RESUMEN
Systemic metabolic signatures of oral diseases have been rarely investigated, and prospective studies do not exist. We analyzed whether signs of current or past infectious/inflammatory oral diseases are associated with circulating metabolites. Two study populations were included: the population-based Health-2000 (n = 6,229) and Parogene (n = 452), a cohort of patients with an indication to coronary angiography. Health-2000 participants (n = 4,116) provided follow-up serum samples 11 y after the baseline. Serum concentrations of 157 metabolites were determined with a nuclear magnetic resonance spectroscopy-based method. The associations between oral parameters and metabolite concentrations were analyzed using linear regression models adjusted for age, sex, number of teeth, smoking, presence of diabetes, and education (in Health-2000 only). The number of decayed teeth presented positive associations with low-density lipoprotein diameter and the concentrations of pyruvate and citrate. Negative associations were found between caries and the unsaturation degree of fatty acids (FA) and relative proportions of docosahexaenoic and omega-3 FAs. The number of root canal fillings was positively associated with very low-density lipoprotein parameters, such as diameter, cholesterol, triglycerides, and number of particles. Deepened periodontal pockets were positively associated with concentrations of cholesterol, triglycerides, pyruvate, leucine, valine, phenylalanine, and glycoprotein acetyls and negatively associated with high-density lipoprotein (HDL) diameter, FA unsaturation degree, and relative proportions of omega-6 and polyunsaturated FAs. Bleeding on probing (BOP) was associated with increased concentrations of triglycerides and glycoprotein acetyls, as well as decreased proportions of omega-3 and omega-6 FAs. Caries at baseline predicted alterations in apolipoprotein B-containing lipoproteins and HDL-related metabolites in the follow-up, and both caries and BOP were associated with changes in HDL-related metabolites and omega-3 FAs in the follow-up. Signs of current or past infectious/inflammatory oral diseases, especially periodontitis, were associated with metabolic profiles typical for inflammation. Oral diseases may represent a modifiable risk factor for systemic chronic inflammation and thus cardiometabolic disorders.
Asunto(s)
Colesterol , Ácidos Grasos , Humanos , Estudios Prospectivos , Triglicéridos , Lipoproteínas LDL , Inflamación , Glicoproteínas , PiruvatosRESUMEN
The products from the 193 nm irradiation of triphenylsulfonium nonaflate (TPS) embedded in a poly(methyl methacrylate) (PMMA) film have been characterized. The analysis of the photoproduct formation was performed using chromatographic techniques including HPLC, GPC and GC-MS as well as UV-vis and NMR spectroscopic methods. Two previously unreported TPS photoproducts, triphenylene and dibenzothiophene, were detected; additionally, GPC and DOSY-NMR spectroscopic analyses after irradiation suggested that TPS fragments had been incorporated into the polymer film. The irradiation of acetonitrile solutions containing 10% w/v PMMA and 1% w/v TPS in a 1 cm-path-length cuvette showed only a trace amount of triphenylene or dibenzothiophene, indicating that topochemical factors were important for the formation of these molecules. The accumulated evidence indicates that both products were formed by in-cage, secondary photochemical reactions: 2-(phenylthio)biphenyl to triphenylene, and diphenylsulfide to dibenzothiophene.
