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1.
Blood ; 140(9): 1020-1037, 2022 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-35737916

RESUMEN

Acute lung injury, referred to as the acute chest syndrome, is a major cause of morbidity and mortality in patients with sickle cell disease (SCD), which often occurs in the setting of a vaso-occlusive painful crisis. P-selectin antibody therapy reduces hospitalization of patients with SCD by ∼50%, suggesting that an unknown P-selectin-independent mechanism promotes remaining vaso-occlusive events. In patients with SCD, intraerythrocytic polymerization of mutant hemoglobin promotes ischemia-reperfusion injury and hemolysis, which leads to the development of sterile inflammation. Using intravital microscopy in transgenic, humanized mice with SCD and in vitro studies with blood from patients with SCD, we reveal for the first time that the sterile inflammatory milieu in SCD promotes caspase-4/11-dependent activation of neutrophil-gasdermin D (GSDMD), which triggers P-selectin-independent shedding of neutrophil extracellular traps (NETs) in the liver. Remarkably, these NETs travel intravascularly from liver to lung, where they promote neutrophil-platelet aggregation and the development of acute lung injury. This study introduces a novel paradigm that liver-to-lung embolic translocation of NETs promotes pulmonary vascular vaso-occlusion and identifies a new GSDMD-mediated, P-selectin-independent mechanism of lung injury in SCD.


Asunto(s)
Lesión Pulmonar Aguda , Anemia de Células Falciformes , Trampas Extracelulares , Proteínas de Unión a Fosfato , Proteínas Citotóxicas Formadoras de Poros , Daño por Reperfusión , Lesión Pulmonar Aguda/etiología , Animales , Hígado , Pulmón/irrigación sanguínea , Ratones , Ratones Transgénicos , Selectina-P , Proteínas de Unión a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Daño por Reperfusión/complicaciones
2.
Blood ; 137(19): 2676-2680, 2021 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-33619560

RESUMEN

Sickle cell disease (SCD) is caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, vasoocclusion, and intense hemolysis. P-selectin inhibition has been shown to prevent vasoocclusive events in patients with SCD; however, the chronic effect of P-selectin inhibition in SCD remains to be determined. Here, we used quantitative liver intravital microscopy in our recently generated P-selectin-deficient SCD mice to show that chronic P-selectin deficiency attenuates liver ischemia but fails to prevent hepatobiliary injury. Remarkably, we find that this failure in resolution of hepatobiliary injury in P-selectin-deficient SCD mice is associated with the increase in cellular senescence and reduced epithelial cell proliferation in the liver. These findings highlight the importance of investigating the long-term effects of chronic P-selectin inhibition therapy on liver pathophysiology in patients with SCD.


Asunto(s)
Anemia de Células Falciformes/patología , Isquemia/patología , Hígado/irrigación sanguínea , Selectina-P/deficiencia , Anemia de Células Falciformes/fisiopatología , Animales , Arteriopatías Oclusivas/etiología , Arteriopatías Oclusivas/patología , Senescencia Celular , Células Epiteliales/patología , Hemo-Oxigenasa 1/análisis , Hemólisis , Hígado/patología , Hígado/fisiopatología , Proteínas de la Membrana/análisis , Ratones , Ratones Noqueados , Modelos Animales , Selectina-P/genética
3.
Hepatology ; 72(6): 2165-2181, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32190913

RESUMEN

BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.


Asunto(s)
Anemia de Células Falciformes/complicaciones , Bilis/metabolismo , Colestasis/etiología , Insuficiencia Hepática/etiología , Hígado/patología , Adolescente , Adulto , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/genética , Animales , Conductos Biliares Intrahepáticos/diagnóstico por imagen , Conductos Biliares Intrahepáticos/patología , Colestasis/patología , Colestasis/prevención & control , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Hemoglobina Falciforme/genética , Insuficiencia Hepática/patología , Insuficiencia Hepática/prevención & control , Humanos , Microscopía Intravital , Hígado/diagnóstico por imagen , Masculino , Ratones , Persona de Mediana Edad , FN-kappa B/antagonistas & inhibidores , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Adulto Joven
4.
Am J Respir Crit Care Med ; 201(1): 33-46, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31498653

RESUMEN

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet-neutrophil aggregate-dependent vasoocclusion is known to occur in the lung and contribute to acute chest syndrome, the etiological mechanisms that trigger acute chest syndrome are largely unknown.Objectives: To identify the innate immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vasoocclusion and injury in SCD.Methods:In vivo imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system was performed. SCD mice were systemically challenged with nanogram quantities of LPS to trigger lung vasoocclusion.Measurements and Main Results: Platelet-inflammasome activation led to generation of IL-1ß and caspase-1-carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation, and platelet-neutrophil aggregation were enhanced by the presence of LPS at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1ß pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro.Conclusions: These results are the first to identify that platelet-inflammasome-dependent shedding of IL-1ß and caspase-1-carrying platelet EVs promote lung vasoocclusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome-dependent innate immune pathway to prevent acute chest syndrome.


