RESUMEN
Activation of AT1 (type 1 Ang) receptors stimulates cardiomyocyte hypertrophy in vitro. Accordingly, it has been suggested that regression of cardiac hypertrophy associated with renin-Ang system blockade is due to inhibition of cellular actions of Ang II in the heart, above and beyond their effects to reduce pressure overload. We generated 2 distinct mouse lines with cell-specific deletion of AT1A receptors, from cardiomyocytes. In the first line (C-SMKO), elimination of AT1A receptors was achieved using a heterologous Cre recombinase transgene under control of the Sm22 promoter, which expresses in cells of smooth muscle lineage including cardiomyocytes and vascular smooth muscle cells of conduit but not resistance vessels. The second line (R-SMKO) utilized a Cre transgene knocked-in to the Sm22 locus, which drives expression in cardiac myocytes and vascular smooth muscle cells in both conduit and resistance arteries. Thus, although both groups lack AT1 receptors in the cardiomyocytes, they are distinguished by presence (C-SMKO) or absence (R-SMKO) of peripheral vascular responses to Ang II. Similar to wild-types, chronic Ang II infusion caused hypertension and cardiac hypertrophy in C-SMKO mice, whereas both hypertension and cardiac hypertrophy were reduced in R-SMKOs. Thus, despite the absence of AT1A receptors in cardiomyocytes, C-SMKOs develop robust cardiac hypertrophy. By contrast, R-SMKOs developed identical levels of hypertrophy in response to pressure overload-induced by transverse aortic banding. Our findings suggest that direct activation of AT1 receptors in cardiac myocytes has minimal influence on cardiac hypertrophy induced by renin-Ang system activation or pressure overload.
Asunto(s)
Angiotensina II/farmacología , Cardiomegalia/genética , Hipertensión/genética , Miocitos Cardíacos/metabolismo , Receptor de Angiotensina Tipo 1/genética , Sistema Renina-Angiotensina/efectos de los fármacos , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/metabolismo , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Resistencia Vascular/efectos de los fármacosRESUMEN
Chronic inflammation contributes to cardiovascular disease. Increased levels of the inflammatory cytokine, TNF-α, are often present in conditions associated with cardiovascular disease risk, and TNF-α induces a number of pro-atherogenic effects in macrovascular endothelial cells, including expression of adhesion molecules and chemokines, and lipoprotein uptake and transcytosis to the subendothelial tissue. However, little is known about the roles of acyl-CoA synthetases (ACSLs), enzymes that esterify free fatty acids into their acyl-CoA derivatives, or about the effects of TNF-α on ACSLs in endothelial cells. Therefore, we investigated the effects of TNF-α on ACSLs and downstream lipids in cultured human coronary artery endothelial cells and human umbilical vein endothelial cells. We demonstrated that TNF-α induces ACSL1, ACSL3, and ACSL5, but not ACSL4, in both cell types. TNF-α also increased oleoyl-CoA levels, consistent with the increased ACSL3 expression. RNA-sequencing demonstrated that knockdown of ACSL3 had no marked effects on the TNF-α transcriptome. Instead, ACSL3 was required for TNF-α-induced lipid droplet formation in cells exposed to oleic acid. These results demonstrate that increased acyl-CoA synthesis as a result of ACSL3 induction is part of the TNF-α response in human macrovascular endothelial cells.
Asunto(s)
Coenzima A Ligasas/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Gotas Lipídicas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Células Cultivadas , Coenzima A Ligasas/genética , Células Endoteliales/enzimología , Femenino , Humanos , Gotas Lipídicas/metabolismo , MasculinoRESUMEN
AIMS: To assess the role of oxidative stress in mediating adverse outcomes in metabolic syndrome (MetS) and resultant cardiovascular autonomic neuropathy (CAN), and to evaluate the effects of lifestyle interventions on measures of oxidative stress and CAN in subjects with MetS. METHODS: Pilot study in 25 non-diabetic subjects with MetS (age 49±10years, 76% females) participating in a 24-week lifestyle intervention (supervised aerobic exercise/Mediterranean diet), and 25 age-matched healthy controls. CAN was assessed by cardiovascular reflex tests, heart rate variability (HRV) and PET imaging with sympathetic analog [11C] meta-hydroxyephedrine ([11C]HED). Specific oxidative fingerprints were measured by liquid-chromatography/mass-spectrometry (LC/MS). RESULTS: At baseline, MetS subjects had significantly higher oxidative stress markers [3-nitrotyrosine (234±158 vs. 54±47µmol/mol tyrosine), ortho-tyrosine (59±38 vs. 18±10µmol/molphenylalanine, all P<0.0001], and impaired HRV at rest and during deep breathing (P=0.039 and P=0.021 respectively) compared to controls. Twenty-four-week lifestyle intervention significantly reduced all oxidative stress markers (all P<0.01) but did not change any of the CAN measures. CONCLUSIONS: Subjects with MetS present with signs of CAN and increased oxidative stress in the absence of diabetes. The 24-week lifestyle intervention was effective in ameliorating oxidative stress, but did not improve measures of CAN. Larger clinical trials with longer duration are required to confirm these findings.
