RESUMEN
Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2ß) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2ß in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2ß showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2ß in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2ß as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.
Asunto(s)
Células Endoteliales , Fosfatidilinositol 3-Quinasas , Uniones Adherentes/metabolismo , Animales , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Permeabilidad Capilar , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Blood-brain barrier (BBB) disruption and transendothelial trafficking of immune cells into the central nervous system (CNS) are pathophysiological hallmarks of neuroinflammatory disorders like multiple sclerosis (MS). Recent evidence suggests that the kallikrein-kinin and coagulation system might participate in this process. Here, we identify plasma kallikrein (KK) as a specific direct modulator of BBB integrity. Levels of plasma prekallikrein (PK), the precursor of KK, were markedly enhanced in active CNS lesions of MS patients. Deficiency or pharmacologic blockade of PK renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by a remarkable reduction of BBB disruption and CNS inflammation. In vitro analysis revealed that KK modulates endothelial cell function in a protease-activated receptor-2-dependent manner, leading to an up-regulation of the cellular adhesion molecules Intercellular Adhesion Molecule 1 and Vascular Cell Adhesion Molecule 1, thereby amplifying leukocyte trafficking. Our study demonstrates that PK is an important direct regulator of BBB integrity as a result of its protease function. Therefore, KK inhibition can decrease BBB damage and cell invasion during neuroinflammation and may offer a strategy for the treatment of MS.
Asunto(s)
Bradiquinina/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Calicreínas/metabolismo , Receptor PAR-2/metabolismo , Animales , Barrera Hematoencefálica , Western Blotting , Bradiquinina/fisiología , Encefalomielitis Autoinmune Experimental/fisiopatología , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Humanos , Calicreínas/antagonistas & inhibidores , Calicreínas/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Esclerosis Múltiple/metabolismo , Receptor PAR-2/fisiologíaRESUMEN
Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood-brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/enzimología , Isquemia Miocárdica/enzimología , NADPH Oxidasa 4/genética , Animales , Benzoxazoles/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/patología , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Isquemia Encefálica/prevención & control , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Inhibidores Enzimáticos/farmacología , Femenino , Arteria Femoral/lesiones , Regulación de la Expresión Génica , Miembro Posterior/irrigación sanguínea , Miembro Posterior/efectos de los fármacos , Miembro Posterior/metabolismo , Miembro Posterior/patología , Humanos , Masculino , Ratones , Ratones Noqueados , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Isquemia Miocárdica/prevención & control , NADPH Oxidasa 4/antagonistas & inhibidores , NADPH Oxidasa 4/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/farmacología , Especificidad de Órganos , Pirazoles/farmacología , Piridonas/farmacología , Ratas , Transducción de Señal , Triazoles/farmacologíaRESUMEN
Background and Purpose- The selection of appropriate neurological scores and tests is crucial for the evaluation of stroke consequences. The validity and reliability of neurological deficit scores and tests has repeatedly been questioned in ischemic stroke models in the past. Methods- In 198 male mice exposed to transient intraluminal middle cerebral artery occlusion, we examined the validity and reliability of 11 neurological scores (Bederson score 0-3, Bederson score 0-4, Bederson score 0-5, modified neurological severity [0-14], subjective overall impression [0-10], or simple neurological tests: grip test, latency to move body length test, pole test, wire hanging test, negative geotaxis test, and elevated body swing test) in the acute stroke phase, that is, after 24 hours. Combinations of neurological scores or tests for predicting infarct volume were statistically analyzed. Results- Infarct volume was left skewed (median [Q1-Q3], 78.4 [54.8-101.3] mm3). Among all tests, the Bederson (0-5; r=0.63, P<0.001), modified neurological severity (r=0.80, P<0.001), and subjective overall impression (r=-0.63, P<0.001) scores had the highest test validities, using infarct volume as external reference. Subjective overall impression had the best agreement between 5 raters (Kendall W=0.11, P<0.001). The Bederson (0-5) score discriminated infarct volume in mice with small (≤50 mm3; r=0.33, P=0.027) and large (>50 mm3; r=0.48, P<0.001) brain infarcts, all other tests only in mice with large infarcts. Combining subjective overall impression with Bederson (0-5) score explained 47.6% of the variance of infarct volume. Conclusions- Despite their simplicity, the Bederson (0-5) score, modified neurological severity score, and subjective overall impression have reasonable validity and reliability in the acute stroke phase. The Bederson (0-5) score equally distinguishes infarct volume in small and large infarcts. Visual Overview- An online visual overview is available for this article.