Asunto(s)
Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/inmunología , Vesículas Extracelulares/inmunología , Inflamasomas/inmunología , Lesión Pulmonar/etiología , Lesión Pulmonar/fisiopatología , Agregación Plaquetaria/inmunología , Síndrome Torácico Agudo/etiología , Síndrome Torácico Agudo/fisiopatología , Anemia de Células Falciformes/fisiopatología , Animales , Humanos , Ratones , Ratones Transgénicos , Modelos Animales , Neutrófilos/inmunología
5.
Physiol Rep ; 12(1): e15902, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38163670

RESUMEN

Although zinc deficiency (secondary to malnutrition) has long been considered an important contributor to morbidity and mortality of infectious disease (e.g. diarrhea disorders), epidemiologic data (including randomized controlled trials with supplemental zinc) for such a role in lower respiratory tract infection are somewhat ambiguous. In the current study, we provide the first preclinical evidence demonstrating that although diet-induced acute zinc deficiency (Zn-D: ~50% decrease) did not worsen infection induced by either influenza A (H1N1) or methicillin-resistant staph aureus (MRSA), Zn-D mice were sensitive to the injurious effects of superinfection of H1N1 with MRSA. Although the mechanism underlying the sensitivity of ZnD mice to combined H1N1/MRSA infection is unclear, it was noteworthy that this combination exacerbated lung injury as shown by lung epithelial injury markers (increased BAL protein) and decreased genes related to epithelial integrity in Zn-D mice (surfactant protein C and secretoglobins family 1A member 1). As bacterial pneumonia accounts for 25%-50% of morbidity and mortality from influenza A infection, zinc deficiency may be an important pathology component of respiratory tract infections.


Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Desnutrición , Staphylococcus aureus Resistente a Meticilina , Neumonía Bacteriana , Animales , Ratones , Neumonía Bacteriana/complicaciones , Staphylococcus aureus , Zinc
6.
Diabetes Metab Res Rev ; 28(3): 228-35, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22057777

RESUMEN

BACKGROUND: In diabetes mellitus, increased formation of reactive oxygen species due to high level of glucose in both blood plasma and tissues creates oxidative stress and damages the tissues. Antioxidants together with the antioxidant enzymes are very important in order to protect the cells against oxidative damage. METHODS: Differential expressions of both mRNA and proteins of major antioxidant enzymes in streptozotocin-induced diabetic rat kidneys were measured with the help of real-time polymerase chain reaction and western blot analysis, respectively. Furthermore, effects of two strong antioxidants α-lipoic acid, vitamin C and their combination on the regulation of both expressions and the activities of antioxidant enzymes were also studied. RESULTS: In diabetic rat kidney tissue, both catalase and glutathione peroxidase activities were reduced (although mRNA expression for both was greatly increased) when compared with controls. No significant change was observed in superoxide dismutase (SOD) activity. Alpha-lipoic acid increased catalase activity towards the control values. Combined administration of alpha-lipoic acid and vitamin C increased the activities of both catalase and SOD, demonstrating a posttranslational effect. Glutathione concentrations were decreased in diabetic kidney; alpha-lipoic acid treatment partially restored the glutathione levels. CONCLUSIONS: All data showed the importance of post-transcriptional and translational regulation of the antioxidant enzyme activities against oxidative stress that is associated with diabetes.


Asunto(s)
Antioxidantes/metabolismo , Diabetes Mellitus Experimental/enzimología , Animales , Ácido Ascórbico/administración & dosificación , Ácido Ascórbico/farmacología , Catalasa/metabolismo , Combinación de Medicamentos , Glutatión/metabolismo , Glutatión Peroxidasa/metabolismo , Riñón/efectos de los fármacos , Riñón/enzimología , Masculino , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Ácido Tióctico/administración & dosificación , Ácido Tióctico/farmacología
7.
Blood Adv ; 6(12): 3729-3734, 2022 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-35427414

RESUMEN

Hemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.