Asunto(s)
Enfermedades del Sistema Nervioso Autónomo/etiología , Enfermedades Cardiovasculares/etiología , Síndrome Metabólico/complicaciones , Síndrome Metabólico/terapia , Estrés Oxidativo , Conducta de Reducción del Riesgo , Programas de Reducción de Peso/métodos , Adulto , Sistema Nervioso Autónomo/fisiopatología , Enfermedades del Sistema Nervioso Autónomo/epidemiología , Enfermedades Cardiovasculares/epidemiología , Sistema Cardiovascular/fisiopatología , Estudios Transversales , Dieta Mediterránea , Ejercicio Físico/fisiología , Femenino , Humanos , Estilo de Vida , Masculino , Síndrome Metabólico/epidemiología , Síndrome Metabólico/fisiopatología , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Proyectos Piloto , Factores de Riesgo , Resultado del TratamientoRESUMEN
Acyl-CoA thioesterase 7 (ACOT7) is an intracellular enzyme that converts acyl-CoAs to FFAs. ACOT7 is induced by lipopolysaccharide (LPS); thus, we investigated downstream effects of LPS-induced induction of ACOT7 and its role in inflammatory settings in myeloid cells. Enzymatic thioesterase activity assays in WT and ACOT7-deficient macrophage lysates indicated that endogenous ACOT7 contributes a significant fraction of total acyl-CoA thioesterase activity toward C20:4-, C20:5-, and C22:6-CoA, but contributes little activity toward shorter acyl-CoA species. Lipidomic analyses revealed that LPS causes a dramatic increase, primarily in bis(monoacylglycero)phosphate species containing long (≥C20) polyunsaturated acyl-chains in macrophages, and that the limited effect observed by ACOT7 deficiency is restricted to glycerophospholipids containing 20-carbon unsaturated acyl-chains. Furthermore, ACOT7 deficiency did not detectably alter the ability of LPS to induce cytokines or prostaglandin E2 production in macrophages. Consistently, although ACOT7 was induced in macrophages from diabetic mice, hematopoietic ACOT7 deficiency did not alter the stimulatory effect of diabetes on systemic inflammation or atherosclerosis in LDL receptor-deficient mice. Thus, inflammatory stimuli induce ACOT7 and remodeling of phospholipids containing unsaturated long (≥C20)-acyl chains in macrophages, and, although ACOT7 has preferential thioesterase activity toward these lipid species, loss of ACOT7 has no major detrimental effect on macrophage inflammatory phenotypes.≥.
