RESUMEN
Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one-pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against α-thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1â mg kg-1 . The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions, in addition to an active site.
Asunto(s)
Anticoagulantes/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Proteínas y Péptidos Salivales/uso terapéutico , Trombosis/tratamiento farmacológico , Amblyomma/química , Animales , Anopheles/química , Anticoagulantes/síntesis química , Anticoagulantes/metabolismo , Dominio Catalítico , Humanos , Masculino , Ratones Endogámicos C57BL , Inhibidores de Agregación Plaquetaria/síntesis química , Inhibidores de Agregación Plaquetaria/metabolismo , Unión Proteica , Ingeniería de Proteínas , Proteínas y Péptidos Salivales/síntesis química , Proteínas y Péptidos Salivales/metabolismo , Trombina/química , Trombina/metabolismo , Moscas Tse-Tse/químicaRESUMEN
Neutrophil infiltration to ischemic tissues following reperfusion worsens injury. A key driver of neutrophil recruitment and activation is the complement factor C5a, which signals through two receptors, C5aR1 and C5aR2. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to investigate the underexplored role of C5aR2 in neutrophil mobilization, recruitment, and disease outcomes. We show that intestinal IR induces rapid neutrophil mobilization along with a concomitant reduction in plasma C5a levels that is driven by both C5aR1 and C5aR2. Intestinal IR in C5aR2-/- mice led to worsened intestinal damage and increased neutrophil infiltration. Inhibition of C5aR1 signaling in C5aR2-/- mice with PMX53 prevented neutrophil accumulation and reduced IR pathology, suggesting a key requirement for enhanced neutrophil C5aR1 activation in the absence of C5aR2 signaling. Interestingly, C5aR2 deficiency also reduced circulating neutrophil numbers after IR, as well as following G-CSF-mediated bone marrow mobilization, which was independent of C5aR1, demonstrating that C5aR2 has unique and distinct functions from C5aR1 in neutrophil egress. Despite enhanced tissue injury in C5aR2-/- IR mice, there were significant reductions in intestinal proinflammatory cytokines, highlighting complicated dual protective/pathogenic roles for C5aR2 in pathophysiology. Collectively, we show that C5aR2 is protective in intestinal IR by inhibiting C5aR1-mediated neutrophil recruitment to the ischemic tissue. This is despite the potentially local pathogenic effects of C5aR2 in increasing intestinal proinflammatory cytokines and enhancing circulating neutrophil numbers in response to mobilizing signals. Our data therefore suggest that this balance between the dual pro- and anti-inflammatory roles of C5aR2 ultimately dictates disease outcomes.
Asunto(s)
Isquemia Mesentérica/inmunología , Infiltración Neutrófila , Receptor de Anafilatoxina C5a/metabolismo , Daño por Reperfusión/inmunología , Animales , Complemento C5a/análisis , Complemento C5a/metabolismo , Modelos Animales de Enfermedad , Humanos , Yeyuno/citología , Yeyuno/inmunología , Yeyuno/patología , Masculino , Isquemia Mesentérica/sangre , Isquemia Mesentérica/complicaciones , Isquemia Mesentérica/patología , Ratones , Ratones Noqueados , Receptor de Anafilatoxina C5a/genética , Daño por Reperfusión/sangre , Daño por Reperfusión/patologíaRESUMEN
Free fatty acid 2 receptor (FFA2) is highly expressed on neutrophils and, when activated by its cognate ligand acetate, generates potent anti-inflammatory activities. The roles of FFA2 and acetate have not been explored in ischemia-reperfusion injury (IRI). We therefore examined the function of FFA2 and the therapeutic potential of acetate to reduce tissue injury in an acute model of intestinal IRI. The superior mesenteric artery of wild-type (WT) and FFA2-/- mice was briefly occluded then reperfused following treatment with acetate or vehicle. The absence of FFA2 resulted in intestinal injury similar to that observed in WT mice, indicating a minimal causal role for FFA2 in this model. Acetate treatment to WT mice prior to ischemia profoundly protected the intestine from IRI-induced damage. Amelioration of IRI was also observed, although to a lesser extent, when acetate was administered to FFA2-/- mice demonstrating that certain protective effects of acetate were FFA2-independent. Remarkably, despite the lack of tissue damage following IRI, acetate-treated mice had markedly increased neutrophil infiltration to the reperfused intestine which was dependent on FFA2. These studies reveal a minimal causal role for FFA2 in intestinal IRI but highlight the novel therapeutic potential for acetate in the amelioration of ischemia-mediated tissue damage.
Asunto(s)
Acetatos/metabolismo , Intestinos/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/fisiología , Neutrófilos/metabolismo , Daño por Reperfusión/metabolismoRESUMEN
Hematophagous organisms produce a suite of salivary proteins which interact with the host's coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure-activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.