Asunto(s)
Hemofilia A , Animales , Endotelio , Terapia Genética , Vectores Genéticos/genética , Hemofilia A/genética , Hemofilia A/metabolismo , Hemofilia A/terapia , Humanos , Hígado/metabolismo , Ratones , Ratones Noqueados
9.
Exp Hematol ; 84: 1-6.e1, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32243995

RESUMEN

Sickle cell disease (SCD) is a monogenic disorder estimated to affect more than three million people worldwide. Acute systemic painful vaso-occlusive episode (VOE) is the primary reason for emergency medical care among SCD patients. VOE may also progress to acute chest syndrome (ACS), a type of acute lung injury and one of the primary reasons for mortality among SCD patients. Recently, P-selectin monoclonal antibodies were found to attenuate VOE in SCD patients and lung vaso-occlusion in transgenic humanized SCD mice, highlighting the therapeutic benefit of P-selectin inhibition in SCD. Here, we use quantitative fluorescence intravital lung microscopy (qFILM) to illustrate that tandem P-selectin-glycoprotein ligand-immunoglobulin (TSGL-Ig) fusion molecule containing four P-selectin binding sites, significantly attenuated intravenous (IV) oxyhemoglobin triggered lung vaso-occlusion in SCD mice. These findings highlight the therapeutic potential of TSGL-Ig in preventing VOE and ACS in SCD.


Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Inmunoglobulinas/farmacología , Enfermedades Pulmonares/tratamiento farmacológico , Selectina-P/farmacología , Proteínas Recombinantes de Fusión/farmacología , Enfermedades Vasculares/tratamiento farmacológico , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Anemia de Células Falciformes/patología , Animales , Femenino , Humanos , Inmunoglobulinas/genética , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/patología , Masculino , Ratones , Selectina-P/genética , Ratas , Proteínas Recombinantes de Fusión/genética , Enfermedades Vasculares/genética , Enfermedades Vasculares/metabolismo
10.
JCI Insight ; 5(14)2020 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-32544100

RESUMEN

Patients with hereditary or acquired hemolytic anemias have a high risk of developing in situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice to assess the pathogenesis of pulmonary thrombosis following deionized water-induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbß3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravascular ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet P2Y12 purinergic receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to our knowledge to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation, leading to thrombosis in the precapillary pulmonary arterioles, and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis-triggered pulmonary thrombosis.


Asunto(s)
Anemia Hemolítica/genética , Hemólisis/genética , Arteria Pulmonar/metabolismo , Receptores Purinérgicos P2Y12/genética , Trombosis/genética , Adenosina Difosfato/metabolismo , Anemia Hemolítica/metabolismo , Anemia Hemolítica/patología , Animales , Arteriolas/metabolismo , Arteriolas/patología , Coagulación Sanguínea/genética , Plaquetas/metabolismo , Humanos , Pulmón/metabolismo , Pulmón/patología , Ratones , Arteria Pulmonar/patología , Trombina/genética , Trombina/metabolismo , Trombosis/metabolismo , Trombosis/patología
11.
JCI Insight ; 2(1): e89761, 2017 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-28097236

RESUMEN

In patients with sickle cell disease (SCD), the polymerization of intraerythrocytic hemoglobin S promotes downstream vaso-occlusive events in the microvasculature. While vaso-occlusion is known to occur in the lung, often in the context of systemic vaso-occlusive crisis and the acute chest syndrome, the pathophysiological mechanisms that incite lung injury are unknown. We used intravital microscopy of the lung in transgenic humanized SCD mice to monitor acute vaso-occlusive events following an acute dose of systemic lipopolysaccharide sufficient to trigger events in SCD but not control mice. We observed cellular microembolism of precapillary pulmonary arteriolar bottlenecks by neutrophil-platelet aggregates. Blood from SCD patients was next studied under flow in an in vitro microfluidic system. Similar to the pulmonary circulation, circulating platelets nucleated around arrested neutrophils, translating to a greater number and duration of neutrophil-platelet interactions compared with normal human blood. Inhibition of platelet P-selectin with function-blocking antibody attenuated the neutrophil-platelet interactions in SCD patient blood in vitro and resolved pulmonary arteriole microembolism in SCD mice in vivo. These results establish the relevance of neutrophil-platelet aggregate formation in lung arterioles in promoting lung vaso-occlusion in SCD and highlight the therapeutic potential of targeting platelet adhesion molecules to prevent acute chest syndrome.


Asunto(s)
Anemia de Células Falciformes/complicaciones , Arteriolas/patología , Pulmón/patología , Selectina-P/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Síndrome Torácico Agudo/fisiopatología , Síndrome Torácico Agudo/prevención & control , Anemia de Células Falciformes/fisiopatología , Animales , Arteriolas/citología , Plaquetas/metabolismo , Plaquetas/patología , Embolia/patología , Embolia/fisiopatología , Femenino , Humanos , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/ultraestructura , Masculino , Ratones , Ratones Transgénicos , Neutrófilos/metabolismo , Neutrófilos/patología , Selectina-P/metabolismo , Adhesividad Plaquetaria/fisiología , Receptor Toll-Like 4/metabolismo
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