Asunto(s)
Infarto de la Arteria Cerebral Media/complicaciones , Enfermedades del Sistema Nervioso/diagnóstico , Enfermedades del Sistema Nervioso/etiología , Examen Neurológico/métodos , Animales , Masculino , Ratones , Reproducibilidad de los ResultadosRESUMEN
Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.
Asunto(s)
Quinasa de la Caseína II/fisiología , Fragmentos de Péptidos/fisiología , Activación Plaquetaria , Trombopoyesis , Trombosis/patología , Animales , Plaquetas , Señalización del Calcio , Quinasa de la Caseína II/deficiencia , Megacariocitos/metabolismo , Megacariocitos/patología , Megacariocitos/ultraestructura , Ratones , Ratones Noqueados , Fragmentos de Péptidos/deficiencia , Trombosis/etiología , Trombosis/metabolismoRESUMEN
OBJECTIVE: Activation of platelets by subendothelial collagen results in an increase of cytosolic Ca(2+) concentration ([Ca(2+)]i) and is followed by platelet activation and thrombus formation that may lead to vascular occlusion. The present study determined the role of phosphoinositide-dependent protein kinase 1 (PDK1) in collagen-dependent platelet Ca(2+) signaling and ischemic stroke in vivo. APPROACH AND RESULTS: Platelet activation with collagen receptor glycoprotein VI agonists collagen-related peptide or convulxin resulted in a significant increase in PDK1 activity independent of second-wave signaling. PDK1 deficiency was associated with reduced platelet phospholipase Cγ2-dependent inositol-1,4,5-trisphosphate production and intracellular [Ca(2+)]i in response to stimulation with collagen-related peptide or convulxin. The defective increase of [Ca(2+)]i resulted in a substantial defect in activation-dependent platelet secretion and aggregation on collagen-related peptide stimulation. Furthermore, Rac1 activation and spreading, adhesion to collagen, and thrombus formation under high arterial shear rates were significantly diminished in PDK1-deficient platelets. Mice with PDK1-deficient platelets were protected against arterial thrombotic occlusion after FeCl3-induced mesenteric arterioles injury and ischemic stroke in vivo. These mice had significantly reduced brain infarct volumes, with a significantly increased survival of 7 days after transient middle cerebral artery occlusion without increase of intracerebral hemorrhage. Tail bleeding time was prolonged in pdk1(-/-) mice, reflecting an important role of PDK1 in primary hemostasis. CONCLUSIONS: PDK1 is required for Ca(2+)-dependent platelet activation on stimulation of collagen receptor glycoprotein VI, arterial thrombotic occlusion, and ischemic stroke in vivo.
Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Plaquetas/enzimología , Señalización del Calcio , Colágeno/metabolismo , Infarto de la Arteria Cerebral Media/enzimología , Activación Plaquetaria , Trombosis/enzimología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/deficiencia , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Animales , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Infarto de la Arteria Cerebral Media/sangre , Infarto de la Arteria Cerebral Media/patología , Inositol 1,4,5-Trifosfato/sangre , Ratones Noqueados , Neuropéptidos/sangre , Fenotipo , Fosfolipasa C gamma/sangre , Adhesividad Plaquetaria , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/deficiencia , Glicoproteínas de Membrana Plaquetaria/genética , Trombosis/sangre , Trombosis/patología , Factores de Tiempo , Proteína de Unión al GTP rac1/sangreRESUMEN
OBJECTIVE: It is known that both platelets and coagulation strongly influence infarct progression after ischemic stroke, but the mechanisms and their interplay are unknown. Our aim was to assess the contribution of the procoagulant platelet surface, and thus platelet-driven thrombin generation, to the progression of thromboinflammation in the ischemic brain. APPROACH AND RESULTS: We present the characterization of a novel platelet and megakaryocyte-specific TMEM16F (anoctamin 6) knockout mouse. Reflecting Scott syndrome, platelets from the knockout mouse had a significant reduction in procoagulant characteristics that altered thrombin and fibrin generation kinetics. In addition, knockout mice showed significant defects in hemostasis and arterial thrombus formation. However, infarct volumes in a model of ischemic stroke were comparable with wild-type mice. CONCLUSIONS: Platelet TMEM16F activity contributes significantly to hemostasis and thrombosis but not cerebral thromboinflammation. These results highlight another key difference between the roles of platelets and coagulation in these processes.
Asunto(s)
Plaquetas/metabolismo , Enfermedades de las Arterias Carótidas/sangre , Encefalitis/sangre , Encefalitis/genética , Hemostasis , Infarto de la Arteria Cerebral Media/sangre , Fosfatidilserinas/sangre , Proteínas de Transferencia de Fosfolípidos/sangre , Trombina/metabolismo , Trombosis/sangre , Animales , Anoctaminas , Coagulación Sanguínea , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Encefalitis/patología , Fibrina/metabolismo , Infarto de la Arteria Cerebral Media/genética , Infarto de la Arteria Cerebral Media/patología , Cinética , Megacariocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Transferencia de Fosfolípidos/deficiencia , Proteínas de Transferencia de Fosfolípidos/genética , Activación Plaquetaria , Transducción de Señal , Trombosis/genética , Trombosis/patologíaAsunto(s)
Isquemia Encefálica/patología , Encéfalo/patología , Inflamación/patología , Accidente Cerebrovascular/patología , Plaquetas/metabolismo , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Colágeno/metabolismo , Humanos , Inflamación/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Accidente Cerebrovascular/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calcium-activated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenation-induced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.
Asunto(s)
Barrera Hematoencefálica/enzimología , Calcio/metabolismo , NADPH Oxidasa 5/metabolismo , Accidente Cerebrovascular/enzimología , Animales , Barrera Hematoencefálica/patología , Humanos , Ratones , Ratones Transgénicos , NADPH Oxidasa 5/genética , Especies Reactivas de Oxígeno/metabolismo , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patologíaRESUMEN
Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease-disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy.
RESUMEN
Treatment of neuropathic pain remains challenging as the etiology is heterogeneous and pathomechanisms are incompletely understood. One possible mechanism is oxidative stress due to unphysiological reactive oxygen species (ROS) formation. The only know dedicated enzymatic source of ROS are NADPH oxidases of which the type 4 isoform (NOX4) has been suggested to be involved in the subacute and chronic phase of neuropathic pain. Here, we aim to translate this finding into a treatment strategy by examining the efficacy of the NOX1/4-specific inhibitor GKT136901 using the chronic constriction injury (CCI) mouse model of neuropathic pain. Unexpectedly, post-nerve lesion treatment using GKT136901 was ineffective to reduce pain-related behavior after CCI. We therefore re-investigated the role of NOX4 using an independent KO mouse model. Early after CCI we found an increase in pro-inflammatory cytokines, ROS formation and the oxidative stress marker nitrotyrosine in the lesioned nerve together with an upregulated Nox4 gene expression. In NOX4 KO mice, mechanical allodynia was markedly reduced from day 4 after nerve injury as were all ROS related and acute biomarkers. In addition, we observed a reduction in the CCI-induced upregulation of pro-inflammatory cytokines in the sciatic nerve and dorsal root ganglia along with NOX4-deficiency. Thus, we conclude that NOX4 is involved in the development of neuropathic pain states by producing oxidative stress and subsequent cytokine dysregulation at the lesion site. This appears at very early stages immediately after nerve injury explaining ineffectiveness of post-acute pharmacological NOX inhibition. We suggest that future target validation of NOX4 should now focus on defining the possible therapeutic window in human neuropathic pain.