RESUMEN
BACKGROUND: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. METHODS: Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. RESULTS: LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. CONCLUSIONS: In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Lisofosfolípidos/farmacología , Microglía/efectos de los fármacos , Proteína Quinasa C/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Animales Recién Nacidos , Ácidos Carboxílicos/farmacología , Células Cultivadas , Corteza Cerebral/citología , Citocinas/genética , Citocinas/metabolismo , Inhibidores Enzimáticos/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Fosforilación , Proteína Quinasa C/genética , Pirimidinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores del Ácido Lisofosfatídico/antagonistas & inhibidoresRESUMEN
Focused ultrasound (FUS) in the presence of microbubbles transiently and reversibly opens the blood-brain barrier (BBB) in rodents and humans, thereby providing a time window for increased drug delivery into brain tissue. To get insight into the underlying mechanisms that govern ultrasound (US)-mediated opening of the BBB, in vitro models are a useful alternative. In this paper, we have utilized an in vitro BBB model that consists of primary porcine brain endothelial cells (PBECs). PBEC monolayers are grown on permeable membranes, which allow assessment of key features of BBB function as well as US treatment. This experimental model is characterized by low permeability for both small molecules and proteins, has a high transendothelial electrical resistance, and expresses tight junctions and efflux pumps. Here, we compare the effects of inertial and stable cavitation in the presence of SonoVue microbubbles on PBEC monolayers' electrical resistance and permeability properties. Our results point out the fragility of PBEC monolayers, which enhances results variability. In particular, we show that handling of the inserts, such as medium change and transfer to the US setup, modifies the cellular response, and immunostaining of the monolayers introduces damage and cell detachment within the US-exposed monolayers. Our results indicate that stable cavitation might have a more pronounced impact on cell permeability as compared with inertial cavitation in vitro. This paper might contribute to further development of experimental setups that are suitable to characterize the impact of FUS and microbubbles on BBB properties in vitro.
Asunto(s)
Barrera Hematoencefálica , Células Endoteliales , Microburbujas , Ondas Ultrasónicas , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiología , Barrera Hematoencefálica/efectos de la radiación , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Células Endoteliales/efectos de la radiación , Modelos Biológicos , Sonicación , PorcinosRESUMEN
Trophoblast invasion into uterine tissues represents a hallmark of first trimester placental development. As expression of serum amyloid A4 (SAA4) occurs in tumorigenic and invasive tissues we here investigated whether SAA4 is present in trophoblast-like human AC1-M59/Jeg-3 cells and trophoblast preparations of human first trimester and term placenta. SAA4 mRNA was expressed in non-stimulated and cytokine-treated AC1-M59/Jeg-3 cells. In purified trophoblast cells SAA4 mRNA expression was upregulated at weeks 10 and 12 of pregnancy. Western-blot and immunohistochemical staining of first trimester placental tissue revealed pronounced SAA4 expression in invasive trophoblast cells indicating a potential role of SAA4 during invasion.
Asunto(s)
Proteína Amiloide A Sérica/metabolismo , Trofoblastos/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Embarazo , Primer Trimestre del Embarazo , Tercer Trimestre del EmbarazoRESUMEN
BACKGROUND AND PURPOSE: Endothelins (ETs) and their G protein-coupled receptors exert key physiological functions during normal and aberrant placental development. Trophoblast cells mediate the contact between the embryo and the mother, by establishing a transient organ, the placenta. Choriocarcinoma cells display many of the biochemical and morphological characteristics of in utero invasive trophoblast cells and may therefore be used as a suitable model to study epithelial tumour progression of foetal-derived cells. EXPERIMENTAL APPROACH: The present study aimed at investigating ET receptor-mediated activation of the mitogen-activated protein kinase (MAPK) pathway in human choriocarcinoma. KEY RESULTS: Both JAR and Jeg-3 choriocarcinoma cell lines expressed ET receptor subtype B (ET(B)) but not ET(A) receptor transcripts. ET(B) receptor engagement by ET-1 and ET-3 resulted in a similar time- and concentration-dependent phosphorylation of p42/44 MAPK, also known as extracellular regulated kinase 1/2. Using specific pharmacological antagonists/inhibitors, we showed that ET-1/-3-mediated signal transduction by the ET(B) receptor is transmitted via G(i)- and G(q)-dependent pathways through activation of the Src (G(i)) and protein kinase C (G(q)) axis that converge at Ras/Raf, leading to downstream activation of p42/44. On a functional level, ET(B) engagement and subsequent phosphorylation of p42/44 resulted in enhanced transcription of the immediate early response genes c-fos and c-jun, a process commonly assumed to be mediated by the ET(A) receptor, and increased cell growth and relative cell area. CONCLUSIONS AND IMPLICATIONS: As human choriocarcinoma cells secrete ETs, pharmacological antagonism of ETs and/or ET(B) receptor-mediated signal transduction could represent a likely target therapy for choriocarcinoma.
