RESUMEN
Follicular helper T (TFH) cells are a specialized subset of CD4+ T cells that essentially support germinal center responses where high-affinity and long-lived humoral immunity is generated. The regulation of TFH cell survival remains unclear. Here we report that TFH cells show intensified lipid peroxidation and altered mitochondrial morphology, resembling the features of ferroptosis, a form of programmed cell death that is driven by iron-dependent accumulation of lipid peroxidation. Glutathione peroxidase 4 (GPX4) is the major lipid peroxidation scavenger and is necessary for TFH cell survival. The deletion of GPX4 in T cells selectively abrogated TFH cells and germinal center responses in immunized mice. Selenium supplementation enhanced GPX4 expression in T cells, increased TFH cell numbers and promoted antibody responses in immunized mice and young adults after influenza vaccination. Our findings reveal the central role of the selenium-GPX4-ferroptosis axis in regulating TFH homeostasis, which can be targeted to enhance TFH cell function in infection and following vaccination.
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Ferroptosis/fisiología , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Selenio/farmacología , Células T Auxiliares Foliculares/fisiología , Adolescente , Adulto , Animales , Supervivencia Celular/inmunología , Niño , Femenino , Centro Germinal/citología , Centro Germinal/inmunología , Homeostasis/efectos de los fármacos , Homeostasis/genética , Humanos , Inmunidad Humoral/inmunología , Vacunas contra la Influenza/inmunología , Peroxidación de Lípido/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/fisiología , Ovalbúmina , Células T Auxiliares Foliculares/inmunología , Vacunación , Adulto JovenRESUMEN
Activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome has been reported in diabetic complications including diabetic kidney disease (DKD). However, it remains unknown if NLRP3 inhibition is renoprotective in a clinically relevant interventional approach with established DKD. We therefore examined the effect of the NLRP3-specific inhibitor MCC950 in streptozotocin-induced diabetic mice to measure the impact of NLRP3 inhibition on renal inflammation and associated pathology in DKD. We identified an adverse effect of MCC950 on renal pathology in diabetic animals. Indeed, MCC950-treated diabetic animals showed increased renal inflammation and macrophage infiltration in association with enhanced oxidative stress as well as increased mesangial expansion and glomerulosclerosis when compared with vehicle-treated diabetic animals. Inhibition of the inflammasome by MCC950 in diabetic mice led to renal up-regulation of markers of inflammation (Il1ß, Il18 and Mcp1), fibrosis (Col1, Col4, Fn1, α-SMA, Ctgf and Tgfß1) and oxidative stress (Nox2, Nox4 and nitrotyrosine). In addition, enhanced glomerular accumulation of pro-inflammatory CD68 positive cells and pro-oxidant factor nitrotyrosine was identified in the MCC950-treated diabetic compared with vehicle-treated diabetic animals. Collectively, in this interventional model of established DKD, NLRP3 inhibition with MCC950 did not show renoprotective effects in diabetic mice. On the contrary, diabetic mice treated with MCC950 exhibited adverse renal effects particularly enhanced renal inflammation and injury including mesangial expansion and glomerulosclerosis.
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Nefropatías Diabéticas/patología , Furanos/farmacología , Indenos/farmacología , Inflamasomas/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Sulfonamidas/farmacología , Animales , Diabetes Mellitus Experimental , Fibrosis , Furanos/efectos adversos , Indenos/efectos adversos , Inflamación/tratamiento farmacológico , Masculino , Ratones Noqueados para ApoE , Estrés Oxidativo/efectos de los fármacos , Sulfonamidas/efectos adversosRESUMEN
Cardiovascular disease (CVD) remains the leading cause of death worldwide. An ongoing challenge remains the development of novel pharmacotherapies to treat CVD, particularly atherosclerosis. Effective mechanism-informed development and translation of new drugs requires a deep understanding of the known and currently unknown biological mechanisms underpinning atherosclerosis, accompanied by optimization of traditional drug discovery approaches. Current animal models do not precisely recapitulate the pathobiology underpinning human CVD. Accordingly, a fundamental limitation in early-stage drug discovery has been the lack of consensus regarding an appropriate experimental in vivo model that can mimic human atherosclerosis. However, when coupled with a clear understanding of the specific advantages and limitations of the model employed, preclinical animal models remain a crucial component for evaluating pharmacological interventions. Within this perspective, we will provide an overview of the mechanisms and modalities of atherosclerotic drugs, including those in the preclinical and early clinical development stage. Additionally, we highlight recent preclinical models that have improved our understanding of atherosclerosis and associated clinical consequences and propose model adaptations to facilitate the development of new and effective treatments.
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Aterosclerosis , Enfermedades Cardiovasculares , Animales , Humanos , Aterosclerosis/tratamiento farmacológico , Descubrimiento de Drogas , Modelos AnimalesRESUMEN
The acute phase protein serum amyloid A (SAA) is associated with endothelial dysfunction and early-stage atherogenesis. Stimulation of vascular cells with SAA increases gene expression of pro-inflammation cytokines and tissue factor (TF). Activation of the transcription factor, nuclear factor kappa-B (NFκB), may be central to SAA-mediated endothelial cell inflammation, dysfunction and pro-thrombotic responses, while targeting NFκB with a pharmacologic inhibitor, BAY11-7082, may mitigate SAA activity. Human carotid artery endothelial cells (HCtAEC) were pre-incubated (1.5 h) with 10 µM BAY11-7082 or vehicle (control) followed by SAA (10 µg/mL; 4.5 h). Under these conditions gene expression for TF and Tumor Necrosis Factor (TNF) increased in SAA-treated HCtAEC and pre-treatment with BAY11-7082 significantly (TNF) and marginally (TF) reduced mRNA expression. Intracellular TNF and interleukin 6 (IL-6) protein also increased in HCtAEC supplemented with SAA and this expression was inhibited by BAY11-7082. Supplemented BAY11-7082 also significantly decreased SAA-mediated leukocyte adhesion to apolipoprotein E-deficient mouse aorta in ex vivo vascular flow studies. In vascular function studies, isolated aortic rings pre-treated with BAY11-7082 prior to incubation with SAA showed improved endothelium-dependent vasorelaxation and increased vascular cyclic guanosine monophosphate (cGMP) content. Together these data suggest that inhibition of NFκB activation may protect endothelial function by inhibiting the pro-inflammatory and pro-thrombotic activities of SAA.
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Aorta/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Leucocitos/metabolismo , FN-kappa B/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animales , Aorta/patología , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Biomarcadores , Adhesión Celular , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Mediadores de Inflamación , Leucocitos/inmunología , RatasRESUMEN
Supplemental oxygen (O2) increases the risk of lung injury in preterm infants, owing to an immature antioxidant system. Our objective was to determine whether impairing antioxidant defense by decreasing glutathione peroxidase 1 (GPx1) gene expression increases the injurious effects of hyperoxia (Hyp). GPx1+/+ and GPx1-/- C57Bl/6J mice were exposed to 21% O2 (Air) or 40% O2 (Hyp) from birth to postnatal day 7 (P7d); they were euthanized on P7d or maintained in air until adulthood [postnatal day 56 (P56d)] to assess short-term and long-term effects, respectively. We assessed lung architecture, three markers of pulmonary oxidative stress (P7d, P56d), macrophages in lung tissue (P7d), immune cells in bronchoalveolar lavage fluid (BALF; P56d), and GPx1-4 and catalase gene expression in lung tissue (P7d, P56d). On P7d, macrophages were decreased by lack of GPx1 expression and further decreased by hyperoxia. GPx1 expression was increased in GPx1+/+Hyp mice and decreased in both GPx1-/- groups. On P56d, heme oxygenase-1 was increased by hyperoxia when GPx1 was absent. There were significantly more immune cells from Hyp groups than from the GPx1+/+Air group and a greater proportion of lymphocytes in GPx1-/-Hyp mice. GPx1 expression was significantly decreased in GPx1-/- mice; GPx2-4 and catalase expression was increased in GPx1-/-Hyp mice compared with other groups. Tissue fraction was decreased in GPx1-/-Air mice; bronchiolar smooth muscle was decreased in GPx1-/- mice. GPx1 does not clearly exacerbate hyperoxia-induced increases in oxidative stress or lung injury but may alter pulmonary immune function. Increased expression of GPx2-4 and catalase in GPx1-/-Hyp mice suggests gene redundancy within the model.
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Progresión de la Enfermedad , Regulación Enzimológica de la Expresión Génica , Glutatión Peroxidasa/genética , Hiperoxia/enzimología , Hiperoxia/genética , Lesión Pulmonar/enzimología , Lesión Pulmonar/genética , Aldehídos/metabolismo , Animales , Animales Recién Nacidos , Antioxidantes/metabolismo , Femenino , Glutatión Peroxidasa/metabolismo , Hemo-Oxigenasa 1/metabolismo , Pulmón/inmunología , Pulmón/patología , Lesión Pulmonar/inmunología , Lesión Pulmonar/patología , Masculino , Ratones Endogámicos C57BL , Estrés Oxidativo , Tirosina/análogos & derivados , Tirosina/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
OBJECTIVES: To study the effect of a lack of antioxidant defenses during lethal pneumonia induced by Klebsiella pneumonia, compared to wild-type mice. SETTING: Laboratory experiments. SUBJECTS: C57Bl6 and glutathione peroxidase 1 knockout mice. INTERVENTION: Murine acute pneumonia model induced by Klebsiella pneumonia. MEASUREMENTS AND MAIN RESULTS: We show here that despite a lack of one of the major antioxidant defense enzymes, glutathione peroxidase 1 knockout mice are protected during lethal pneumonia induced by Klebsiella pneumonia, compared to wild-type mice. Furthermore, this protective effect was suppressed when antioxidant defenses were restored. Infected glutathione peroxidase 1 mice showed an early and significant, albeit transient, increase in the activity of the NOD-like receptor family, pyrin domain containing 3 inflammasome when compared with wild-type mice. The key role of the NOD-like receptor family, pyrin domain containing 3 inflammasome during acute pneumonia was confirmed in vivo when the protective effect was suppressed by treating glutathione peroxidase 1 mice with an interleukin-1 receptor antagonist. Additionally we report, in vitro, that increased concentrations of active caspase-1 and interleukin-1ß are related to an increased concentration of hydrogen peroxide in bacterially infected glutathione peroxidase 1 macrophages and that restoring hydrogen peroxide antioxidant defenses suppressed this effect. CONCLUSIONS: Our findings demonstrate that, contrary to current thinking, an early intervention targeting NOD-like receptor family, pyrin domain containing 3 inflammasome activity induces a timely and efficient activation of the innate immune response during acute infection. Our findings also demonstrate a role for hydrogen peroxide in the mechanisms tightly regulating NOD-like receptor family, pyrin domain containing 3 activation.
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Peróxido de Hidrógeno/metabolismo , Inflamasomas/fisiología , Choque Séptico/fisiopatología , Animales , Antioxidantes/uso terapéutico , Western Blotting , Modelos Animales de Enfermedad , Femenino , Glutatión Peroxidasa/metabolismo , Infecciones por Klebsiella/fisiopatología , Klebsiella pneumoniae , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía Bacteriana/patología , Neumonía Bacteriana/fisiopatología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Choque Séptico/patología , Glutatión Peroxidasa GPX1RESUMEN
BACKGROUND: Vascular dysfunction is a pivotal event in the development of diabetes-associated vascular disease. Increased inflammation and oxidative stress are major contributors to vascular dysfunction. Nrf2, a master regulator of several anti-oxidant genes and a suppressor of inflammatory NF-κB, has potential as a target to combat oxidative stress and inflammation. The aim of this study was to investigate the effects of a novel Nrf2 activator, the bardoxolone methyl derivative dh404, on endothelial function in vitro and in vivo. METHODS: dh404 at 3 mg/kg was administered to male Akita mice, an established diabetic mouse model of insulin insufficiency and hyperglycemia, from 6 weeks of age. At 26 weeks of age, vascular reactivity was assessed by wire myography, pro-inflammatory expression was assessed in the aortas by qRT-PCR and immunohistochemistry, and systemic and vascular oxidative stress measurements were determined. Additionally, studies in human aortic endothelial cells (HAECs) derived from normal and diabetic patients in the presence or absence of dh404 included assessment of pro-inflammatory genes by qRT-PCR and western blotting. Oxidative stress was assessed by three methods; L-012, DCFDA and amplex red. Static adhesion assays were performed to determine the leukocyte-endothelial interaction in the presence or absence of dh404. RESULTS: Dh404 significantly attenuated endothelial dysfunction in diabetic Akita mice characterized by reduced contraction in response to phenylephrine and the downregulation of inflammatory genes (VCAM-1, ICAM-1, p65, IL-1ß) and pro-oxidant genes (Nox1 and Nox2). Furthermore, reduced systemic and vascular oxidative stress levels were observed in diabetic Akita mice. dh404 exhibited cytoprotective effects in diabetic HAECs in vitro, reflected by significant upregulation of Nrf2-responsive genes, NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), reduction of oxidative stress markers (O 2·- and H2O2), inhibition of inflammatory genes (VCAM-1 and the p65 subunit of NF-κB) and attenuation of leukocyte-endothelial interactions (P < 0.05 for all in vitro and in vivo parameters; one or two-way ANOVA as appropriate with post hoc testing). CONCLUSION: These studies demonstrate that upregulation of Nrf2 by dh404 represents a novel therapeutic strategy to limit diabetes-associated vascular injury.
Asunto(s)
Diabetes Mellitus/metabolismo , Diabetes Mellitus/prevención & control , Endotelio Vascular/metabolismo , Factor 2 Relacionado con NF-E2/agonistas , Factor 2 Relacionado con NF-E2/biosíntesis , Ácido Oleanólico/análogos & derivados , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatología , Células Cultivadas , Diabetes Mellitus/fisiopatología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ácido Oleanólico/farmacología , Ácido Oleanólico/uso terapéutico , Técnicas de Cultivo de ÓrganosRESUMEN
An imbalance in oxidative stress and antioxidant defense mechanisms contributes to the development of ischaemic retinopathies such as diabetic retinopathy and retinopathy of prematurity (ROP). Currently, the therapeutic utility of targeting key transcription factors to restore this imbalance remains to be determined. We postulated that dh404, an activator of nuclear factor erythroid-2 related factor 2 (Nrf2), the master regulator of oxidative stress responses, would attenuate retinal vasculopathy by mechanisms involving protection against oxidative stress-mediated damage to glia. Oxygen-induced retinopathy (OIR) was induced in neonatal C57BL/6J mice by exposure to hyperoxia (phase I) followed by room air (phase II). dh404 (1 mg/kg/every second day) reduced the vaso-obliteration of phase I OIR and neovascularization, vascular leakage and inflammation of phase II OIR. In phase I, the astrocytic template and vascular endothelial growth factor (VEGF) expression necessary for physiological angiogenesis are compromised resulting in vaso-obliteration. These events were attenuated by dh404 and related to dh404's ability to reduce the hyperoxia-induced increase in reactive oxygen species (ROS) and markers of cell damage as well as boost the Nrf2-responsive antioxidants in cultured astrocytes. In phase II, neovascularization and vascular leakage occurs following gliosis of Müller cells and their subsequent increased production of angiogenic factors. dh404 reduced Müller cell gliosis and vascular leakage in OIR as well as the hypoxia-induced increase in ROS and angiogenic factors with a concomitant increase in Nrf2-responsive antioxidants in cultured Müller cells. In conclusion, agents such as dh404 that reduce oxidative stress and promote antioxidant capacity offer a novel approach to lessen the vascular and glial cell damage that occurs in ischaemic retinopathies.
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Antioxidantes/farmacología , Células Ependimogliales/efectos de los fármacos , Factor 2 Relacionado con NF-E2/agonistas , Ácido Oleanólico/análogos & derivados , Estrés Oxidativo/efectos de los fármacos , Retina/efectos de los fármacos , Neovascularización Retiniana/prevención & control , Retinopatía de la Prematuridad/prevención & control , Proteínas Angiogénicas/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Ependimogliales/metabolismo , Células Ependimogliales/patología , Hiperoxia/complicaciones , Mediadores de Inflamación/metabolismo , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , Ácido Oleanólico/farmacología , Ratas Sprague-Dawley , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/etiología , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Retinopatía de la Prematuridad/etiología , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Oxidative stress is an important contributor to glial and vascular cell damage in ischemic retinopathies. We hypothesized that ebselen via its ability to reduce reactive oxygen species (ROS) and augment nuclear factor-like 2 (Nrf2) anti-oxidants would attenuate hypoxia-induced damage to macroglial Müller cells and also lessen retinal vasculopathy. Primary cultures of rat Müller cells were exposed to normoxia (21% O2), hypoxia (0.5% O2) and ebselen (2.5 µM) for up to 72 h. Oxygen-induced retinopathy (OIR) was induced in C57BL/6J mice while control mice were housed in room air. Mice received vehicle (saline, 5% dimethyl sulfoxide) or ebselen (10 mg/kg) each day between postnatal days 6-18. In cultured Müller cells, flow cytometry for dihydroethidium revealed that ebselen reduced the hypoxia-induced increase in ROS levels, whilst increasing the expression of Nrf2-regulated anti-oxidant genes, heme oxygenase 1, glutathione peroxidase-1, NAD(P)H dehydrogenase quinone oxidoreductase 1 and glutamate-cysteine ligase. Moreover, in Müller cells, ebselen reduced the hypoxia-induced increase in protein levels of pro-angiogenic and pro-inflammatory factors including vascular endothelial growth factor, interleukin-6, monocyte chemoattractant-protein 1 and intercellular adhesion molecule-1, and the mRNA levels of glial fibrillary acidic protein (GFAP), a marker of Müller cell injury. Ebselen improved OIR by attenuating capillary vaso-obliteration and neovascularization and a concomitant reduction in Müller cell gliosis and GFAP. We conclude that ebselen protects against hypoxia-induced injury of retinal Müller cells and the microvasculature, which is linked to its ability to reduce oxidative stress, vascular damaging factors and inflammation. Agents such as ebselen may be potential treatments for retinopathies that feature oxidative stress-mediated damage to glia and the microvasculature.
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Antioxidantes/farmacología , Azoles/farmacología , Células Ependimogliales/efectos de los fármacos , Gliosis/tratamiento farmacológico , Hipoxia/metabolismo , Compuestos de Organoselenio/farmacología , Estrés Oxidativo/efectos de los fármacos , Degeneración Retiniana/prevención & control , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Ependimogliales/metabolismo , Citometría de Flujo , Proteína Ácida Fibrilar de la Glía , Gliosis/metabolismo , Isoindoles , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Degeneración Retiniana/metabolismo , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/prevención & control , Vasos Retinianos/efectos de los fármacos , Superóxidos/metabolismo , Lesiones del Sistema Vascular/prevención & controlRESUMEN
Despite the wealth of pre-clinical support for a role for reactive oxygen and nitrogen species (ROS/RNS) in the aetiology of diabetic complications, enthusiasm for antioxidant therapeutic approaches has been dampened by less favourable outcomes in large clinical trials. This has necessitated a re-evaluation of pre-clinical evidence and a more rational approach to antioxidant therapy. The present review considers current evidence, from both pre-clinical and clinical studies, to address the benefits of antioxidant therapy. The main focus of the present review is on the effects of direct targeting of ROS-producing enzymes, the bolstering of antioxidant defences and mechanisms to improve nitric oxide availability. Current evidence suggests that a more nuanced approach to antioxidant therapy is more likely to yield positive reductions in end-organ injury, with considerations required for the types of ROS/RNS involved, the timing and dosage of antioxidant therapy, and the selective targeting of cell populations. This is likely to influence future strategies to lessen the burden of diabetic complications such as diabetes-associated atherosclerosis, diabetic nephropathy and diabetic retinopathy.
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Vasos Sanguíneos/metabolismo , Angiopatías Diabéticas/metabolismo , Nefropatías Diabéticas/metabolismo , Riñón/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/uso terapéutico , Azoles/uso terapéutico , Vasos Sanguíneos/efectos de los fármacos , Angiopatías Diabéticas/tratamiento farmacológico , Angiopatías Diabéticas/etiología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/etiología , Retinopatía Diabética/tratamiento farmacológico , Retinopatía Diabética/etiología , Retinopatía Diabética/metabolismo , Diseño de Fármacos , Activación Enzimática , Activadores de Enzimas/uso terapéutico , Humanos , Isoindoles , Riñón/efectos de los fármacos , Terapia Molecular Dirigida , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Compuestos de Organoselenio/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Péptidos/uso terapéuticoRESUMEN
BACKGROUND: In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. METHODS AND RESULTS: Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E-deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E-deficient mice with GKT137831 attenuated atherosclerosis development. CONCLUSIONS: These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.
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Aterosclerosis/enzimología , Aterosclerosis/etiología , Diabetes Mellitus Experimental/enzimología , NADH NADPH Oxidorreductasas/fisiología , NADPH Oxidasas/fisiología , Animales , Aterosclerosis/patología , Células Cultivadas , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Células Endoteliales/enzimología , Células Endoteliales/patología , Humanos , Mediadores de Inflamación/fisiología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , NADPH Oxidasa 1 , Técnicas de Cultivo de Órganos , Isoformas de Proteínas/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The replacement, refinement, and reduction (3Rs) guidelines are the cornerstone of animal welfare practice for medical research. Nowadays, no animal research can be performed without being approved by an animal ethics committee. Therefore, we should expect that any published article would respect and promote the highest standard of animal welfare. However, in the previous issue of Critical Care, Bara and Joffe reported an unexpected finding: animal welfare is extremely poorly reported in critical care research publications involving animal models.This may have a significant negative impact on the reliability of the results and on future funding for our research.The ability of septic shock animal models to translate into clinical studies has been a challenge. Therefore, every means to improve the quality of these models should be pursued. Animal welfare issues should be seen as an additional benefit to achieve this goal. It is therefore critical to draw conclusions from this study to improve the standard of animal welfare in critical care research. This has already been achieved in other fields of research, and we should follow their example.
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Experimentación Animal/ética , Bienestar del Animal/ética , Cuidados Críticos/ética , AnimalesRESUMEN
In this study, a wound dressing of electrospun polycaprolactone (PCL) fibers incorporating the antimicrobial peptide (AMP) nisin was fabricated. Nisin was physically adsorbed to the PCL fibers and tested for antibacterial activity against both Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). The PCL fibers had an average diameter of 1.16 µm ± 0.42 µm and no significant change in diameter occurred after nisin adsorption. X-ray photoelectron spectroscopy (XPS) analysis of the fibers detected nitrogen indicative of adsorbed nisin and the signal was used to quantify the levels of coverage on the fiber surfaces. In vitro nisin release studies showed a burst release profile with 80 % of the nisin being released from the fibers within 30 min. Air plasma pre-treatment of the PCL fibers to render them hydrophilic improved nisin loading and release. Antibacterial testing was performed using minimum inhibitory concentration (MIC) and surface attachment assays. The released nisin remained active against both Gram positive S. aureus and Gram negative P. aeruginosa, which has previously been difficult to achieve with single polymer fiber systems. Mammalian cell culture of the nisin coated fibers with L-929 mouse fibroblasts and human epidermal keratinocytes (HEKa) showed that the nisin did not have a significant effect on the biocompatibility of the PCL fibers. The results presented here demonstrate that the physical adsorption, which is a post-treatment, overcomes the potential limitations of harsh chemicals and fabrication conditions of electrospinning from organic solvents and provides a drug loading system having effective antibacterial properties in wound dressings.
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Nisina , Infecciones Estafilocócicas , Ratones , Animales , Humanos , Nisina/farmacología , Nisina/química , Staphylococcus aureus , Pseudomonas aeruginosa , Antibacterianos/farmacología , Antibacterianos/química , MamíferosRESUMEN
Diabetes mellitus (DM) is an independent risk factor for micro- and macrovascular complications such as nephropathy and atherosclerosis respectively, which are the major causes of premature morbidity and mortality in Type 1 and Type 2 diabetic patients. Endothelial dysfunction is the critical first step of vascular disease and is characterized by reduced bioavailability of the essential endothelial vasodilator, nitric oxide (NO), coupled with an elevation in inflammation and oxidative stress. A novel pathway to bolster NO activity is to upregulate soluble guanylate cyclase (sGC), an enzyme responsible for mediating the protective actions of NO. Two classes of sGC modulators exist, activators and stimulators, with differing sensitivity to oxidative stress. In this study, we investigated the therapeutic effects of the sGC stimulator BAY 41-2272 (Bay 41) and the sGC activator BAY 60-2770 (Bay 60) on endpoints of atherosclerosis and renal disease as well as inflammation and oxidative stress in diabetic Apolipoprotein E knockout (ApoE-/-) mice. We hypothesized that under oxidative conditions known to accompany diabetes, sGC activation might be more efficacious than sGC stimulation in limiting diabetic vascular complications. We demonstrate that Bay 60 not only significantly decreased nitrotyrosine staining (P < 0.01) and F4/80 positive cells by 75% (P < 0.05), but it also significantly reduced total plaque area (P < 0.05) and improved endothelial function (P < 0.01). Our data suggest an important anti-atherogenic role for Bay 60 accompanied by reduced oxidative stress and inflammation under diabetic settings. Treatment with the stimulator Bay 41, on the other hand, had minimal effects or caused no changes with respect to cardiovascular or renal pathology. In the kidneys, treatment with Bay 60 significantly lessened urinary albuminuria, mesangial expansion and nitrotyrosine staining under diabetic conditions. In summary, our head-to-head comparator is the first preclinical study to show that a sGC activator is more efficacious than a sGC stimulator for the treatment of diabetes-associated vascular and renal complications.
RESUMEN
Epidemiologic data suggest that the prevalence of hypertension in patients with diabetes mellitus is â¼1.5-2.0 times greater than in matched non-diabetic patients. This co-existent disease burden exacerbates cardiac and vascular injury, leading to structural and functional changes to the myocardium, impaired cardiac function and heart failure. Oxidative stress and persistent low-grade inflammation underlie both conditions, and are identified as major contributors to pathological cardiac remodelling. There is an urgent need for effective therapies that specifically target oxidative stress and inflammation to protect against cardiac remodelling. Animal models are a valuable tool for testing emerging therapeutics, however, there is a notable lack of appropriate animal models of co-morbid diabetes and hypertension. In this study, we describe a novel preclinical mouse model combining diabetes and hypertension to investigate cardiac and vascular pathology of co-morbid disease. Type 1 diabetes was induced in spontaneously hypertensive, 8-week old, male Schlager (BPH/2) mice via 5 consecutive, daily injections of streptozotocin (55 mg/kg in citrate buffer; i.p.). Non-diabetic mice received citrate buffer only. After 10 weeks of diabetes induction, cardiac function was assessed by echocardiography prior to post-mortem evaluation of cardiomyocyte hypertrophy, interstitial fibrosis and inflammation by histology, RT-PCR and flow cytometry. We focussed on the oxidative and inflammatory stress pathways that contribute to cardiovascular remodelling. In particular, we demonstrate that markers of inflammation (monocyte chemoattractant protein; MCP-1), oxidative stress (urinary 8-isoprostanes) and fibrosis (connective tissue growth factor; CTGF) are significantly increased, whilst diastolic dysfunction, as indicated by prolonged isovolumic relaxation time (IVRT), is elevated in this diabetic and hypertensive mouse model. In summary, this pre-clinical mouse model provides researchers with a tool to test therapeutic strategies unique to co-morbid diabetes and hypertension, thereby facilitating the emergence of novel therapeutics to combat the cardiovascular consequences of these debilitating co-morbidities.
Asunto(s)
Diabetes Mellitus , Cardiomiopatías Diabéticas , Hipertensión , Masculino , Ratones , Animales , Remodelación Ventricular , Miocardio/metabolismo , Hipertensión/patología , Modelos Animales de Enfermedad , Estrés Oxidativo , Fibrosis , Inflamación/patología , Morbilidad , Citratos/farmacología , Cardiomiopatías Diabéticas/patología , Diabetes Mellitus/metabolismoRESUMEN
New medicines are urgently required to treat the fatal neuromuscular disease Duchenne muscular dystrophy (DMD). Dimethyl fumarate (DMF) is a potent immunomodulatory small molecule nuclear erythroid 2-related factor 2 activator with current clinical utility in the treatment of multiple sclerosis and psoriasis that could be effective for DMD and rapidly translatable. Here, we tested 2 weeks of daily 100 mg/kg DMF versus 5 mg/kg standard-care prednisone (PRED) treatment in juvenile mdx mice with early symptomatic DMD. Both drugs modulated seed genes driving the DMD disease program and improved force production in fast-twitch muscle. However, only DMF showed pro-mitochondrial effects, protected contracting muscles from fatigue, improved histopathology, and augmented clinically compatible muscle function tests. DMF may be a more selective modulator of the DMD disease program than PRED, warranting follow-up longitudinal studies to evaluate disease-modifying impact.
Asunto(s)
Dimetilfumarato , Distrofia Muscular de Duchenne , Animales , Ratones , Ratones Endogámicos mdx , Dimetilfumarato/farmacología , Dimetilfumarato/uso terapéutico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Prednisona , Músculos/patologíaRESUMEN
Diabetes is a chronic metabolic disorder associated with the accelerated development of macrovascular (atherosclerosis and coronary artery disease) and microvascular complications (nephropathy, retinopathy and neuropathy), which remain the principal cause of mortality and morbidity in this population. Current understanding of cellular and molecular pathways of diabetes-driven vascular complications, as well as therapeutic interventions has arisen from studying disease pathogenesis in animal models. Diabetes-associated vascular complications are multi-faceted, involving the interaction between various cellular and molecular pathways. Thus, the choice of an appropriate animal model to study vascular pathogenesis is important in our quest to identify innovative and mechanism-based targeted therapies to reduce the burden of diabetic complications. Herein, we provide up-to-date information on available mouse models of both Type 1 and Type 2 diabetic vascular complications as well as experimental analysis and research outputs. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.
Asunto(s)
Aterosclerosis , Enfermedades Cardiovasculares , Enfermedad de la Arteria Coronaria , Diabetes Mellitus , Angiopatías Diabéticas , Animales , Angiopatías Diabéticas/etiología , Modelos Animales de Enfermedad , RatonesRESUMEN
Stents are lifesaving mechanical devices that re-establish essential blood flow to the coronary circulation after significant vessel occlusion due to coronary vessel disease or thrombolytic blockade. Improvements in stent surface engineering over the last 20 years have seen significant reductions in complications arising due to restenosis and thrombosis. However, under certain conditions such as diabetes mellitus (DM), the incidence of stent-mediated complications remains 2-4-fold higher than seen in non-diabetic patients. The stents with the largest market share are designed to target the mechanisms behind neointimal hyperplasia (NIH) through anti-proliferative drugs that prevent the formation of a neointima by halting the cell cycle of vascular smooth muscle cells (VSMCs). Thrombosis is treated through dual anti-platelet therapy (DAPT), which is the continual use of aspirin and a P2Y12 inhibitor for 6-12 months. While the most common stents currently in use are reasonably effective at treating these complications, there is still significant room for improvement. Recently, inflammation and redox stress have been identified as major contributing factors that increase the risk of stent-related complications following percutaneous coronary intervention (PCI). The aim of this review is to examine the mechanisms behind inflammation and redox stress through the lens of PCI and its complications and to establish whether tailored targeting of these key mechanistic pathways offers improved outcomes for patients, particularly those where stent placement remains vulnerable to complications. In summary, our review highlights the most recent and promising research being undertaken in understanding the mechanisms of redox biology and inflammation in the context of stent design. We emphasize the benefits of a targeted mechanistic approach to decrease all-cause mortality, even in patients with diabetes.