RESUMEN
Ferroptosis is a regulated form of cell death, the mechanism of which is still to be understood. 15-lipoxygenase (15LOX) complex with phosphatidylethanolamine (PE)-binding protein 1 (PEBP1) catalyzes the generation of pro-ferroptotic cell death signals, hydroperoxy-polyunsaturated PE. We focused on gaining new insights into the molecular basis of these pro-ferroptotic interactions using computational modeling and liquid chromatography-mass spectrometry experiments. Simulations of 15LOX-1/PEBP1 complex dynamics and interactions with lipids revealed that association with the membrane triggers a conformational change in the complex. This conformational change facilitates the access of stearoyl/arachidonoyl-PE (SAPE) substrates to the catalytic site. Furthermore, the binding of SAPE promotes tight interactions within the complex and induces further conformational changes that facilitate the oxidation reaction. The reaction yields two hydroperoxides as products, 15-HpETE-PE and 12-HpETE-PE, at a ratio of 5:1. A significant effect of PEBP1 is observed only on the predominant product. Moreover, combined experiments and simulations consistently demonstrate the significance of PEBP1 P112E mutation in generating ferroptotic cell death signals.
Asunto(s)
Araquidonato 15-Lipooxigenasa , Ferroptosis , Proteínas de Unión a Fosfatidiletanolamina , Muerte Celular , Ferroptosis/fisiología , Oxidación-Reducción , Araquidonato 15-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/fisiología , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/fisiología , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Humanos , Animales , PorcinosRESUMEN
BACKGROUND: The epithelium is increasingly recognized as a pathologic contributor to asthma and its phenotypes. Although delayed wound closure by asthmatic epithelial cells is consistently observed, underlying mechanisms remain poorly understood, partly due to difficulties in studying dynamic physiologic processes involving polarized multilayered cell systems. Although type-2 immunity has been suggested to play a role, the mechanisms by which repair is diminished are unclear. OBJECTIVES: This study sought to develop and utilize primary multilayered polarized epithelial cell systems, derived from patients with asthma, to evaluate cell migration in response to wounding under type-2 and untreated conditions. METHODS: A novel wounding device for multilayered polarized cells, along with time-lapse live cell/real-time confocal imaging were evaluated under IL-13 and untreated conditions. The influence of inhibition of 15 lipoxygenase (15LO1), a type-2 enzyme, on the process was also addressed. Cell migration patterns were analyzed by high-dimensional frequency modulated Möbius for statistical comparisons. RESULTS: IL-13 stimulation negatively impacts wound healing by altering the total speed, directionality, and acceleration of individual cells. Inhibition 15LO1 partially improved the wound repair through improving total speed. CONCLUSIONS: Migration abnormalities contributed to markedly slower wound closure of IL-13 treated cells, which was modestly reversed by 15LO1 inhibition, suggesting its potential as an asthma therapeutic target. These novel methodologies offer new ways to dynamically study cell movements and identify contributing pathologic processes.
Asunto(s)
Asma/etiología , Araquidonato 15-Lipooxigenasa/fisiología , Asma/diagnóstico por imagen , Asma/tratamiento farmacológico , Asma/inmunología , Movimiento Celular , Células Cultivadas , Células Epiteliales/fisiología , Humanos , Interleucina-13/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
To rigorously explore the role of omega-3 polyunsaturated fatty acids (PUFA) in the treatment of diabetic peripheral neuropathy (DPN), we have created a transgenic mouse utilizing a Cre-lox promoter to control overexpression of human 15-lipoxygenase-1 (15-LOX-1). In this study, we sought to determine the effect of treating type 2 diabetic wild-type mice and transgenic mice ubiquitously overexpressing 15-LOX-1 with menhaden oil on endpoints related to DPN. Wild-type and transgenic mice on a C57Bl/6J background were divided into three groups. Two of each of these groups were used to create a high-fat diet/streptozotocin model for type 2 diabetes. The remaining mice were control groups. Four weeks later, one set of diabetic mice from each group was treated with menhaden oil for twelve weeks and then evaluated using DPN-related endpoints. Studies were also performed using dorsal root ganglion neurons isolated from wild-type and transgenic mice. Wild-type and transgenic diabetic mice developed DPN as determined by slowing of nerve conduction velocity, decreased sensory nerve fibers in the skin and cornea, and impairment of thermal and mechanical sensitivity of the hindpaw compared to their respective control mice. Although not significant, there was a trend for the severity of these DPN-related deficits to be less in the diabetic transgenic mice compared to the diabetic wild-type mice. Treating diabetic wild-type and transgenic mice with menhaden oil improved the DPN-related endpoints with a trend for greater improvement or protection by menhaden oil observed in the diabetic transgenic mice. Treating dorsal root ganglion neurons with docosahexanoic acid but not eicosapentaenoic acid significantly increased neurite outgrowth with greater efficacy observed with neurons isolated from transgenic mice. Targeting pathways that will increase the production of the anti-inflammatory metabolites of omega-3 PUFA may be an efficacious approach to developing an effective treatment for DPN.
Asunto(s)
Araquidonato 15-Lipooxigenasa/fisiología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Neuropatías Diabéticas/tratamiento farmacológico , Aceites de Pescado/uso terapéutico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Animales , Araquidonato 15-Lipooxigenasa/genética , Diabetes Mellitus Tipo 2/complicaciones , Neuropatías Diabéticas/etiología , Ácidos Docosahexaenoicos/sangre , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades del Sistema Nervioso Periférico/etiologíaRESUMEN
Arachidonic acid 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes, which has previously been implicated in the maturational breakdown of intracellular organelles and plasma membrane remodeling during reticulocyte-erythrocyte transition. Conventional Alox15-/- mice are viable, develop normally but do not exhibit a major defective erythropoietic phenotype. To characterize the putative in vivo relevance of Alox15 for red blood cell development, we explored the impact of systemic inactivation of the Alox15 gene on mouse erythropoiesis. We found that Alox15-/- mice exhibited reduced erythrocyte counts, elevated reticulocyte counts and red cell hyperchromia. The structure of the plasma membrane of Alox15-/- erythrocytes is altered and a significant share of the red cells was present as echinocytes and/or acanthocytes. An increased share of the Alox15-/- erythrocytes cells were annexin V positive, which indicates a loss of plasma membrane asymmetry. Erythrocytes of Alox15-/- mice were more susceptible to osmotic hemolysis and exhibited a reduced ex vivo life span. When we transgenically expressed human ALOX15 in Alox15-/- mice under the control of the aP2 promoter the defective erythropoietic system was rescued and the impaired osmotic resistance was normalized. Together these data suggest the involvement Alox15 in the maturational remodeling of the plasma membrane during red cell development.
Asunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/administración & dosificación , Araquidonato 15-Lipooxigenasa/fisiología , Eritropoyesis , Hiperpigmentación/prevención & control , Reticulocitosis , Transgenes , Animales , Hemólisis , Hiperpigmentación/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FenotipoRESUMEN
Lipid mediators play important roles in regulating inflammatory responses and tissue homeostasis. Since 12/15-lipoxygenase (12/15-LOX)-derived lipid mediators such as lipoxin A4 (LXA4 ) and protectin D1 (PD1) protect against corneal epithelial cell damage, the major cell types that express 12/15-LOX and contribute to the corneal wound healing process are of particular interest. Here, we found that eosinophils were the major cell type expressing 12/15-LOX during the corneal wound healing process. Eosinophils were recruited into the conjunctiva after corneal epithelium wounding, and eosinophil-deficient and/or eosinophil-specific 12/15-LOX knockout mice showed delayed corneal wound healing compared with wild-type mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based mediator lipidomics revealed that a series of 12/15-LOX-derived mediators were significantly decreased in eosinophil-deficient mice and topical application of 17-hydroxydocosahexaenoic acid (17-HDoHE), a major 12/15-LOX-derived product, restored the phenotype. These results indicate that 12/15-LOX-expressing eosinophils, by locally producing pro-resolving mediators, significantly contribute to the corneal wound healing process in the eye.
Asunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Lesiones de la Cornea/patología , Eosinófilos/citología , Cicatrización de Heridas , Animales , Córnea/patología , Eosinófilos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.
Asunto(s)
Arginasa/biosíntesis , Citocromo P-450 CYP1A1/fisiología , Janus Quinasa 1/antagonistas & inhibidores , Macrófagos Peritoneales/efectos de los fármacos , Peritonitis/prevención & control , Factor de Transcripción STAT6/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacología , Traslado Adoptivo , Animales , Araquidonato 15-Lipooxigenasa/fisiología , Arginasa/genética , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , Endotoxinas/toxicidad , Humanos , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Ácidos Hidroxieicosatetraenoicos/genética , Ácidos Hidroxieicosatetraenoicos/farmacología , Interleucina-4/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/trasplante , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Peritonitis/inducido químicamente , Células RAW 264.7 , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/genética , Células THP-1 , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NOâ¢-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO⢠donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO⢠donors and/or suppression of NO⢠production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.
Asunto(s)
Ferroptosis/fisiología , Macrófagos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Araquidonato 15-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/fisiología , Muerte Celular , Femenino , Hierro/metabolismo , Hierro/fisiología , Leucotrienos/metabolismo , Peroxidación de Lípido/fisiología , Peróxidos Lipídicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Óxido Nítrico Sintasa de Tipo II/fisiología , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
PURPOSE: Lipoxygenases (LOX) have been implicated in carcinogenesis, however both pro- and anti-carcinogenic effects have been reported in different cancer models. Using transgenic mice, which specifically overexpress human 15-lipoxygenase (ALOX15) in endothelial cells (EC), we previously demonstrated significant inhibition of tumor development. In the Lewis lung carcinoma (LLC) model, the primary tumor developed similarly in both wild type (WT) and ALOX15 overexpressing mice. However, metastases development was significantly inhibited in the transgenic mice. Here, we explored the molecular basis for the anti-metastatic effect of endothelial cell specific ALOX15 overexpression. MATERIALS/METHODS: We used ALOX15 overexpressing mice, and in-vitro cell model to evaluate the molecular effect of ALOX15 on EC and LLC cells. RESULTS: When LLC cells were injected in WT and ALOX15 overexpressing mice, we observed a higher degree of apoptosis and necrosis in primary and metastatic tumors of ALOX15 overexpressing animals. These anti-carcinogenic and anti-metastatic effects were paralleled by augmented expression of cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) and of the peroxisome proliferators-activated receptor (PPAR)γ and by downregulation of the steady state concentrations of connexin26 mRNA. Consistent with these in vivo effects, ALOX15 overexpression in LLC and HeLa cancer cells in vitro significantly reduced cell viability in culture. In contrast, similar treatment of non-cancerous B2B epithelial cells did not impact cell viability. CONCLUSIONS: Taken together, our data suggests that endothelial cell specific overexpression of ALOX15 promotes apoptosis and necrosis in primary and metastatic tumors in mice, by upregulation of P21 and PPARγ expression in adjacent cancer cells.
Asunto(s)
Apoptosis , Araquidonato 15-Lipooxigenasa/fisiología , Biomarcadores de Tumor/metabolismo , Carcinoma Pulmonar de Lewis/patología , Modelos Animales de Enfermedad , Células Endoteliales/patología , Animales , Biomarcadores de Tumor/genética , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Proliferación Celular , Células Endoteliales/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Tumorales CultivadasRESUMEN
AIMS/INTRODUCTION: Diabetes is an important risk factor for atherosclerotic disease. The initiating factor of atherosclerosis is local endothelial cell injury. The arachidonic acid metabolite, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), might be involved in this process. In recent years, some studies have discussed the effect of 12(S)-HETE on vascular endothelial cell function. In the present study, we investigated the effect of 12(S)-HETE on vascular endothelial cell function in high-glucose conditions and the mechanisms involved. MATERIALS AND METHODS: Human umbilical vein endothelial cells were cultured in conventional M199 medium and high-glucose M199 medium. Human umbilical vein endothelial cells were stimulated with 12(S)-HETE and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (a 12/15-lipoxygenases inhibitor). A type 1 diabetes mellitus model was established in C57BL/6 or 12/15-lipoxygenases knockout mice with streptozotocin. Aortic tissue was harvested for subsequent testing. The transmembrane transport of dextran and human acute monocytic leukaemia cell line (THP-1) cells was measured. The adherens junction protein, IkBα, nuclear factor kappa Bp65 (P65), intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression and phosphorylation, and the binding/dissociation of endothelial cell components were observed. RESULTS: Transendothelial migration of dextran and THP-1 cells was significantly increased by stimulation of human umbilical vein endothelial cell monolayers with high glucose and 12(S)-HETE (P < 0.05). High glucose and 12(S)-HETE altered the vascular endothelial cadherin and ß-catenin phosphorylation level, and promoted the dissociation of ß-catenin and vascular endothelial cadherin. Expression levels of P-Ikbα, P-P65, intercellular adhesion molecule 1 and vascular cell adhesion protein 1 were elevated in high glucose and 12(S)-HETE treated cells and diabetic mice compared with controls (P < 0.05). CONCLUSIONS: The lipoxygenases metabolite, 12(S)-HETE, can impair vascular endothelial permeability by altering adherens junction phosphorylation levels, and affecting the binding and dissociation of its components in high-glucose conditions.
Asunto(s)
Uniones Adherentes/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Endotelio Vascular/patología , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/toxicidad , Ácidos Hidroxieicosatetraenoicos/toxicidad , Lesiones del Sistema Vascular/etiología , Animales , Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Proteínas Portadoras/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/fisiopatología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Edulcorantes/toxicidad , Factor de Transcripción ReIA/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Lesiones del Sistema Vascular/metabolismo , Lesiones del Sistema Vascular/patologíaRESUMEN
15-lipoxygenase is involved in the generation of specialized pro-resolving lipid mediators that play essential roles in resolution and inflammatory responses. Here, we investigated anti-inflammatory role of Alox15 in skin homeostasis. We demonstrated that knockout (KO) of Alox15 led to hair loss and disrupted the structural integrity of the dorsal skin. Alox15 KO resulted in loss of hair follicle stem cells and abnormal transition of dermal adipocytes into fibroblasts. Alox15 deficiency increased infiltration of proinflammatory macrophages and upregulated proinflammatory and necroptotic signaling in dermal adipose tissue in the dorsal skin. Lipidomic analysis revealed severe loss of resolvin D2 in the dorsal skin of Alox15 KO mice compared to wild type controls. Treatment with resolvin D2 reduced skin inflammation in Alox15 KO mice. Collectively, these results indicate that Alox15-mediated production of resolvin D2 is required to maintain skin integrity by suppressing dermal inflammation.
Asunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Ácidos Docosahexaenoicos/metabolismo , Inflamación , Piel/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Alopecia , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Muerte Celular , Fibroblastos/citología , Técnicas de Inactivación de Genes , Folículo Piloso/patología , Homeostasis , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
OBJECTIVE: 12/15-Lipoxygenase (12/15-LOX) catalyzes the generation of various anti-inflammatory lipid mediators, and has been implicated in several inflammatory and degenerative diseases. However, there is currently no evidence that 12/15-LOX has a role in osteoarthritis (OA). The aim of this study was to investigate the role of 12/15-LOX in the pathogenesis of OA. METHODS: The development of aging-associated and destabilization of the medial meniscus (DMM)-induced OA were compared in 12/15-LOX-deficient (12/15-LOX-/-) and wild-type (WT) mice. The extent of cartilage damage was evaluated by histology. The expression of OA markers was evaluated by immunohistochemistry and RT-PCR. Cartilage explants were stimulated with IL-1α in the absence or presence of the 12/15-LOX metabolites, 15-hydroxyeicosatetraenoic acids (15-HETE), 13-hydroxyoctadecadienoic acid (13-HODE) or lipoxin A4 (LXA4), and the levels of matrix metalloproteinases-13 (MMP-13), Nitric oxide (NO) and prostaglandin E2 (PGE2) were determined. The effect of LXA4 on the progression of OA was evaluated in wild type (WT) mice. RESULTS: The expression of 12/15-LOX in cartilage increased during the progression of DMM-induced OA and with aging in WT mice. Cartilage degeneration was more severe in 12/15-LOX-/- mice compared to WT mice in both models of OA, and this was associated with increased expression of MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs, aggrecanases (ADAMTS5), inducible NO synthases (iNOS), and mPGES-1. Treatment of cartilage explants with 12/15-LOX metabolites, suppressed IL-1α-induced production of MMP-13, NO and PGE2, with LXA4 being the most potent. Intra-peritoneal injection of LXA4 reduced the severity of DMM-induced cartilage degradation. CONCLUSIONS: These data suggest an important role of 12/15-LOX in the pathogenesis of OA. They also suggest that activation of this pathway may provide a novel strategy for prevention and treatment of OA.
Asunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Artritis Experimental/enzimología , Osteoartritis/enzimología , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/genética , Artritis Experimental/etiología , Artritis Experimental/prevención & control , Cartílago Articular/metabolismo , Progresión de la Enfermedad , Mediadores de Inflamación/metabolismo , Inestabilidad de la Articulación/complicaciones , Lipoxinas/uso terapéutico , Masculino , Ratones Noqueados , Osteoartritis/etiología , Osteoartritis/prevención & control , Lesiones de Menisco Tibial/complicaciones , Técnicas de Cultivo de Tejidos , Regulación hacia ArribaRESUMEN
OBJECTIVE In this study, the authors investigated the involvement of 15( S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in the regulation of peroxisome proliferator-activated receptor-γ (PPARγ) after intracerebral hemorrhage (ICH) and its effects on hemorrhage-induced inflammatory response and oxidative stress in an experimental rodent model. METHODS To simulate ICH in a rat model, the authors injected autologous whole blood into the right striatum of male Sprague-Dawley rats. The distribution and expression of 12/15-lipoxygenase (12/15-LOX) were determined by immunohistochemistry and Western blot analysis, respectively. Immunofluorescent double labeling was used to study the cellular localization of 12/15-LOX, and 15(S)-HETE was measured with a 15(S)-HETE enzyme immunoassay kit. Neurological deficits in the animals were assessed through behavioral testing, and apoptotic cell death was determined with terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling. RESULTS Rats with ICH had increased expression of 12/15-LOX predominantly in neurons and also in oligodendrocytes, astrocytes, and microglia. Moreover, ICH elevated production of 15(S)-HETE in the brain area ipsilateral to the blood injection. The PPARγ agonist, exogenous 15(S)-HETE, significantly increased PPARγ protein levels and increased PPARγ-regulated gene (i.e., catalase) expression in the ICH rats. Reduced expression of the gene for the proinflammatory protein nuclear factor κB coincided with decreased neuron damage and improved functional recovery from ICH. A PPARγ antagonist, GW9662, reversed the effects of exogenous 15(S)-HETE on the PPARγ-regulated genes. CONCLUSIONS The induction of 15(S)-HETE during simulated ICH suggests generation of endogenous signals of neuroprotection. The effects of exogenous 15(S)-HETE on brain hemorrhage-induced inflammatory responses and oxidative stress might be mediated via PPARγ.
Asunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Hemorragia Cerebral/metabolismo , Ácidos Hidroxieicosatetraenoicos/fisiología , Neuroprotección , PPAR gamma/fisiología , Animales , Encéfalo , Hemorragia Cerebral/tratamiento farmacológico , Ácidos Hidroxieicosatetraenoicos/farmacología , Inflamación/tratamiento farmacológico , Masculino , Estrés Oxidativo/efectos de los fármacos , PPAR gamma/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Regulación hacia ArribaRESUMEN
BACKGROUND: The 12/15-lipoxygenase (12/15-LO) enzyme is upregulated in the brains of patients with Alzheimer's disease (AD), and its expression levels influence the onset of the AD-like phenotype in mouse models. However, whether targeting this pathway after the neuropathology and behavioral impairments have been established remains to be investigated. METHODS: Triple transgenic (3xTg) mice received either PD146176-a selective and specific pharmacological inhibitor of 12/15-LO-or placebo starting at 12 months of age for 12 weeks. They were then assessed for the effect of the treatment on neuropathologies and behavioral impairments. RESULTS: At the end of the study, mice in the control group showed a worsening of memory and learning abilities, whereas mice receiving PD146176 were undistinguishable from wild-type mice. The same group also had significantly lower amyloid beta levels and deposition, less tau neuropathology, increased synaptic integrity, and autophagy activation. Ex vivo and in vitro genetic and pharmacological studies found that the mechanism involved in these effects was the activation of neuronal autophagy. CONCLUSIONS: Our findings provide new insights into the disease-modifying action of 12/15-LO pharmacological inhibition and establish it as a viable therapeutic approach for patients with AD.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Péptidos beta-Amiloides/metabolismo , Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Autofagia , Encéfalo/enzimología , Disfunción Cognitiva/enzimología , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/psicología , Animales , Autofagia/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , Encefalitis/metabolismo , Miedo/efectos de los fármacos , Miedo/fisiología , Fluorenos/administración & dosificación , Recuerdo Mental/efectos de los fármacos , Recuerdo Mental/fisiología , Ratones , Ratones Transgénicos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Células Tumorales CultivadasRESUMEN
PURPOSE OF INVESTIGATION: To investigate the presence of 15-lipoxygenase-1 (15-LOX-1) expression and its potential role in the pathogenesis of endometrial hyperplasia and endometrial adenocarcinomas. MATERIALS AND METHODS: The authors investigated the presence of 15-LOX-1 expression in samples from patients diagnosed with normal endometrium (n = 12), endometrial hyperplasia (n = 12), and endometrial cancer (n = 12). The immunohistochemical stainings were scored by three independent pathologists. A Western blot of 15- LOX-1 determined the presence of protein expression in normal endometrium. A Kolmogorov-Smirnov test was used to evaluate the data's distribution pattern. For pairwise comparisons of the combined scores between groups, the Mann-Whitney U test was used. RESULTS: Based on the combined scores for 15-LOX-1 expression, strong immunochemistry staining was observed in samples diagnosed with normal endometrium. There was a significant difference in 15-LOX-1 expression between normal endometrium and endometrial adenocarcinoma (p = 0.03). Comparing tissues from normal endometrium and endometrial hyperplasia, there was a decline in the expression from normal endometrium to endometrial hyperplasia. However, the difference was not statistically significant. CONCLUSION: The present results show that a decrease of 15-LOX-1 expression in the endometrial tumorigenesis process, starting from normal endometrium to hyperplasia and endometrial cancer, might be a trigger. Further studies are required to determine its potential use as a marker in a larger randomized multicenter study.
Asunto(s)
Araquidonato 15-Lipooxigenasa/fisiología , Hiperplasia Endometrial/etiología , Neoplasias Endometriales/etiología , Adulto , Anciano , Araquidonato 15-Lipooxigenasa/análisis , Hiperplasia Endometrial/enzimología , Neoplasias Endometriales/enzimología , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana EdadAsunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Quimiotaxis de Leucocito , Ácidos Docosahexaenoicos/fisiología , Ileus/inmunología , Yeyuno/inmunología , Músculo Liso/inmunología , Neutrófilos/inmunología , Complicaciones Posoperatorias/inmunología , AnimalesRESUMEN
Resolution of inflammation is an active counter-regulatory mechanism involving polyunsaturated fatty acid-derived proresolving lipid mediators. Postoperative intestinal motility disturbances, clinically known as postoperative ileus, occur frequently after abdominal surgery and are mediated by a complex inflammation of the intestinal muscularis externa. Herein, we tested the hypothesis that proresolving lipid mediators are involved in the resolution of postoperative ileus. In a standardized experimental model of postoperative ileus, we detected strong expression of 12/15-lipoxygenase within the postoperative muscularis externa of C57BL/6 mice, predominately located within CX3CR1(+)/Ly6C(+) infiltrating monocytes rather than Ly6G(+) neutrophils. Mass spectrometry analyses demonstrated that a 12/15-lipoxygenase increase was accompanied by production of docosahexaenoic acid-derived lipid mediators, particularly protectin DX and resolvin D2, and their common precursor 17-hydroxy docosahexaenoic acid. Perioperative administration of protectin DX, but not resolvin D2 diminished blood-derived leukocyte infiltration into the surgically manipulated muscularis externa and improved the gastrointestinal motility. Flow cytometry analyses showed impaired Ly6G(+)/Ly6C(+) neutrophil extravasation after protectin DX treatment, whereas Ly6G(-)/Ly6C(+) monocyte numbers were not affected. 12/15-lipoxygenase-deficient mice, lacking endogenous protectin DX synthesis, demonstrated increased postoperative leukocyte levels. Preoperative intravenous administration of a docosahexaenoic acid-rich lipid emulsion reduced postoperative leukocyte infiltration in wild-type mice but failed in 12/15-lipoxygenase-deficient mice mice. Protectin DX application reduced leukocyte influx and rescued 12/15-lipoxygenase-deficient mice mice from postoperative ileus. In conclusion, our results show that 12/15-lipoxygenase mediates postoperative ileus resolution via production of proresolving docosahexaenoic acid-derived protectin DX. Perioperative, parenteral protectin DX or docosahexaenoic acid supplementation, as well as modulation of the 12/15-lipoxygenase pathway, may be instrumental in prevention of postoperative ileus.
Asunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Quimiotaxis de Leucocito , Ácidos Docosahexaenoicos/fisiología , Ileus/inmunología , Yeyuno/inmunología , Músculo Liso/inmunología , Neutrófilos/inmunología , Complicaciones Posoperatorias/inmunología , Animales , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/deficiencia , Quimiotaxis de Leucocito/fisiología , Ácidos Docosahexaenoicos/biosíntesis , Ácidos Docosahexaenoicos/deficiencia , Ácidos Docosahexaenoicos/uso terapéutico , Evaluación Preclínica de Medicamentos , Emulsiones , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Grasos Omega-3/uso terapéutico , Motilidad Gastrointestinal/efectos de los fármacos , Ileus/enzimología , Ileus/etiología , Ileus/prevención & control , Inflamación , Yeyuno/metabolismo , Yeyuno/patología , Ratones , Ratones Endogámicos C57BL , Modelos Inmunológicos , Músculo Liso/metabolismo , Músculo Liso/patología , Complicaciones Posoperatorias/enzimología , Complicaciones Posoperatorias/prevención & control , Organismos Libres de Patógenos EspecíficosRESUMEN
BACKGROUND AND PURPOSE: 15-Lipoxygenase (15-LOX) activity is associated with inflammation and immune regulation. The objectives of the present study were to investigate the expression of 15-LOX-1 and 15-LOX-2 and evaluate the enzymes' roles in the polarization of human lung macrophages (LMs) in response to LPS and Th2 cytokines (IL-4/-13). EXPERIMENTAL APPROACH: LMs were isolated from patients undergoing surgery for carcinoma. The cells were cultured with a 15-LOX inhibitor (PD146176 or ML351), a COX inhibitor (indomethacin), a 5-LOX inhibitor (MK886) or vehicle and then stimulated with LPS (10 ng · mL(-1)), IL-4 (10 ng · mL(-1)) or IL-13 (50 ng · mL(-1)) for 24 h. Levels of ALOX15 (15-LOX-1) and ALOX15B (15-LOX-2) transcripts were determined by real-time quantitative PCR. Immunoassays were used to measure levels of LPS-induced cytokines (TNF-α, CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL10) and Th2 cytokine-induced chemokines (CCL13, CCL18 and CCL22) in the culture supernatant. KEY RESULTS: Stimulation of LMs with LPS was associated with increased expression of ALOX15B, whereas stimulation with IL-4/IL-13 induced the expression of ALOX15. PD146176 and ML351 (10 µM) reduced the release of the chemokines induced by LPS and Th2 cytokines. The effects of these 15-LOX inhibitors were maintained in the presence of indomethacin and MK886. Furthermore, indomethacin revealed the inhibitory effect of PD146176 on TNF-α release. CONCLUSIONS AND IMPLICATIONS: Inhibition of the 15-LOX pathways is involved in the down-regulation of the in vitro production of chemokines in LMs. Our results suggest that the 15-LOX pathways have a role in the pathogenesis of inflammatory lung disorders and may thus constitute a potential drug target.
Asunto(s)
Araquidonato 15-Lipooxigenasa/fisiología , Quimiocinas/biosíntesis , Inhibidores de la Lipooxigenasa/farmacología , Macrófagos Alveolares/metabolismo , Anciano , Células Cultivadas , Femenino , Humanos , Macrófagos Alveolares/efectos de los fármacos , Masculino , Persona de Mediana EdadRESUMEN
DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell-driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase-meditated (12/15-LO-mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO-encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO-deficient DCs to 12/15-LO-derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO-deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune encephalomyelitis in mice lacking 12/15-LO resulted in an exacerbated Th17-driven autoimmune disease. Together, our data reveal that 12/15-LO controls maturation of DCs and implicate enzymatic lipid oxidation in shaping the adaptive immune response.
Asunto(s)
Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Células Dendríticas/citología , Inmunidad Adaptativa , Animales , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/genética , Diferenciación Celular , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/enzimología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Ácidos Grasos/metabolismo , Femenino , Humanos , Tejido Linfoide/enzimología , Tejido Linfoide/inmunología , Tejido Linfoide/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Fosfolípidos/metabolismo , Células Th17/inmunologíaRESUMEN
Alzheimer's disease (AD) is a chronic neurodegenerative condition characterized by progressive memory loss. Mutations in genes involved in the production of amyloid-ß (Aß) are linked to the early-onset variant of AD. However, the most common form, sporadic AD, is considered to be the result of an interaction between environmental risk factors and various genes. Among them, recent work has highlighted the potential role that the 12/15-lipoxygenase (12/15LO) pathway may play in AD pathogenesis. 12/15LO is widely distributed in the central nervous system, and its levels are upregulated in patients with AD or mild cognitive impairments. Studies using animal models have implicated 12/15LO in the molecular pathology of AD, including the metabolism of Aß and tau, synaptic integrity, and cognitive functions. We provide an overview of this pathway and its relevance to AD pathogenesis, discuss the mechanism(s) involved, and provide an assessment of how targeting 12/15LO could lead to novel AD therapeutics.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/fisiología , Araquidonato 15-Lipooxigenasa/fisiología , Humanos , Memoria , Transmisión Sináptica , Proteínas tau/metabolismoRESUMEN
Cancer stem cells (CSCs) are responsible for the initiation and maintenance of some types of cancer, suggesting that inhibition of these cells may limit disease progression and relapse. Unfortunately, few CSC-specific genes have been identified. Here, we determined that the gene encoding arachidonate 15-lipoxygenase (Alox15/15-LO) is essential for the survival of leukemia stem cells (LSCs) in a murine model of BCR-ABL-induced chronic myeloid leukemia (CML). In the absence of Alox15, BCR-ABL was unable to induce CML in mice. Furthermore, Alox15 deletion impaired LSC function by affecting cell division and apoptosis, leading to an eventual depletion of LSCs. Moreover, chemical inhibition of 15-LO function impaired LSC function and attenuated CML in mice. The defective CML phenotype in Alox15-deficient animals was rescued by depleting the gene encoding P-selectin, which is upregulated in Alox15-deficient animals. Both deletion and overexpression of P-selectin affected the survival of LSCs. In human CML cell lines and CD34+ cells, knockdown of Alox15 or inhibition of 15-LO dramatically reduced survival. Loss of Alox15 altered expression of PTEN, PI3K/AKT, and the transcription factor ICSBP, which are known mediators of cancer pathogenesis. These results suggest that ALOX15 has potential as a therapeutic target for eradicating LSCs in CML.