Asunto(s)
Anticoagulantes/química , Proteínas de Insectos/química , Proteínas y Péptidos Salivales/química , Trombina/química , Secuencia de Aminoácidos/genética , Coagulación Sanguínea/genética , Biología Computacional , Biblioteca de Genes , Humanos , Proteínas de Insectos/genética , Procesamiento Proteico-Postraduccional/genética , Proteínas y Péptidos Salivales/genética , Relación Estructura-Actividad , Trombina/antagonistas & inhibidores , Trombina/genética , Tirosina/químicaRESUMEN
Multiphoton fluorescence microscopy (MPM), using near infrared excitation light, provides increased penetration depth, decreased detection background, and reduced phototoxicity. Using stimulated emission depletion (STED) approach, MPM can bypass the diffraction limitation, but it requires both spatial alignment and temporal synchronization of high power (femtosecond) lasers, which is limited by the inefficiency of the probes. Here, we report that upconversion nanoparticles (UCNPs) can unlock a new mode of near-infrared emission saturation (NIRES) nanoscopy for deep tissue super-resolution imaging with excitation intensity several orders of magnitude lower than that required by conventional MPM dyes. Using a doughnut beam excitation from a 980 nm diode laser and detecting at 800 nm, we achieve a resolution of sub 50 nm, 1/20th of the excitation wavelength, in imaging of single UCNP through 93 µm thick liver tissue. This method offers a simple solution for deep tissue super resolution imaging and single molecule tracking.
RESUMEN
Gut ischemia is common in critically ill patients, promoting thrombosis and inflammation in distant organs. The mechanisms linking hemodynamic changes in the gut to remote organ thrombosis remain ill-defined. We demonstrate that gut ischemia in the mouse induces a distinct pulmonary thrombotic disorder triggered by neutrophil macroaggregates. These neutrophil aggregates lead to widespread occlusion of pulmonary arteries, veins, and the microvasculature. A similar pulmonary neutrophil-rich thrombotic response occurred in humans with the acute respiratory distress syndrome. Intravital microscopy during gut ischemia-reperfusion injury revealed that rolling neutrophils extract large membrane fragments from remnant dying platelets in multiple organs. These platelet fragments bridge adjacent neutrophils to facilitate macroaggregation. Platelet-specific deletion of cyclophilin D, a mitochondrial regulator of cell necrosis, prevented neutrophil macroaggregation and pulmonary thrombosis. Our studies demonstrate the existence of a distinct pulmonary thrombotic disorder triggered by dying platelets and neutrophil macroaggregates. Therapeutic targeting of platelet death pathways may reduce pulmonary thrombosis in critically ill patients.
Asunto(s)
Tracto Gastrointestinal/irrigación sanguínea , Tracto Gastrointestinal/patología , Isquemia/complicaciones , Pulmón/patología , Neutrófilos/patología , Trombosis/etiología , Trombosis/patología , Animales , Plaquetas/metabolismo , Agregación Celular , Membrana Celular/metabolismo , Peptidil-Prolil Isomerasa F , Ciclofilinas/deficiencia , Ciclofilinas/metabolismo , Tracto Gastrointestinal/fisiopatología , Humanos , Isquemia/fisiopatología , Pulmón/irrigación sanguínea , Pulmón/fisiopatología , Ratones Endogámicos C57BL , Fosfatidilserinas/metabolismo , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/fisiopatología , Circulación EsplácnicaRESUMEN
The inflammatory response is characterized by increased endothelial permeability, which permits the passage of fluid and inflammatory cells into interstitial spaces. The Eph/ephrin receptor ligand system plays a role in inflammation through a signaling cascade, which modifies Rho-GTPase activity. We hypothesized that blocking Eph/ephrin signaling using an EphA4-Fc would result in decreased inflammation and tissue injury in a model of ischemia/reperfusion (I/R) injury. Mice undergoing intestinal I/R pretreated with the EphA4-Fc had significantly reduced intestinal injury compared to mice injected with the control Fc. This reduction in I/R injury was accompanied by significantly reduced neutrophil infiltration, but did not affect intestinal inflammatory cytokine generation. Using microdialysis, we identified that intestinal I/R induced a marked increase in systemic vascular leakage, which was completely abrogated in EphA4-Fc-treated mice. Finally, we confirmed the direct role of Eph/ephrin signaling in endothelial leakage by demonstrating that EphA4-Fc inhibited tumor necrosis factor-α-induced vascular permeability in human umbilical vein endothelial cells. This study identifies that Eph/ephrin interaction induces proinflammatory signaling in vivo by inducing vascular leak and neutrophil infiltration, which results in tissue injury in intestinal I/R. Therefore, therapeutic targeting of Eph/ephrin interaction using inhibitors, such as EphA4-Fc, may be a novel method to prevent tissue injury in acute inflammation by influencing endothelial integrity and by controlling vascular leak.