RESUMEN
Background: Neutrophil extracellular trap (NET) production has been implicated in the pathogenesis of thromboinflammatory conditions such as Sickle Cell Disease (SCD), contributing to heightened risk for ischemic stroke. NETs are catalyzed by the enzyme Peptidyl Arginine Deiminase 4 (PAD4) and neutrophil derived reactive oxygen species (ROS), especially NADPH oxidase (NOX) which interacts with PAD4 and is therefore critical for neutrophil function. However, the role that NOX-dependent ROS and NETs play in the accelerated cerebral microvascular thrombosis associated with thromboinflammatory conditions, such as SCD, has not been fully elucidated and is the aim of this study. Methods: The in-vitro effects of targeting PAD4 and NOX were examined using physiologically relevant NET assays with neutrophils isolated from healthy volunteers (control) and SCD patients. In addition, in-vivo intravascular effects of targeting PAD4 and NOX in the cerebral microcirculation of C57BL/6 and sickle transgenic mice (STM) were assessed using a photoactivation thrombosis model (light/dye) coupled with real-time fluorescence intravital microscopy. Results: We found that targeting PAD4 and NOX in human neutrophils significantly inhibited ionomycin dependent H3cit+ neutrophils. Targeting PAD4 and NOX in-vivo resulted in prolonged blood flow cessation in cerebrovascular arterioles as well as venules. Moreover, we were able to replicate the effects of PAD4 and NOX targeting in a clinical model of accelerated thromboinflammation by increasing blood flow cessation times in cerebral microvessels in STM. These findings concurred with the clinical setting i.e. neutrophils isolated from SCD patients, which possessed an attenuation of H3cit+ neutrophil production on targeting PAD4 and NOX. Conclusions: Taken together, our compelling data suggests that PAD4 and NOX play a significant role in neutrophil driven thromboinflammation. Targeting PAD4 and NOX limits pathological H3cit+ neutrophils, which may further explain attenuation of cerebral thrombosis. Overall, this study presents a viable pre-clinical model of prevention and management of thromboinflammatory complications such as ischemic stroke.
Asunto(s)
Neutrófilos , Trombosis , Ratones , Animales , Humanos , NADPH Oxidasas , Especies Reactivas de Oxígeno , Tromboinflamación , Inflamación , Arginina Deiminasa Proteína-Tipo 4/farmacología , Trombosis/patología , Ratones Endogámicos C57BLRESUMEN
Osteosarcoma is the most common type of bone malignancy, and the pathogenesis has not been entirely elucidated yet. An important deimination modification enzyme PADI4 (peptidylarginine deiminase 4) has attracted much attention in recent years for its important function in several kinds of human tumors. However, the role of PADI4 on osteosarcoma tumorigenesis remains largely unrevealed. Here, we first assessed the effect of PADI4 on osteosarcoma proliferation by the CCK8 method and colony formation assay. Ectopically expressing PADI4 positively regulates the colony formation capacity of both U2OS and Saos-2 cells. Furthermore, we explored the related mechanism and showed that PADI4 could stimulate Wnt/ß-catenin and MEK/ERK signaling in both U2OS and Saos-2 cells. Then, we detected expression of PADI4 in human tissues of osteosarcoma and revealed that differential expression of PADI4 was associated with tumorigenesis of osteosarcoma. Last, we performed the in vivo experiment in nude mice and results also showed PADI4 could affect the tumor growth. In conclusion, this work revealed that PADI4 could upregulate the proliferation of osteosarcoma, mainly via the Wnt/ß-catenin and MEK/ERK signaling pathway. This study gives us new insight into the regulation mechanism of osteosarcoma proliferation and highlights PADI4 as a promising target for osteosarcoma diagnosis and treatment.
Asunto(s)
Neoplasias Óseas/metabolismo , Proliferación Celular/efectos de los fármacos , Osteosarcoma/metabolismo , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Arginina Deiminasa Proteína-Tipo 4/farmacología , Adolescente , Adulto , Anciano , Animales , Neoplasias Óseas/patología , Carcinogénesis , Línea Celular Tumoral , Colecistoquinina , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Osteosarcoma/patología , Fragmentos de Péptidos , Arginina Deiminasa Proteína-Tipo 4/genética , Desiminasas de la Arginina Proteica , Transducción de Señal , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto Joven , beta Catenina/metabolismoRESUMEN
Background: Excessive production of neutrophil extracellular traps (NETs) in sepsis contributes to vascular occlusion by acting as a scaffold and stimulus for thrombus formation. Removal of extracellular DNA, the major structural component of NETs, by DNase I may reduce host injury. Objectives: (1) To determine how heparin variants (unfractionated heparin, enoxaparin, Vasoflux, and fondaparinux) affect DNase I activity, (2) to measure temporal changes in circulating DNA and DNase I in septic patients. Methods: DNAhistone complexes were treated with DNase I ± heparin variants and visualized via agarose gels. We compared the ability of DNase I ± heparin variants to digest NETs released by phorbol 12-myristate 13-acetate-stimulated neutrophils versus DNAhistone complexes released by necrotic HEK293 cells. Plasma DNA and DNase I levels were measured longitudinally in 76 septic patients. Results: Heparin enhances DNase I-mediated digestion of DNAhistone complexes in a size-dependent manner that does not require the antithrombin-binding region. In contrast, DNase I alone was able to degrade the DNAhistone component of NETs presumably due to peptidylarginine deiminase 4 (PAD4)-mediated histone citrullination that weakens DNAhistone interactions. In purified systems, PAD4 treatment of DNAhistone complexes enhanced the ability of DNase I to degrade histone-bound DNA. In septic patients, endogenous DNase I levels remained persistently low over 28 days, and there were no significant correlations between DNA and DNase I levels. Conclusion: Heparin enhances DNA-mediated digestion of DNAhistone complexes in a size-dependent manner that is independent of its anticoagulant properties. Citrullination of histones by PAD4 renders DNAhistone complexes susceptible to DNase I digestion. Endogenous DNase I levels are persistently decreased in septic patients, which supports the potential utility of DNase I as a therapy for sepsis.
Asunto(s)
Desoxirribonucleasa I/sangre , Heparina/farmacología , Arginina Deiminasa Proteína-Tipo 4/farmacología , Sepsis/sangre , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
We determined that renal proximal tubular (PT) NF-κB essential modulator (NEMO) plays a direct and critical role in ischemic acute kidney injury (AKI) using mice lacking renal PT NEMO and by targeted renal PT NEMO inhibition with mesoscale nanoparticle-encapsulated NEMO binding peptide (NBP MNP). We subjected renal PT NEMO-deficient mice, WT mice, and C57BL/6 mice to sham surgery or 30 minutes of renal ischemia and reperfusion (IR). C57BL/6 mice received NBP MNP or empty MNP before renal IR injury. Mice treated with NBP MNP and mice deficient in renal PT NEMO were protected against ischemic AKI, having decreased renal tubular necrosis, inflammation, and apoptosis compared with control MNP-treated or WT mice, respectively. Recombinant peptidylarginine deiminase type 4 (rPAD4) targeted kidney PT NEMO to exacerbate ischemic AKI in that exogenous rPAD4 exacerbated renal IR injury in WT mice but not in renal PT NEMO-deficient mice. Furthermore, rPAD4 upregulated proinflammatory cytokine mRNA and NF-κB activation in freshly isolated renal proximal tubules from WT mice but not from PT NEMO-deficient mice. Taken together, our studies suggest that renal PT NEMO plays a critical role in ischemic AKI by promoting renal tubular inflammation, apoptosis, and necrosis.