Asunto(s)
Coriocarcinoma/genética , Endotelina-1/farmacología , Endotelina-3/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Expresión Génica/efectos de los fármacos , Genes fos/efectos de los fármacos , Genes jun/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Receptor de Endotelina B/genética , Western Blotting , Recuento de Células , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cartilla de ADN , Humanos , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sales de Tetrazolio , TiazolesRESUMEN
Myeloperoxidase (MPO), a member of the haem peroxidase-cyclooxygenase superfamily, is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in innate immune defence mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, detailed information on the structure of ferric MPO and its complexes with low- and high-spin ligands is available. This, together with a thorough understanding of reaction mechanisms including redox properties of intermediates, enables a rationale attempt in developing specific MPO inhibitors that still maintain MPO activity during host defence and bacterial killing but interfere with pathophysiologically persistent activation of MPO. The various approaches to inhibit enzyme activity of MPO and to ameliorate adverse effects of MPO-derived oxidants will be discussed. Emphasis will be put on mechanism-based inhibitors and high-throughput screening of compounds as well as the discussion of physiologically useful HOCl scavengers.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Peroxidasa/antagonistas & inhibidores , Peroxidasa/fisiología , Animales , Cloruros/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Depuradores de Radicales Libres/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Modelos Moleculares , Oxidantes/metabolismo , Peroxidasa/química , Peroxidasa/metabolismoRESUMEN
We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Lipasa/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatidilcolinas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos , Lisofosfolipasa/metabolismoRESUMEN
Alpha-tocopheryl-succinate (alphaTS) is a synthetic, anti-neoplastic derivative of alpha-tocopherol. Here we studied the effects of free and high-density lipoprotein subclass 3 (HDL3)-associated alphaTS on the growth of human (A549) and mouse Lewis (LL2) lung carcinoma cells. Both free and HDL3-associated alphaTS inhibited A549 growth in a time- and concentration-dependent manner. Treatment of A549 cells with alphaTS-enriched HDL3 led to DNA fragmentation and a time-dependent decrease in immunoreactivity of poly(ADP-ribose)polymerase. Uptake experiments revealed a high capacity for selective alphaTS uptake in excess of holoparticle endocytosis. Overexpression of scavenger receptor class B, type I (SR-BI), the prime receptor mediating selective lipid uptake, in A549 cells resulted in significantly increased selective alphaTS uptake, a finding associated with complete cellular growth arrest. The present in vitro findings were verified in an in vivo model: tumor inoculation in C57BL6 was performed with either wild-type, beta-galactosidase- or SR-BI-overexpressing LL2 cells. After tumor inoculation, the animals received six consecutive intravenous injections of alphaTS. This experimental setup resulted in significantly reduced tumor burden in animals that were inoculated with SR-BI-overexpressing LL2 cells but not in animals inoculated with wild-type or beta-galactocidase-transfected cells. Based on our in vitro and in vivo findings, we propose that SR-BI could provide a novel route for HDL3-mediated drug delivery of anti-neoplastic drugs.