Asunto(s)
Macrófagos/metabolismo , Palmitoil-CoA Hidrolasa/biosíntesis , Fosfolípidos/metabolismo , Animales , Citocinas/biosíntesis , Dinoprostona/metabolismo , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glicerofosfolípidos/metabolismo , Inflamación/enzimología , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Palmitoil-CoA Hidrolasa/deficiencia , Palmitoil-CoA Hidrolasa/genética , Palmitoil-CoA Hidrolasa/metabolismoRESUMEN
OBJECTIVES: Recent evidence indicates that high-density lipoprotein (HDL) exerts vasculoprotective activities by promoting activating transcription factor 3 (ATF3), leading to downregulation of toll-like receptor (TLR)-induced inflammatory responses. Systemic lupus erythematosus (SLE) is associated with increased cardiovascular disease risk not explained by the Framingham risk score. Recent studies have indicated oxidised HDL as a possible contributor. We investigated the potential mechanisms by which lupus HDL may lose its anti-inflammatory effects and promote immune dysregulation. METHODS: Control macrophages were challenged with control and SLE HDL in vitro and examined for inflammatory markers by real-time qRT-PCR, confocal microscopy, ELISA and flow cytometry. Lupus-prone mice were treated with an HDL mimetic (ETC-642) in vivo and inflammatory cytokine levels measured by real-time qRT-PCR and ELISA. RESULTS: Compared with control HDL, SLE HDL activates NFκB, promotes inflammatory cytokine production and fails to block TLR-induced inflammation in control macrophages. This failure of lupus HDL to block inflammatory responses is due to an impaired ability to promote ATF3 synthesis and nuclear translocation. This inflammation is dependent on lectin-like oxidised low-density lipoprotein receptor 1 (LOX1R) binding and rho-associated, coiled-coil containing protein kinase 1 and 2 (ROCK1/2) kinase activity. HDL mimetic-treated lupus mice showed significant ATF3 induction and proinflammatory cytokine abrogation. CONCLUSIONS: Lupus HDL promotes proinflammatory responses through NFκB activation and decreased ATF3 synthesis and activity in an LOX1R-dependent and ROCK1/2-dependent manner. HDL mimetics should be explored as potential therapies for inflammation and SLE cardiovascular risk.
Asunto(s)
Factor de Transcripción Activador 3/biosíntesis , Citocinas/genética , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacología , Lupus Eritematoso Sistémico/sangre , ARN Mensajero/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/farmacología , Factor de Transcripción Activador 3/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Amidas/farmacología , Animales , Células Cultivadas , Femenino , Humanos , Macrófagos , Ratones , FN-kappa B/metabolismo , Oxidación-Reducción , Péptidos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Receptores Depuradores de Clase A/genética , Receptores Depuradores de Clase E/genética , Receptores Depuradores de Clase E/metabolismo , Esfingomielinas/farmacología , Bazo/citología , Receptores Toll-Like/metabolismo , Transcripción Genética/efectos de los fármacos , Quinasas Asociadas a rho/metabolismoRESUMEN
RATIONALE: Interstitial lung diseases (ILDs) are associated with oxidative stress. Plasma biomarkers that are directly linked to oxidative stress responses in this disease have not been identified. Stable oxidation products of tyrosine residues in proteins may reflect the oxidative microenvironment in the lung or a systemic inflammatory state. OBJECTIVES: To determine if levels of protein tyrosine oxidation are elevated in plasma of patients with ILD compared with an age- and sex-matched healthy control cohort. METHODS: Three tyrosine oxidation products (3-chlorotyrosine, 3-nitrotyrosine, and o,o'-dityrosine) were quantified by tandem mass spectrometry in cellular models, a mouse model of injury-induced fibrosis, and in plasma of healthy control subjects and patients with ILD (n = 42 in each group). MEASUREMENTS AND MAIN RESULTS: Plasma levels of 3-chlorotyrosine, 3-nitrotyrosine, and o,o'-dityrosine were markedly elevated in patients with ILD compared with control subjects with receiver operating characteristic curves separating these groups of 0.872, 0.893, and 0.997, respectively. In a murine model of lung fibrosis, levels of all three oxidative tyrosine modifications were increased in plasma and lung tissue. Cellular models support a critical role for a heme peroxidase and enzymatic sources of reactive oxygen species in the generation of these oxidized products. CONCLUSIONS: We demonstrate an increase in oxidized tyrosine moieties within proteins in the circulating plasma of patients with ILD. These data support the potential for development of oxidative stress-related biomarkers in early diagnosis, prognostication, and/or in evaluating responsiveness to emerging therapies for ILD.
