tiazolidinedionas/farmacología , Activación Transcripcional/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular , Células Endoteliales/efectos de los fármacos , Humanos , Pioglitazona , Rosiglitazona , Activación Transcripcional/efectos de los fármacosRESUMEN
Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells.