RESUMEN
Elevated serum amyloid A (SAA) levels may promote endothelial dysfunction, which is linked to cardiovascular and renal pathologies. We investigated the effect of SAA on vascular and renal function in apolipoprotein E-deficient (ApoE-/-) mice. Male ApoE-/- mice received vehicle (control), low-level lipopolysaccharide (LPS), or recombinant human SAA by i.p. injection every third day for 2 weeks. Heart, aorta and kidney were harvested between 3 days and 18 weeks after treatment. SAA administration increased vascular cell adhesion molecule (VCAM)-1 expression and circulating monocyte chemotactic protein (MCP)-1 and decreased aortic cyclic guanosine monophosphate (cGMP), consistent with SAA inhibiting nitric oxide bioactivity. In addition, binding of labeled leukocytes to excised aorta increased as monitored using an ex vivo leukocyte adhesion assay. Renal injury was evident 4 weeks after commencement of SAA treatment, manifesting as increased plasma urea, urinary protein, oxidized lipids, urinary kidney injury molecule (KIM)-1 and multiple cytokines and chemokines in kidney tissue, relative to controls. Phosphorylation of nuclear-factor-kappa-beta (NFκB-p-P65), tissue factor (TF), and macrophage recruitment increased in kidneys from ApoE-/- mice 4 weeks after SAA treatment, confirming that SAA elicited a pro-inflammatory and pro-thrombotic phenotype. These data indicate that SAA impairs endothelial and renal function in ApoE-/- mice in the absence of a high-fat diet.
Asunto(s)
Vasos Sanguíneos/metabolismo , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Aorta/fisiopatología , Apolipoproteínas E/deficiencia , Biomarcadores , Vasos Sanguíneos/patología , Vasos Sanguíneos/fisiopatología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Células Endoteliales/metabolismo , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Pruebas de Función Renal , Lípidos/sangre , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Peroxidasa/metabolismo , Proteína Amiloide A Sérica/metabolismoRESUMEN
Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a large enhancement of fluorescence at the K(α) line for Se (11.2-keV) following treatment with ebselen (10 µM) at time periods from 60 to 240 min. Drug uptake was quantified and ebselen was shown to induce time-dependent changes in cellular elemental content that were characteristic of oxidative stress with the efflux of K, Cl, and Ca species. The SRIXE cellular Se distribution map revealed that ebselen was predominantly localized to a discreet region of the cell which, by comparison with the K and P elemental maps, is postulated to correspond to the endoplasmic reticulum. On the basis of these findings, it is hypothesized that a major outcome of ebselen redox catalysis is the induction of cellular stress. A mechanism of action of ebselen is proposed that involves the cell responding to drug-induced stress by increasing the expression of antioxidant genes. This hypothesis is supported by the observation that ebselen also regulated the homeostasis of the transition metals Mn, Cu, Fe, and Zn, with increases in transition metal uptake paralleling known induction times for the expression of antioxidant metalloenzymes.
Asunto(s)
Antioxidantes/farmacología , Azoles/química , Azoles/farmacología , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Sincrotrones , Animales , Antioxidantes/química , Línea Celular , Isoindoles , Ratones , Estructura Molecular , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ratas , Espectrometría por Rayos X , Células Tumorales CultivadasRESUMEN
The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25µg/ml) decreased nitric oxide ((â¢)NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10µg/ml) stimulated a Ca(2+) influx linked to apocynin-sensitive superoxide radical anion (O(2)(â¢-)) production. Gene expression for arginase-1, nuclear factor κB (NF-κB), interleukin-8, and tissue factor (TF) increased within 4h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24h. Therefore, in addition to modulating (â¢)NO bioavailability by stimulating O(2)(â¢-) production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, µg/ml) before (not after) SAA treatment ameliorated the Ca(2+) influx and O(2)(â¢-) production; decreased TF, NF-κB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating (â¢)NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.
Asunto(s)
Aorta/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Lipoproteínas HDL/uso terapéutico , Proteína Amiloide A Sérica/efectos adversos , Superóxidos/antagonistas & inhibidores , Animales , Aorta/metabolismo , Aorta/patología , Arginasa/genética , Arginasa/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/patología , Western Blotting , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Expresión Génica , Humanos , Inflamación/metabolismo , Inflamación/patología , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacología , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Óxido Nítrico/análisis , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/farmacología , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo , Tromboplastina/genética , Tromboplastina/metabolismoRESUMEN
Cultured neurons tolerate low H(2)O(2) concentrations (< or =50 microM) through the activity of constitutive antioxidant response elements (ARE). At H(2)O(2) levels (> or =100 microM), neurons increase expression of the gene encoding for inducible hemoxygenase-1 while superoxide dismutase-2 and catalase remain unchanged. Despite this adaptive response, the endogenous antioxidant systems are overwhelmed, leading to decreased viability. Elevating the neuronal cell content of human neuroglobin (Ngb) prior to insult with 100 or 200 microM H(2)O(2) enhanced cell viability and this resulted in a significant decrease in oxidative stress and an increase in the intracellular ATP concentration, whereas in parental cells exposed to the same H(2)O(2)-insult, oxidative stress and ATP increased and decreased, respectively. The mechanism for this increase in ATP involves sustained activation of the mito-K(ATP) channel and an increase in phosphoinositide-3 kinase (PI3K)-mediated phosphorylation of Akt. Pharmacological inhibitors directed toward PI3K (wortmannin and LY294002), or the mito-K(ATP) channel (glybenclamide) inhibited the H(2)O(2)-mediated increase in ATP in cells overexpressing human Ngb and consequently cell viability decreased. Neuroglobin's ability to bolster the intracellular pool of ATP in response to added H(2)O(2) is central to the preservation of cytoskeletal integrity and cell viability.