Asunto(s)
Enfermedades Pulmonares Intersticiales/sangre , Estrés Oxidativo , Tirosina/análogos & derivados , Animales , Biomarcadores/sangre , Células Cultivadas , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Espectrometría de Masas en Tándem , Tirosina/sangreRESUMEN
OBJECTIVE: Oxidative stress and oxidized high-density lipoprotein (HDL) are implicated as risk factors for cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Yet, how HDL is oxidized and rendered dysfunctional in SLE remains unclear. Neutrophil extracellular traps (NETs), the levels of which are elevated in lupus, possess oxidant-generating enzymes, including myeloperoxidase (MPO), NADPH oxidase (NOX), and nitric oxide synthase (NOS). We hypothesized that NETs mediate HDL oxidation, impairing cholesterol efflux capacity (CEC). METHODS: Plasma MPO levels and CEC activity were examined in controls and lupus patients, and 3-chlorotyrosine (MPO specific) and 3-nitrotyrosine (derived from reactive nitrogen species) were quantified in human HDL. Multivariable linear models were used to estimate and test differences between groups. HDL was exposed to NETs from control and lupus neutrophils in the presence or absence of MPO, NOX, NOS inhibitors, and chloroquine (CQ). Murine HDL oxidation was quantified after NET inhibition in vivo. RESULTS: SLE patients displayed higher MPO levels and diminished CEC compared to controls. SLE HDL had higher 3-nitrotyrosine and 3-chlorotyrosine content than control HDL, with site-specific oxidation signatures on apolipoprotein A-I. Experiments with human and murine NETs confirmed that chlorination was mediated by MPO and NOX, and nitration by NOS and NOX. Mice with lupus treated with the NET inhibitor Cl-amidine displayed significantly decreased HDL oxidation. CQ inhibited NET formation in vitro. CONCLUSION: Active NOS, NOX, and MPO within NETs significantly modify HDL, rendering the lipoprotein proatherogenic. Since NET formation is enhanced in SLE, these findings support a novel role for NET-derived lipoprotein oxidation in SLE-associated CVD and identify additional proatherogenic roles of neutrophils and putative protective roles of antimalarials in autoimmunity.
Asunto(s)
Lipoproteínas HDL/metabolismo , Lupus Eritematoso Sistémico/enzimología , Neutrófilos/enzimología , Estrés Oxidativo/fisiología , Adulto , Animales , Enfermedades Cardiovasculares/enzimología , Femenino , Humanos , Lupus Eritematoso Sistémico/sangre , Masculino , Ratones , Persona de Mediana Edad , NADPH Oxidasas/sangre , Óxido Nítrico Sintasa/sangre , Oxidación-Reducción , Peroxidasa/sangreRESUMEN
Podocytes are the key cells affected in nephrotic glomerular kidney diseases, and they respond uniformly to injury with cytoskeletal rearrangement. In nephrotic diseases, such as membranous nephropathy and FSGS, persistent injury often leads to irreversible structural damage, whereas in minimal change disease, structural alterations are mostly transient. The factors leading to persistent podocyte injury are currently unknown. Proteolysis is an irreversible process and could trigger persistent podocyte injury through degradation of podocyte-specific proteins. We, therefore, analyzed the expression and functional consequence of the two most prominent proteolytic systems, the ubiquitin proteasome system (UPS) and the autophagosomal/lysosomal system, in persistent and transient podocyte injuries. We show that differential upregulation of both proteolytic systems occurs in persistent human and rodent podocyte injury. The expression of specific UPS proteins in podocytes differentiated children with minimal change disease from children with FSGS and correlated with poor clinical outcome. Degradation of the podocyte-specific protein α-actinin-4 by the UPS depended on oxidative modification in membranous nephropathy. Notably, the UPS was overwhelmed in podocytes during experimental glomerular disease, resulting in abnormal protein accumulation and compensatory upregulation of the autophagosomal/lysosomal system. Accordingly, inhibition of both proteolytic systems enhanced proteinuria in persistent nephrotic disease. This study identifies altered proteolysis as a feature of persistent podocyte injury. In the future, specific UPS proteins may serve as new biomarkers or therapeutic targets in persistent nephrotic syndrome.
Asunto(s)
Podocitos/metabolismo , Podocitos/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteinuria/metabolismo , Proteinuria/patología , Ubiquitina/metabolismo , Actinina/genética , Actinina/metabolismo , Animales , Autofagia/fisiología , Línea Celular Transformada , Modelos Animales de Enfermedad , Humanos , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Lisosomas/metabolismo , Lisosomas/patología , Complejo de la Endopetidasa Proteasomal/genética , Proteinuria/genética , Ratas Wistar , Transcriptoma , Ubiquitina/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Inflammatory activation of myeloid cells is accompanied by increased glycolysis, which is required for the surge in cytokine production. Although in vitro studies suggest that increased macrophage glucose metabolism is sufficient for cytokine induction, the proinflammatory effects of increased myeloid cell glucose flux in vivo and the impact on atherosclerosis, a major complication of diabetes, are unknown. We therefore tested the hypothesis that increased glucose uptake in myeloid cells stimulates cytokine production and atherosclerosis. Overexpression of the glucose transporter GLUT1 in myeloid cells caused increased glycolysis and flux through the pentose phosphate pathway but did not induce cytokines. Moreover, myeloid-cell-specific overexpression of GLUT1 in LDL receptor-deficient mice was ineffective in promoting atherosclerosis. Thus, increased glucose flux is insufficient for inflammatory myeloid cell activation and atherogenesis. If glucose promotes atherosclerosis by increasing cellular glucose flux, myeloid cells do not appear to be the key targets.