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Receptor EphA4/antagonistas & inhibidores , Daño por Reperfusión/tratamiento farmacológico , Animales , Línea Celular , Humanos , Masculino , Ratones , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The receptor for advanced glycation endproducts (RAGE) can engage a diverse class of ligands and contribute to the immune and inflammatory response to infection and injury. It is known to be a pathogenic receptor in many inflammatory diseases, including ischemia/reperfusion (IR) injuries in several tissues; however, its role has not been investigated in IR injuries of the intestine to date. Mesenteric (or intestinal) IR leads to recruitment of inflammatory cells into intestinal interstitial spaces, which markedly disrupts intestinal mucosa. IR-induced mucosal injury is accompanied by the development of a local and systemic inflammatory response and remote organ injury, and results in high mortality in the clinic. We hypothesized that elimination of RAGE signaling using RAGE(-/-) mice would result in decreased local and remote organ injury and reduced inflammation in a mesenteric IR model, and thus be a target for therapeutic intervention. We found that RAGE ligands including HMGB-1 and C3a were elevated after mesenteric IR indicating the potential for enhanced RAGE activation in this model. However despite this, wild-type and RAGE(-/-) mice both displayed similar degrees of mesenteric injury, neutrophil infiltration, intestinal edema, cytokine generation, neutrophil mobilization, and remote organ injury after mesenteric IR. We, therefore, conclude that despite its role in other organ IR injuries, and the robust production of RAGE ligands after intestinal ischemia, RAGE itself does not directly influence tissue injury and the inflammatory response in mesenteric IR.
RESUMEN
C3a is a key complement activation fragment, yet its neutrophil-expressed receptor (C3aR) still has no clearly defined role. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to explore the role of C3aR in acute tissue injuries. C3aR deficiency worsened intestinal injury, which corresponded with increased numbers of tissue-infiltrating neutrophils. Circulating neutrophils were significantly increased in C3aR(-/-) mice after intestinal ischemia, and C3aR(-/-) mice also mobilized more circulating neutrophils after granulocyte colony-stimulating factor infusion compared with WT mice, indicating a specific role for C3aR in constraining neutrophil mobilization in response to intestinal injury. In support of this role, C3aR(-/-) mice reconstituted with WT bone marrow reversed IR pathology back to WT levels. Complement C5a receptor (C5aR) antagonism in C3aR(-/-) mice also rectified the worsened pathology after intestinal IR injury but had no effect on circulating neutrophils, highlighting the opposing roles of C3a and C5a in disease pathogenesis. Finally, we found that using a potent C3a agonist to activate C3aR in vivo reduced neutrophil mobilization and ameliorated intestinal IR pathology in WT, but not C3aR(-/-), mice. This study identifies a role for C3aR in regulating neutrophil mobilization after acute intestinal injury and highlights C3aR agonism as a potential treatment option for acute, neutrophil-driven pathologies.
Asunto(s)
Intestinos/inmunología , Neutrófilos/inmunología , Receptores de Complemento/inmunología , Daño por Reperfusión/prevención & control , Animales , Apoptosis/inmunología , Citocinas/sangre , Hemoglobinas/metabolismo , Técnicas Histológicas , Intestinos/citología , Ratones , Ratones Noqueados , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Daño por Reperfusión/inmunologíaRESUMEN
The anaphylatoxin C5a generated by activation of the innate immunity complement system is a potent inflammatory peptide mediator through the G-protein-coupled receptor C5aR (CD88) present in immune-inflammatory cells, including monocytes, macrophages, neutrophils, T cells, and mast cells. Inflammatory cells infiltrate and initiate the development of fibrosis in the chronically hypertensive heart. In this study, we have investigated whether treatment with a selective C5aR antagonist prevents cardiovascular remodeling in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Control and DOCA-salt rats were treated with PMX53 (AcF-[OPdChaWR], 1 mg·kg·d oral gavage) for 32 days; structural and functional changes in cardiovascular system were determined. DOCA-salt hypertension increased leukocyte extravasation into ventricular tissue, increasing collagen deposition and ventricular stiffness; PMX53 treatment attenuated these changes, thereby improving cardiac function. Further, treatment with PMX53 suppressed an increased expression of C5aR in the left ventricle from DOCA-salt rats, consistent with the reduced infiltration of inflammatory cells. Vascular endothelial dysfunction in thoracic aortic rings was attenuated by PMX53 treatment, but systolic blood pressure was unchanged in DOCA-salt rats. In the heart, PMX53 treatment attenuated inflammatory cell infiltration, fibrosis, and ventricular stiffness, indicating that C5aR is critically involved in ventricular remodeling by regulating inflammatory responses in the hypertensive heart.