Asunto(s)
Carcinoma/tratamiento farmacológico , Lipoproteínas HDL/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Vitamina E/análogos & derivados , Vitamina E/farmacología , Adenoviridae/genética , Animales , Antígenos CD36 , División Celular , Línea Celular Tumoral , Centrifugación por Gradiente de Densidad , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Endocitosis , Humanos , Hidrólisis , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Nucleosomas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Receptores Inmunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B , Factores de Tiempo , Tocoferoles , Transfección , beta-Galactosidasa/metabolismoRESUMEN
As villous trophoblast does represent the contact zone between foetal and maternal tissues, the present in vitro study was aimed at investigating cholesterol supply from human high density lipoprotein subclass 3 (HDL(3)) to trophoblast cells isolated from human first trimester and term placenta. Binding of (125)I-HDL(3) was specific and saturable with similar K(d)-values for first trimester (54 microg HDL(3)-protein/ml) and term villous trophoblast cells (29 microg HDL(3)-protein/ml). The cell-association of (125)I-HDL(3) was 3-fold higher for term trophoblast cells while the specific cell-association of [(3)H]cholesterol ester(CE)-labelled HDL(3) was higher for first trimester trophoblast preparations. As a consequence, first trimester trophoblast cells have a pronounced capacity for selective CE-uptake from HDL(3). Competition experiments with native and oxidized low-density lipoprotein as well as cAMP-mediated stimulation of cell-association of [(3)H]CE-HDL(3) in both trophoblast preparations suggested the scavenger receptor class B, type I (SR-BI) as a likely receptor mediating this pathway. SR-BI m RNA could be identified by RT-PCR and Northern blot experiments in both trophoblast preparations. Western blot analysis and immunocytochemistry revealed high expression of SR-BI in first trimester trophoblast. A polyclonal antiserum raised against murine SR-BI significantly decreased cell-association of [(3)H]CE-HDL(3) in trophoblast cells. We conclude that human first trimester and term trophoblast cells express SR-BI which could serve as an efficient route for supplying cholesterol esters from maternal lipoproteins to foetal tissues.
Asunto(s)
Ésteres del Colesterol/metabolismo , Vellosidades Coriónicas/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Trofoblastos/metabolismo , Adulto , Animales , Unión Competitiva , Western Blotting , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células CHO , Adhesión Celular/efectos de los fármacos , Vellosidades Coriónicas/efectos de los fármacos , Cricetinae , AMP Cíclico/farmacología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Trabajo de Parto , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL3 , Microscopía Confocal , Embarazo , Primer Trimestre del Embarazo , ARN Mensajero/metabolismo , Receptores Depuradores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Depuradores de Clase B , Trofoblastos/citología , Trofoblastos/efectos de los fármacosRESUMEN
As in other phagocytic cells, the NADPH-oxidase system in microglia is thought to be primarily responsible for the production of superoxide anion radicals (O2(-.), a potentially cytotoxic reactive oxygen species. The assembly of a functional NADPH-oxidase complex at the plasma membrane depends on the phosphorylation and subsequent translocation of several cytosolic subunits. Immunocytochemical and subcellular fractionation experiments performed during the present study revealed that the NADPH-oxidase subunit p67(phox) translocates from the cytosol to the plasma membrane upon stimulation. Pre-incubation of microglia in alpha-tocopherol (alphaTocH) containing medium decreased O2(-.) production in a time- and concentration-dependent manner, findings attributed to attenuated p67(phox) translocation to the plasma membrane. Moreover, alphaTocH-supplementation of the culture medium resulted in decreased microglial protein kinase C (PKC) activities, an effect that could be partially or completely reversed by the addition of protein phosphatase inhibitors (okadaic acid and calyculin A). The addition of the PKC-inhibitor staurosporine inhibited the microglial respiratory burst in a manner comparable to alphaTocH. The addition of okadaic acid or calyculin A completely restored O2(-.) production in alphaTocH-supplemented cells. The present findings suggest that alphaTocH inactivates PKC via a PP1 or PP2A-mediated pathway and, as a consequence, blocks the phosphorylation-dependent translocation of p67(phox) to the plasma membrane. As a result, O2(-.) production by the microglial NADPH-oxidase system is substantially inhibited.
Asunto(s)
Antioxidantes/farmacología , Microglía/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas , Proteínas del Tejido Nervioso/fisiología , Fosfoproteínas Fosfatasas/fisiología , Fosfoproteínas/antagonistas & inhibidores , Estallido Respiratorio/efectos de los fármacos , Superóxidos/metabolismo , alfa-Tocoferol/farmacología , Animales , Membrana Celular/enzimología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Citosol/enzimología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Toxinas Marinas , Ratones , Microglía/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Estallido Respiratorio/fisiología , Estaurosporina/farmacología , PorcinosRESUMEN
Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, generates an array of oxidants proposed to play critical roles in host defense, tissues damage, and foam cell formation. Although neutrophils are the major source for MPO, the enzyme could be identified abundantly in circulating monocytes and monocytes/macrophages in rabbit lesions. MPO is the only enzyme known to generate hypochlorous acid (HOCl) and HOCl-modified lipoproteins have pronounced atherogenic and/or proinflammatory features in vivo and in vitro. Using specific monoclonal antibodies, HOCl-modified (lipo)proteins were detected in atherosclerotic plaques of heterozygous Watanabe heritable hyperlipidemic rabbits and to a lesser extent in a specific strain of New Zealand White rabbits with a high atherosclerotic response to hypercholesterolemia. Colocalization of immunoreactive MPO and HOCl-modified-epitopes in serial sections of rabbit lesions provides convincing evidence for MPO-H2O2-chloride system-mediated oxidation of (lipo)proteins under in vivo conditions. We propose that monocyte-derived MPO could connect chronic inflammatory conditions with arterial lipid/lipoprotein deposition during diet-induced atherogenesis in rabbits.
Asunto(s)
Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Dieta Aterogénica , Ácido Hipocloroso/metabolismo , Lipoproteínas/metabolismo , Animales , Colesterol en la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Humanos , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Inflamación/metabolismo , Lipoproteínas/química , Monocitos/metabolismo , Oxidantes/metabolismo , Peroxidasa/metabolismo , Proteínas/química , Proteínas/metabolismo , ConejosRESUMEN
During reverse cholesterol transport plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and pre-beta-HDL. The acute-phase response is accompanied with dramatic changes in lipid metabolism including alterations in HDL concentration, composition, and thereby its function as a substrate for HDL remodeling proteins in circulation. To evaluate how acute-phase HDL (AP-HDL) functions in PLTP-mediated HDL conversion, we collected plasma samples from patients with severe acute-phase response (n=17), and from healthy controls (n=30). Subsequently, total HDL (1.063Asunto(s)
Reacción de Fase Aguda/sangre
, Proteínas Portadoras/sangre
, Glicoproteínas
, Lipoproteínas HDL/metabolismo
, Proteínas de la Membrana/sangre
, Proteínas de Transferencia de Fosfolípidos
, Transporte Biológico
, Proteínas Portadoras/química
, Proteínas Portadoras/aislamiento & purificación
, Proteínas de Transferencia de Ésteres de Colesterol
, Humanos
, Lipoproteínas HDL/química
, Lipoproteínas HDL/aislamiento & purificación
, Proteínas de la Membrana/química
, Fosfatidilcolina-Esterol O-Aciltransferasa/sangre
, Ultracentrifugación
RESUMEN
The structural integrity of apolipoprotein A-I (apo A-I) is critical to the physiological function of high-density lipoprotein (HDL). Oxidized lipoproteins are thought to be of central importance in atherogenesis, and oxidation products characteristic of myeloperoxidase, a heme protein secreted by activated phagocytes, have been detected in human atherosclerotic tissue. At plasma concentrations of halide ion, hypochlorous acid is a major product of the myeloperoxidase-hydrogen peroxide-chloride system. We therefore investigated the effects of activated human neutrophils, a potent source of myeloperoxidase and hydrogen peroxide, on the protein and lipid components of HDL. Both free and HDL-associated apo A-I exposed to activated human neutrophils underwent extensive degradation as monitored by RP-HPLC and Western blotting with a polyclonal antibody to apo A-I. Replacement of the neutrophils with reagent HOCl resulted in comparable damage (at molar oxidant : HDL subclass 3 ratio = 100) as observed in the presence of activated phagocytes. Apo A-I degradation by activated neutrophils was partially inhibited by the HOCl scavenger methionine, by the heme inhibitor azide, by chloride-free conditions, by the peroxide scavenger catalase, and by a combination of superoxide dismutase (SOD)/catalase, implicating HOCl in the cell-mediated reaction. The addition of a protease inhibitor (3,4-dichloroisocoumarin) further reduced the extent of apo A-I damage. In contrast to the protein moiety, there was little evidence for oxidation of unsaturated fatty acids or cholesterol in HDL3 exposed to activated neutrophils, suggesting that HOCl was selectively damaging apo A-I. Our observations indicate that HOCl generated by myeloperoxidase represents one pathway for protein degradation in HDL3 exposed to activated phagocytes.
Asunto(s)
Cloruros/metabolismo , Peróxido de Hidrógeno/metabolismo , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Western Blotting , Catalasa/metabolismo , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Neutrófilos/enzimología , Superóxido Dismutasa/metabolismoAsunto(s)
Antígenos CD36/aislamiento & purificación , Endotelio Vascular/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de LDL/aislamiento & purificación , Receptores de Lipoproteína , Antígenos CD36/química , Antígenos CD36/metabolismo , Ésteres del Colesterol/metabolismo , Humanos , Ácido Hipocloroso/química , Lipoproteínas HDL/química , Receptores de LDL/química , Receptores de LDL/metabolismo , Receptores de LDL Oxidadas , Receptores Depuradores , Receptores Depuradores de Clase B , Receptores Depuradores de Clase E , Venas Umbilicales/citologíaRESUMEN
Myeloperoxidase (MPO), which is released from cytoplasmic granules of activated phagocytes by a degranulation process, reacts with H(2)O(2) (generated during the oxidative burst) and chloride ions to generate hypochlorous acid/hypochlorite (HOCl/OCl(-)). HOCl, a strong oxidant, in turn reacts with proteins to form HOCl-modified proteins. The presence of these cytotoxic chloramines during inflammatory conditions, eg, atherosclerosis and glomerular and tubulointerstitial injury, suggested that chloramines are powerful oxidants that can have profound biologic effects. In the present study, immunoreactive MPO was identified in fetal membranes and the basal plate and in maternal and fetal blood cells of human placental tissues. Monocytes/macrophages represent the major cell source for MPO in human placental tissues. Immunohistochemical findings revealed that HOCl-modified proteins are present in normal human term placenta but not during the first trimester of pregnancy (Weeks 7 to 12). HOCl-modified proteins were localized in areas formed by fetally derived cells as well as maternal decidual tissues, ie, areas where fetal extravillous trophoblast cells invade the maternal tissue and stimulate the maternal immune system. HOCl-modified proteins, products of the MPO-H(2)O(2)-chloride system in vivo, were not present intracellularly, but immunoreactivity for HOCl-modified proteins was cell-associated and/or present in the extracellular matrix. Extravillous trophoblast cells, which may also exert phagocytic activities, showed no intracellular immunoreactivity for MPO or HOCl-modified proteins. The present findings indicate that the generation of HOCl-modified proteins during normal pregnancy is a physiologic rather than a pathophysiologic process.
Asunto(s)
Ácido Hipocloroso/metabolismo , Peroxidasa/fisiología , Placenta/enzimología , Embarazo/metabolismo , Vellosidades Coriónicas/química , Vellosidades Coriónicas/enzimología , Decidua/química , Decidua/enzimología , Epítopos/inmunología , Membranas Extraembrionarias/química , Membranas Extraembrionarias/enzimología , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Macrófagos/enzimología , Monocitos/enzimología , Oxidación-Reducción , Peroxidasa/análisis , Peroxidasa/inmunología , Placenta/química , Proteínas/metabolismoRESUMEN
In reverse cholesterol transport, plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and prebeta-HDL. During the acute-phase reaction (APR), serum amyloid A (SAA) becomes the predominant apolipoprotein on HDL. Displacement of apo A-I by SAA and subsequent remodeling of HDL during the APR impairs cholesterol efflux from peripheral tissues, and might thereby change substrate properties of HDL for lipid transfer proteins. Therefore, the aim of this work was to study the properties of SAA-containing HDL in PLTP-mediated conversion. Enrichment of HDL by SAA was performed in vitro and in vivo and the SAA content in HDL varied between 32 and 58 mass%. These HDLs were incubated with PLTP, and the conversion products were analyzed for their size, composition, mobility in agarose gels, and apo A-I degradation. Despite decreased apo A-I concentrations, PLTP facilitated the conversion of acute-phase HDL (AP-HDL) more effectively than the conversion of native HDL(3), and large fusion particles with diameters of 10.5, 12.0, and 13.8 nm were generated. The ability of PLTP to release prebeta from AP-HDL was more profound than from native HDL(3). Prebeta-HDL formed contained fragmented apo A-I with a molecular mass of about 23 kDa. The present findings suggest that PLTP-mediated conversion of AP-HDL is not impaired, indicating that the production of prebeta-HDL is functional during the ARP. However, PLTP-mediated in vitro degradation of apo A-I in AP-HDL was more effective than that of native HDL, which may be associated with a faster catabolism of inflammatory HDL.
Asunto(s)
Reacción de Fase Aguda/metabolismo , Proteínas Portadoras/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos , Animales , Colesterol/metabolismo , Lipoproteínas de Alta Densidad Pre-beta , Humanos , Tamaño de la Partícula , Conejos , Proteína Amiloide A Sérica/metabolismoRESUMEN
It is clearly established that an efficient supply to the brain of alpha-tocopherol (alphaTocH), the most biologically active member of the vitamin E family, is of the utmost importance for proper neurological functioning. Although the mechanism of uptake of alphaTocH into cells constituting the blood-brain barrier (BBB) is obscure, we previously demonstrated that high-density lipoprotein (HDL) plays a major role in the supply of alphaTocH to porcine brain capillary endothelial cells (pBCECs). Here we studied whether a porcine analogue of human and rodent scavenger receptor class B, type I mediates selective (without concomitant lipoprotein particle internalization) uptake of HDL-associated alphaTocH in a similar manner to that described for HDL-associated cholesteryl esters (CEs). In agreement with this hypothesis we observed that a major proportion of alphaTocH uptake by pBCECs occurred by selective uptake, exceeding HDL3 holoparticle uptake by up to 13-fold. The observation that selective uptake of HDL-associated CE exceeded HDL3 holoparticle up to fourfold suggested that a porcine analogue of SR-BI (pSR-BI) may be involved in lipid uptake at the BBB. In line with the observation of selective lipid uptake, RT-PCR and northern and western blot analyses revealed the presence of pSR-BI in cells constituting the BBB. Adenovirus-mediated overexpression of the human analogue of SR-BI (hSR-BI) in pBCECs resulted in a fourfold increase in selective HDL-associated alphaTocH uptake. In accordance with the proposed function of SR-BI, selective HDL-CE uptake was increased sixfold in Chinese hamster ovary cells stably transfected with murine SR-BI (mSR-BI). Most importantly stable mSR-BI overexpression mediated a twofold increase in HDL-associated [14C]alphaTocH selective uptake in comparison with control cells. In line with tracer experiments, mass transfer studies with unlabelled lipoproteins revealed that mSR-BI overexpression resulted in a twofold increase in endogenous HDL3-associated alphaTocH uptake. The results of this study indicate that SR-BI promotes the uptake of HDL-associated alphaTocH into cells constituting the BBB and plays an important role during the supply of the CNS with this indispensable micronutrient.
Asunto(s)
Antígenos CD36/biosíntesis , Capilares/metabolismo , Endotelio Vascular/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Vitamina E/farmacocinética , Animales , Barrera Hematoencefálica/fisiología , Encéfalo/irrigación sanguínea , Antígenos CD36/genética , Células CHO , Capilares/citología , Células Cultivadas , Cricetinae , Endotelio Vascular/citología , Lipoproteínas HDL3 , Receptores Depuradores , Receptores Depuradores de Clase B , Porcinos , Transfección , Vitamina E/metabolismoRESUMEN
The serum amyloid A (SAA) superfamily comprises a number of differentially expressed genes with a high degree of homology in mammalian species. SAA4, an apolipoprotein constitutively expressed only in humans and mice, is associated almost entirely with lipoproteins of the high-density range. The presence of SAA4 mRNA and protein in macrophage-derived foam cells of coronary and carotid arteries suggested a specific role of human SAA4 during inflammation including atherosclerosis. Here we underline the importance of ribosome binding site (rbs)-like sequences (also known as Shine-Dalgarno sequences) in the SAA4 cDNA for expression of recombinant SAA4 protein in Escherichia coli. In contrast to rbs sequences coded by the expression vectors, rbs-like sequences in the cDNA of target protein(s) are known to interfere with protein translation via binding to the small 16S ribosome subunit, yielding low or even no expression. Here we show that PCR mutations of two rbs-like sequences in the human SAA4 cDNA promote expression of considerable amounts of recombinant SAA4 in E.coli.
Asunto(s)
Clonación Molecular , Escherichia coli/genética , Mutagénesis , Proteína Amiloide A Sérica/genética , Sitios de Unión/genética , Clonación Molecular/métodos , ADN Complementario , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribosomas/metabolismo , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismoRESUMEN
In addition to their central role in triglyceride storage, fat cells are a primary depot of unesterified cholesterol (FC) in the body. In comparison, peripheral cells contain very little FC. This difference in adipocytes versus peripheral tissues is inconsistent with the current theory of cholesterol homeostasis. Attempting to resolve this discrepancy, we examined intracellular storage sites of FC in murine 3T3-F442A adipocytes. Using the cholesterol-binding antibiotic, filipin, in combination with high resolution fluorescence microscopy, intense fluorescent staining characteristically decorated the periphery of triglyceride droplets (TGD) as well as the plasma membrane (PM) of fat cells. Filipin-staining was not visible inside the lipid droplets. Purification of TGD by subcellular fractionation demonstrated that the rise in total FC content of adipocytes upon differentiation was attributable to an increase in TGD-FC, which contributed up to one third of the total cellular FC. The protein component of purified TGD from cultured adipocytes as well as from murine adipocytes obtained from fresh tissues contained the lumenal endoplasmic reticulum (ER) immunoglobulin binding protein (BiP) and the integral ER membrane protein calnexin. Efflux experiments using the extracellular FC acceptors (&bgr;)-cyclodextrin or apolipoprotein A-I demonstrated that TGD-associated FC was releasable from TGD. Whereas FC efflux from adipocytes was unaffected in the presence of brefeldin A or monensin, the secretion of a control protein, lipoprotein lipase, was effectively reduced. In summary, our findings identify the TGD surface layer as primary intracellular storage site for FC within adipocytes. We suggest that the structural role of ER-resident proteins in this adipocyte TGD envelope has been previously neglected. Our findings support the suggestion that an ER-like structure, albeit of modified lipid composition, constitutes the lipid droplets' surface layer. Finally, the efflux process of FC from adipocytes upon extracellular stimulation with (beta)-cyclodextrin provides evidence for an energy-dependent intracellular trafficking route between the TGD-FC pool and the PM-FC sites which is distinct from the secretory pathway of proteins.