Asunto(s)
Aterosclerosis/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Glucosa/metabolismo , Células Mieloides/metabolismo , Animales , Transporte Biológico Activo , Citocinas/genética , Citocinas/metabolismo , Transportador de Glucosa de Tipo 1/genética , Glucólisis , Inflamación/metabolismo , Ratones , Vía de Pentosa Fosfato , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease.
Asunto(s)
Enfermedad de Fabry/complicaciones , Enfermedad de Fabry/metabolismo , Tirosina/análogos & derivados , Enfermedades Vasculares/etiología , Enfermedades Vasculares/metabolismo , Adolescente , Adulto , Animales , Biomarcadores/metabolismo , Estudios de Casos y Controles , Línea Celular , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Enfermedad de Fabry/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Óxido Nítrico Sintasa de Tipo III/metabolismo , ARN Interferente Pequeño/genética , Tirosina/metabolismo , Adulto Joven , alfa-Galactosidasa/antagonistas & inhibidores , alfa-Galactosidasa/genéticaRESUMEN
AIMS: High circulating long chain fatty acids (LCFAs) are implicated in diabetic neuropathy (DN) development. Expression of the long-chain acyl-CoA synthetase 1 (Acsl1) gene, a gene required for LCFA metabolic activation, is altered in human and mouse diabetic peripheral nerve. We assessed the significance of Acsl1 upregulation in primary cultured Schwann cells. RESULTS: Acsl1 overexpression prevented oxidative stress (nitrotyrosine; hydroxyoctadecadienoic acids [HODEs]) and attenuated cellular injury (TUNEL) in Schwann cells following 12 h exposure to LCFAs (palmitate, linoleate, and oleate, 100 µM). Acsl1 overexpression potentiated the observed increase in medium to long-chain acyl-carnitines following 12 h LCFA exposure. Data are consistent with increased mitochondrial LCFA uptake, largely directed to incomplete beta-oxidation. LCFAs uncoupled mitochondrial oxygen consumption from ATP production. Acsl1 overexpression corrected mitochondrial dysfunction, increasing coupling efficiency and decreasing proton leak. INNOVATION: Schwann cell mitochondrial function is critical for peripheral nerve function, but research on Schwann cell mitochondrial dysfunction in response to hyperlipidemia is minimal. We demonstrate that high levels of a physiologically relevant mixture of LCFAs induce Schwann cell injury, but that improved mitochondrial uptake and metabolism attenuate this lipotoxicity. CONCLUSION: Acsl1 overexpression improves Schwann cell function and survival following high LCFA exposure in vitro; however, the observed endogenous Acsl1 upregulation in peripheral nerve in response to diabetes is not sufficient to prevent the development of DN in murine models of DN. Therefore, targeted improvement in Schwann cell metabolic disposal of LCFAs may improve DN phenotypes.
Asunto(s)
Coenzima A Ligasas/genética , Ácidos Grasos/metabolismo , Expresión Génica , Mitocondrias/metabolismo , Estrés Oxidativo , Células de Schwann/metabolismo , Animales , Células Cultivadas , Neuropatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Ácidos Grasos/farmacología , Humanos , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno , Cultivo Primario de Células , Ratas , Células de Schwann/efectos de los fármacosRESUMEN
The early events leading to the development of rheumatoid arthritis (RA) remain unclear, but formation of autoantibodies to citrullinated protein antigens (ACPAs) is considered a key pathogenic event. Neutrophils isolated from patients with various autoimmune diseases display enhanced neutrophil extracellular trap (NET) formation, a phenomenon that exposes autoantigens in the context of immunostimulatory molecules. We investigated whether aberrant NETosis occurs in RA, determined its triggers, and examined its deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and RA synovial fluid neutrophils compared to neutrophils from healthy controls and from patients with osteoarthritis (OA). Further, netting neutrophils infiltrated RA synovial tissue, rheumatoid nodules, and skin. NETosis correlated with ACPA presence and levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. Indeed, during NETosis, neutrophils externalized the citrullinated autoantigens implicated in RA pathogenesis, and anti-citrullinated vimentin antibodies potently induced NET formation. Moreover, the inflammatory cytokines interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α) induced NETosis in RA neutrophils. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines, and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant adaptive and innate immune responses in the joint and in the periphery, and perpetuate pathogenic mechanisms in this disease.
Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Citrulina/metabolismo , Humanos , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Microscopía Fluorescente , Neutrófilos/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The enzyme acyl-CoA synthetase 1 (ACSL1) is induced by peroxisome proliferator-activated receptor α (PPARα) and PPARγ in insulin target tissues, such as skeletal muscle and adipose tissue, and plays an important role in ß-oxidation in these tissues. In macrophages, however, ACSL1 mediates inflammatory effects without significant effects on ß-oxidation. Thus, the function of ACSL1 varies in different tissues. We therefore investigated the signals and signal transduction pathways resulting in ACSL1 induction in macrophages as well as the consequences of ACSL1 deficiency for phospholipid turnover in LPS-activated macrophages. LPS, Gram-negative bacteria, IFN-γ, and TNFα all induce ACSL1 expression in macrophages, whereas PPAR agonists do not. LPS-induced ACSL1 expression is dependent on Toll-like receptor 4 (TLR4) and its adaptor protein TRIF (Toll-like receptor adaptor molecule 1) but does not require the MyD88 (myeloid differentiation primary response gene 88) arm of TLR4 signaling; nor does it require STAT1 (signal transducer and activator of transcription 1) for maximal induction. Furthermore, ACSL1 deletion attenuates phospholipid turnover in LPS-stimulated macrophages. Thus, the regulation and biological function of ACSL1 in macrophages differ markedly from that in insulin target tissues. These results suggest that ACSL1 may have an important role in the innate immune response. Further, these findings illustrate an interesting paradigm in which the same enzyme, ACSL1, confers distinct biological effects in different cell types, and these disparate functions are paralleled by differences in the pathways that regulate its expression.
Asunto(s)
Coenzima A Ligasas/metabolismo , Bacterias Gramnegativas/metabolismo , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Fosfolípidos/metabolismo , Animales , Células de la Médula Ósea/citología , Femenino , Inmunidad Innata , Interferón gamma/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/citología , Macrófagos Peritoneales/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Transducción de SeñalRESUMEN
OBJECTIVE: Phagocyte-derived myeloperoxidase (MPO) and pro-inflammatory high density lipoprotein (HDL) associate with rheumatoid arthritis (RA), but the link between MPO and HDL has not been systematically examined. In this study, we investigated whether MPO can oxidise HDL and determined MPO-specific oxidative signature by apoA-1 by peptide mapping in RA subjects with and without known cardiovascular disease (CVD). METHODS: Two MPO oxidation products, 3-chlorotyrosine and 3-nitrotyrosine, were quantified by tandem mass spectrometry (MS/MS) in in vitro model system studies and in plasma and HDL derived from healthy controls and RA subjects. MPO levels and cholesterol efflux were determined. Site-specific nitration and chlorination of apoA-1 peptides were quantified by MS/MS. RESULTS: RA subjects demonstrated higher levels of MPO, MPO-oxidised HDL and diminished cholesterol efflux. There was marked increase in MPO-specific 3-chlorotyrosine and 3-nitrotyrosine content in HDL in RA subjects consistent with specific targeting of HDL, with increased nitration in RA subjects with CVD. Cholesterol efflux capacity was diminished in RA subjects and correlated inversely with HDL 3-chlorotyrosine suggesting a mechanistic role for MPO. Nitrated HDL was elevated in RACVD subjects compared with RA subjects without CVD. Oxidative peptide mapping revealed site-specific unique oxidation signatures on apoA-1 for RA subjects with and without CVD. CONCLUSIONS: We report an increase in MPO-mediated HDL oxidation that is regiospecific in RA and accentuated in those with CVD. Decreased cholesterol efflux capacity due to MPO-mediated chlorination is a potential mechanism for atherosclerosis in RA and raises the possibility that oxidant resistant forms of HDL may attenuate this increased risk.
Asunto(s)
Artritis Reumatoide/sangre , Lipoproteínas HDL/sangre , Peroxidasa/fisiología , Adulto , Anciano , Apolipoproteína A-I/sangre , Artritis Reumatoide/complicaciones , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/etiología , Estudios de Casos y Controles , Colesterol/sangre , Femenino , Halogenación/fisiología , Humanos , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Mapeo Peptídico/métodos , Peroxidasa/sangre , Espectrometría de Masas en Tándem/métodos , Tirosina/análogos & derivados , Tirosina/sangreRESUMEN
OBJECTIVE: Saturated fatty acids, such as palmitic and stearic acid, cause detrimental effects in endothelial cells and have been suggested to contribute to macrophage accumulation in adipose tissue and the vascular wall, in states of obesity and insulin resistance. Long-chain fatty acids are believed to require conversion into acyl-CoA derivatives to exert most of their detrimental effects, a reaction catalyzed by acyl-CoA synthetases (ACSLs). The objective of this study was to investigate the role of ACSL1, an ACSL isoform previously shown to mediate inflammatory effects in myeloid cells, in regulating endothelial cell responses to a saturated fatty acid-rich environment in vitro and in vivo. METHODS AND RESULTS: Saturated fatty acids caused increased inflammatory activation, endoplasmic reticulum stress, and apoptosis in mouse microvascular endothelial cells. Forced ACSL1 overexpression exacerbated the effects of saturated fatty acids on apoptosis and endoplasmic reticulum stress. However, endothelial ACSL1 deficiency did not protect against the effects of saturated fatty acids in vitro, nor did it protect insulin-resistant mice fed a saturated fatty acid-rich diet from macrophage adipose tissue accumulation or increased aortic adhesion molecule expression. CONCLUSIONS: Endothelial ACSL1 is not required for inflammatory and apoptotic effects of a saturated fatty acid-rich environment.
Asunto(s)
Apoptosis , Coenzima A Ligasas/metabolismo , Células Endoteliales/enzimología , Ácidos Grasos/metabolismo , Inflamación/enzimología , Obesidad/enzimología , Acilcoenzima A/metabolismo , Tejido Adiposo/inmunología , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Aorta/metabolismo , Bovinos , Células Cultivadas , Coenzima A Ligasas/deficiencia , Coenzima A Ligasas/genética , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico , Células Endoteliales/inmunología , Células Endoteliales/patología , Activación Enzimática , Inflamación/inmunología , Inflamación/patología , Resistencia a la Insulina , Molécula 1 de Adhesión Intercelular/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Obesidad/inmunología , Obesidad/patología , Palmitoil Coenzima A/metabolismo , Interferencia de ARN , Factores de Tiempo , Transfección , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Diabetic neuropathy (DN) is the most common complication of diabetes and is characterized by distal-to-proximal loss of peripheral nerve axons. The idea of tissue-specific pathological alterations in energy metabolism in diabetic complications-prone tissues is emerging. Altered nerve metabolism in type 1 diabetes models is observed; however, therapeutic strategies based on these models offer limited efficacy to type 2 diabetic patients with DN. Therefore, understanding how peripheral nerves metabolically adapt to the unique type 2 diabetic environment is critical to develop disease-modifying treatments. In the current study, we utilized targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) to characterize the glycolytic and tricarboxylic acid (TCA) cycle metabolomes in sural nerve, sciatic nerve, and dorsal root ganglia (DRG) from male type 2 diabetic mice (BKS.Cg-m+/+Lepr(db); db/db) and controls (db/+). We report depletion of glycolytic intermediates in diabetic sural nerve and sciatic nerve (glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate (sural nerve only), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and lactate), with no significant changes in DRG. Citrate and isocitrate TCA cycle intermediates were decreased in sural nerve, sciatic nerve, and DRG from diabetic mice. Utilizing LC/electrospray ionization/MS/MS and HPLC methods, we also observed increased protein and lipid oxidation (nitrotyrosine; hydroxyoctadecadienoic acids) in db/db tissue, with a proximal-to-distal increase in oxidative stress, with associated decreased aconitase enzyme activity. We propose a preliminary model, whereby the greater change in metabolomic profile, increase in oxidative stress, and decrease in TCA cycle enzyme activity may cause distal peripheral nerves to rely on truncated TCA cycle metabolism in the type 2 diabetes environment.
Asunto(s)
Ciclo del Ácido Cítrico , Diabetes Mellitus Tipo 2/metabolismo , Neuropatías Diabéticas/metabolismo , Modelos Animales de Enfermedad , Glucólisis , Estrés Oxidativo , Sistema Nervioso Periférico/metabolismo , Aconitato Hidratasa/metabolismo , Animales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/enzimología , Neuropatías Diabéticas/enzimología , Regulación hacia Abajo , Ganglios Espinales/enzimología , Ganglios Espinales/metabolismo , Peroxidación de Lípido , Masculino , Ratones , Ratones Mutantes , Neuronas/enzimología , Neuronas/metabolismo , Sistema Nervioso Periférico/enzimología , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Nervio Ciático/enzimología , Nervio Ciático/metabolismo , Nervio Sural/enzimología , Nervio Sural/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have a notable increase in atherothrombotic cardiovascular disease (CVD) which is not explained by the Framingham risk equation. In vitro studies indicate that type I interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regard to the development of CVD, has not been characterized. This study was undertaken to examine the role of type I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis. METHODS: Lupus-prone New Zealand mixed 2328 (NZM) mice and atherosclerosis-prone apolipoprotein E- knockout (apoE(-/-) ) mice were compared to mice lacking type I IFN receptor (INZM and apoE(-/-) IFNAR(-/-) mice, respectively) with regard to endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development, and occlusive thrombosis. Similar experiments were performed using NZM and apoE(-/-) mice exposed to an IFNα-containing or empty adenovirus. RESULTS: Loss of type I IFN receptor signaling improved endothelium-dependent vasorelaxation, lipoprotein parameters, EPC numbers and function, and neoangiogenesis in lupus-prone mice, independent of disease activity or sex. Further, acute exposure to IFNα impaired endothelial vasorelaxation and EPC function in lupus-prone and non-lupus-prone mice. Decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis were observed in apoE(-/-) IFNAR(-/-) mice, compared to apoE(-/-) mice, while NZM and apoE(-/-) mice exposed to IFNα developed accelerated thrombosis and platelet activation. CONCLUSION: These results support the hypothesis that type I IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature.
Asunto(s)
Aterosclerosis/metabolismo , Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Placa Aterosclerótica/metabolismo , Trombosis/metabolismo , Cicatrización de Heridas/fisiología , Animales , Aterosclerosis/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Femenino , Lupus Eritematoso Sistémico/fisiopatología , Masculino , Ratones , Vasodilatación/fisiologíaRESUMEN
The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.
Asunto(s)
Aterosclerosis/metabolismo , Coenzima A Ligasas/metabolismo , Diabetes Mellitus/metabolismo , Macrófagos/citología , Alelos , Animales , Glucemia/metabolismo , Trasplante de Médula Ósea , Femenino , Eliminación de Gen , Humanos , Inflamación , Lípidos/química , Masculino , Ratones , Ratones Transgénicos , Modelos Biológicos , Monocitos/citología , Fenotipo , Receptores de LDL/genéticaRESUMEN
Chronic kidney disease (CKD) is a significant public health problem, and progression to end-stage renal disease leads to dramatic increases in morbidity and mortality. The mechanisms underlying progression of disease are poorly defined, and current noninvasive markers incompletely correlate with disease progression. Therefore, there is a great need for discovering novel markers for CKD. We utilized a glycoproteomic profiling approach to test the hypothesis that the urinary glycoproteome profile from subjects with CKD would be distinct from healthy controls. N-linked glycoproteins were isolated and enriched from the urine of healthy controls and subjects with CKD. This strategy identified several differentially expressed proteins in CKD, including a diverse array of proteins with endopeptidase inhibitor activity, protein binding functions, and acute-phase/immune-stress response activity supporting the proposal that inflammation may play a central role in CKD. Additionally, several of these proteins have been previously linked to kidney disease implicating a mechanistic role in disease pathogenesis. Collectively, our observations suggest that the human urinary glycoproteome may serve as a discovery source for novel mechanism-based biomarkers of CKD.
RESUMEN
Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer.