RESUMO
Intracellular lipid droplets (LDs) are ubiquitous organelles found in many cell types. During mitosis, membranous organelles, including mitochondria, are divided into small pieces and transferred to daughter cells; however, the process of LD transfer to daughter cells is not fully elucidated. Herein, we investigated the behavior of LDs during mitosis in HuH7 human hepatoma cells. While fragments of the Golgi apparatus were scattered in the cytosol during mitosis, intracellular LDs retained their size and spherical morphology as they translocated to the two daughter cells. LDs were initially distributed throughout the cell during prophase but positioned outside the spindle in metaphase, aligning at the far sides of the centrioles. A similar distribution of LDs during mitosis was observed in another hepatocarcinoma HepG2 cells. When the spindle was disrupted by nocodazole treatment or never in mitosis gene A-related kinase 2A knockdown, LDs were localized in the area outside the chromosomes, suggesting that spindle formation is not necessary for LD localization at metaphase. The amount of major LD protein perilipin 2 reduced while LDs were enriched in perilipin 3 during mitosis, indicating the potential alteration of LD protein composition. Conclusively, the behavior of LDs during mitosis is distinct from that of other organelles in hepatocytes.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Gotículas Lipídicas/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Metabolismo dos Lipídeos , Mitose , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismoRESUMO
Neutrophil extracellular trap (NET) formation is a unique self-defense mechanism of neutrophils; however, it is also involved in many diseases, including atherosclerosis. Resveratrol and catechin are antioxidants with anti-atherosclerotic properties. Here, we examined the effects of resveratrol, catechin, and other related compounds on NET formation. HL-60-derived neutrophils were pretreated with resveratrol and other compounds before stimulation with phorbol-myristate acetate (PMA). DNA and myeloperoxidase released from neutrophils were determined. Resveratrol suppressed the DNA release from neutrophils in a dose-dependent manner. NET formation was enhanced by 1-palmitoyl-2-oxovaleroyl phosphatidylcholine (POVPC), a truncated form of oxidized phospholipid, and resveratrol suppressed NET formation induced by POVPC and PMA. Furthermore, we designed several analogs of resveratrol or catechin whose conformation was restricted by the inhibition of the free rotation of aromatic rings. The conformationally constrained analogs were more effective at inhibiting NET formation; however, their inhibitory function decreased when compound was a large, hydrophobic analog. The most potent compounds, planar catechin and resveratrol, suppressed myeloperoxidase release from activated neutrophils. In addition, these compounds suppressed DNA release from neutrophils stimulated with calcium ionophore. These results suggest that resveratrol, catechin and their analogs exert anti-NET effects, and that constraining the geometry of these compounds enhanced their inhibitory effects.
RESUMO
A high concentration of low-density lipoproteins (LDLs) in circulation has been well-known as a major risk factor for cardiovascular diseases. The presence of oxidized LDLs (oxLDLs) in atherosclerotic lesions and circulation was demonstrated using anti-oxLDL monoclonal antibodies. The so-called "oxLDL hypothesis", as a mechanism for atherosclerosis development, has been attracting attention for decades. However, the oxLDL has been considered a hypothetical particle since the oxLDL present in vivo has not been fully characterized. Several chemically modified LDLs have been proposed to mimic oxLDLs. Some of the subfractions of LDL, especially Lp(a) and electronegative LDL, have been characterized as oxLDL candidates as oxidized phospholipids that stimulate vascular cells. Oxidized high-density lipoprotein (oxHDL) and oxLDL were discovered immunologically in vivo. Recently, an oxLDL-oxHDL complex was found in human plasma, suggesting the involvement of HDLs in the oxidative modification of lipoproteins in vivo. In this review, we summarize our understanding of oxidized lipoproteins and propose a novel standpoint to understand the oxidized lipoproteins present in vivo.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Humanos , Lipoproteínas LDL , Lipoproteínas , Aterosclerose/etiologia , Lipoproteínas HDL , Doenças Cardiovasculares/complicações , Fatores de RiscoRESUMO
Lipid droplets (LDs) are intracellular organelles that are ubiquitous in many types of cells. The LD core consists of triacylglycerols (TGs) surrounded by a phospholipid monolayer and surface proteins such as perilipin 2 (PLIN2). Although TGs accumulate in the phospholipid bilayer of the endoplasmic reticulum (ER) and subsequently nascent LDs buds from ER, the mechanism by which LD proteins are transported to LD particles is not fully understood. Sar1 is a GTPase known as a regulator of coat protein complex â ¡ (COPâ ¡) vesicle budding, and its role in LD formation was investigated in this study. HuH7 human hepatoma cells were infected with adenoviral particles containing genes coding GFP fused with wild-type Sar1 (Sar1 WT) or a GTPase mutant form (Sar1 H79G). When HuH7 cells were treated with oleic acid, Sar1 WT formed a ring-like structure around the LDs. The transient expression of Sar1 did not significantly alter the levels of TG and PLIN2 in the cells. However, the localization of PLIN2 to the LDs decreased in the cells expressing Sar1 H79G. Furthermore, the effects of Sar1 on PLIN2 localization to the LDs were verified by the suppression of endogenous Sar1 using the short hairpin RNA technique. In conclusion, it was found that Sar1 has some roles in the intracellular distribution of PLIN2 to LDs in liver cells.
Assuntos
Retículo Endoplasmático , Gotículas Lipídicas , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Perilipina-2/genética , Perilipina-2/metabolismo , Fosfolipídeos/metabolismo , Triglicerídeos/metabolismoRESUMO
Neutrophil extracellular traps (NETs) are found in patients with various diseases, including cardiovascular diseases. We previously reported that copper-oxidized low-density lipoprotein (oxLDL) promotes NET formation of neutrophils, and that the resulting NETs increase the inflammatory responses of endothelial cells. In this study, we investigated the effects of high-density lipoproteins (HDL) on NET formation. HL-60-derived neutrophils were treated with phorbol 12-myristate 13-acetate (PMA) and further incubated with oxLDL and various concentrations of HDL for 2 h. NET formation was evaluated by quantifying extracellular DNA and myeloperoxidase. We found that the addition of native HDL partially decreased NET formation of neutrophils induced by oxLDL. This effect of HDL was lost when HDL was oxidized. We showed that oxidized phosphatidylcholines and lysophosphatidylcholine, which are generated in oxLDL, promoted NET formation of PMA-primed neutrophils, and NET formation by these products was completely blocked by native HDL. Furthermore, we found that an electronegative subfraction of LDL, LDL(-), which is separated from human plasma and is thought to be an in vivo oxLDL, was capable of promoting NET formation. These results suggest that plasma lipoproteins and their oxidative modifications play multiple roles in promoting NET formation, and that HDL acts as a suppressor of this response.
Assuntos
Armadilhas Extracelulares , Lipoproteínas HDL , Humanos , Fosfolipídeos , Células Endoteliais , Lipoproteínas LDL/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Oxidized LDL (oxLDL) is a known risk factor for atherogenesis. This study aimed to reveal structural features of oxLDL present in human circulation related to atherosclerosis. When LDL was fractionated on an anion-exchange column, in vivo-oxLDL, detected by the anti-oxidized PC (oxPC) mAb, was recovered in flow-through and electronegative LDL [LDL(-)] fractions. The amount of the electronegative in vivo-oxLDL, namely oxLDL in the LDL(-) fraction, present in patients with acute MI was 3-fold higher than that observed in healthy subjects. Surprisingly, the LDL(-) fraction contained apoA1 in addition to apoB, and HDL-sized particles were observed with transmission electron microscopy. In LDL(-) fractions, acrolein adducts were identified at all lysine residues in apoA1, with only a small number of acrolein-modified residues identified in apoB. The amount of oxPC adducts of apoB was higher in the LDL(-) than in the L1 fraction, as determined using Western blotting. The electronegative in vivo-oxLDL was immunologically purified from the LDL(-) fraction with an anti-oxPC mAb. The majority of PC species were not oxidized, whereas oxPC and lysoPC did not accumulate. Here, we propose that there are two types of in vivo-oxLDL in human circulating plasma and the electronegative in vivo-oxLDL accompanies oxidized HDL.
Assuntos
Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/sangue , Infarto do Miocárdio/sangue , Infarto do Miocárdio/metabolismo , Doença Aguda , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The pellicle, the acellular organic material deposited on the surface of tooth enamel, has been thought to be derived from saliva. In this study, protein compositions of the pellicle, gingival crevicular fluid, and saliva collected from healthy adults were compared to elucidate the origin of pellicle proteins. RESULTS: The pellicle, gingival crevicular fluid, and saliva from the parotid gland or mixed gland were collected; subsequently, protein expression in samples from the respective individual was compared by SDS-PAGE and mass spectrometry. Following SDS-PAGE, proteins in the major bands were identified by mass spectrometry. The band pattern of pellicle proteins appeared different from those of gingival crevicular fluid, or saliva samples. Using mass spectrometry, 13 proteins in these samples were identified. The relative abundance of the proteins was quantitatively analyzed using mass spectrometry coupled with stable isotope labeling and by western blot. Cystatin S and α-amylase detected in pellicle were enriched in saliva samples, but not in gingival crevicular fluid, by western blot, and their abundance ratios were high in saliva and low in gingival crevicular fluid when analyzed by stable isotope labeling. Serotransferrin, however, was found only in the pellicle and gingival crevicular fluid by western blot and its abundance ratio was low in saliva. CONCLUSIONS: Our study revealed that the gingival crevicular fluid appears to contribute to pellicle formation in addition to saliva.
Assuntos
Película Dentária/química , Líquido do Sulco Gengival/química , Proteínas/análise , Saliva/química , Adulto , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Espectrometria de MassasRESUMO
AIM: Laser microdissection (LMD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) enable clinicians to analyse proteins from tissue sections. In nephrology, these methods are used to diagnose diseases of abnormal protein deposition, such as amyloidosis, but they are seldom applied to the diagnosis and pathophysiological understanding of human glomerular diseases. METHODS: Renal biopsy specimens were obtained from five patients with IgA nephropathy (IgAN), five patients with membranous nephropathy (MN) and five kidney transplant donors (as controls). From 10-µm-thick sections of formalin-fixed, paraffin-embedded specimens, 0.3-mm2 samples of glomerular tissue were subjected to LMD. The samples were analysed by LC-MS/MS and investigated clinically and histologically. RESULTS: From the control glomeruli, we identified more than 300 types of proteins. In patients with IgAN, we detected significant increases not only in IgA1 and in C3, but also in the factors related to oxidative stress and cell proliferation in comparison to the controls. In patients with MN, levels of IgG1, IgG4, C3, C4a and phospholipase-A2-receptor were significantly elevated in comparison to the controls, as were the aforementioned factors related to oxidative stress and cell proliferations detected in IgAN. CONCLUSION: Application of LMD and LC-MS/MS to renal biopsy specimens enabled us to identify not only pathognomonic proteins for the diagnosis, but also several factors possibly involved in the pathogenesis of human glomerular diseases.
Assuntos
Cromatografia Líquida/métodos , Glomerulonefrite por IGA/diagnóstico , Glomérulos Renais/patologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Biópsia , Feminino , Seguimentos , Glomerulonefrite por IGA/metabolismo , Humanos , Glomérulos Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
Neutrophil extracellular traps (NETs) significantly contribute to various pathophysiological conditions, including cardiovascular diseases. NET formation in the vasculature exhibits inflammatory and thrombogenic activities on the endothelium. NETs are induced by various stimulants such as exogenous damage-associated molecular patterns (DAMPs). Oxidatively modified low-density lipoprotein (oxLDL) has been physiologically defined as a subpopulation of LDL that comprises various oxidative modifications in the protein components and oxidized lipids, which could act as DAMPs. oxLDL has been recognized as a crucial initiator and accelerator of atherosclerosis through foam cell formation by macrophages; however, recent studies have demonstrated that oxLDL stimulates neutrophils to induce NET formation and enhance NET-mediated inflammatory responses in vascular endothelial cells, thereby suggesting that oxLDL may be involved in cardiovascular diseases through neutrophil activation. As NETs comprise myeloperoxidase and proteases, they have the potential to mediate oxidative modification of LDL. This review summarizes recent updates on the analysis of NETs, their implications for cardiovascular diseases, and prospects for a possible link between NET formation and oxidative modification of lipoproteins.
Assuntos
Armadilhas Extracelulares/metabolismo , Lipoproteínas LDL/metabolismo , Neutrófilos/metabolismo , Aterosclerose/metabolismo , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/metabolismo , Células Endoteliais/metabolismo , Armadilhas Extracelulares/imunologia , Células Espumosas/metabolismo , Humanos , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Neutrófilos/imunologia , Oxirredução , Peroxidase/metabolismoRESUMO
AIM: Gingivitis commonly progresses to periodontitis in permanent dentition but rarely in deciduous teeth. Little is known about the biochemical differences between gingiva of deciduous and permanent teeth. Here, we compared the protein profiles of gingival crevicular fluids (GCF) from the gingiva of deciduous and permanent teeth. MATERIALS AND METHODS: Forty children with mixed dentition (Hellman's dental age IIIA) were selected and GCF samples were collected from deciduous cuspids and central incisors in the maxilla. Pairs of GCF samples were labelled using isobaric tags to permit quantitative comparison of protein abundance in the samples using liquid chromatography-electron spray ionization-tandem mass spectrometry. RESULTS: Sixty-two proteins were upregulated in deciduous teeth GCF and 54 in permanent teeth GCF. In particular, neutrophil-derived proteins, including myeloperoxidase and lactoferrin, were repeatedly higher in deciduous teeth GCF than in permanent teeth GCF. These differences were verified using ELISA (p < 0.01). In contrast, immunoglobulin components were upregulated in permanent teeth GCF. CONCLUSIONS: Neutrophil-related proteins were enriched in deciduous teeth GCF and immunoglobulins in permanent teeth GCF. This suggests that neutrophil accumulation plays a protective role in innate immunity against bacterial infection in gingival tissue of deciduous teeth.
Assuntos
Dentição Mista , Líquido do Sulco Gengival/química , Proteômica , Criança , Feminino , Humanos , MasculinoRESUMO
Lipid droplets (LDs) are functional subcellular organelles involved in multiple intracellular processes. LDs are found in nearly all types of eukaryotic cells, but their properties are highly variable in different types of tissues. Steroidogenic cells synthesize steroid hormones de novo from the cholesterol deposited in cytosolic LDs. However, the roles of LD proteins in steroidogenesis under pituitary hormone stimulation have not been well elucidated. The protein profile of isolated LDs from the mouse Leydig tumor cell line MLTC-1 was distinct from that of hepatic cells or macrophages. By proteomic analysis of the components using mass spectrometry, two enzymes for steroidogenesis, 3ß-hydroxysteroid dehydrogenase type 1 (3ßHSD1) and 17 ß-hydroxysteroid dehydrogenase type 11 (17ßHSD11), were identified in two strong bands in the LD fractions. The LD fraction of MLTC-1 cells also included CYP11A1 and CYP17, suggesting that the LDs contain all the enzymes needed for testosterone synthesis. The steroidogenesis in Leydig cells is activated by luteinizing hormone through a PKA-dependent pathway. Stimulation of MLTC-1 cells with luteinizing hormone or 8-bromo-cAMP caused drastic changes in the morphology of the LDs in the MLTC-1 cells. Upon stimulation, large perinuclear LDs are turned into much smaller LDs and dispersed throughout the cytosol. These results raise the possibility that LDs are involved in a regulatory pathway of steroidogenesis, not just by serving as a storage depot for cholesterol esters, but also by providing enzymes and generating sites for enzymatic activity.
Assuntos
Citosol/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Intersticiais do Testículo/enzimologia , Hormônio Luteinizante/farmacologia , Testosterona/biossíntese , Animais , Linhagem Celular Tumoral , Células Intersticiais do Testículo/citologia , Masculino , CamundongosRESUMO
Second generation antipsychotics are useful for the treatment of schizophrenia, but concerns have been raised about the side effects of diabetes mellitus and obesity. Olanzapine, especially, is associated with more weight gain than the others. It has been reported that olanzapine promotes adipocyte-differentiation in rodents both in vivo and in vitro. In this study the effects of antipsychotics on human adipocytes were investigated by using human mesenchymal stem cells (hMSCs). When hMSCs were differentiated and treated with various antipsychotics, olanzapine and clozapine increased intracellular lipids. Olanzapine induced lipid accumulation in a dose-dependent manner. Proteomic analysis revealed that PLIN4 and several enzymes for lipid metabolism were increased in the hMSCs after olanzapine treatment. During adipocyte differentiation, olanzapine increased the protein expression of PLIN1, PLIN2 and PLIN4. These proteins are known to be associated with the initial stage of lipid droplet formation. Immunocytochemistry showed that olanzapine increased and enlarged the lipid droplets coated with PLIN1 and PLIN2 while PLIN4 was largely distributed in the cytosol. mRNA expression of PLIN2, but not PLIN1 or PLIN4, was increased by olanzapine. On the other hand, olanzapine did not alter the mRNA level of transcription regulators involved in adipocyte-differentiation or adipokines. The present study shows that olanzapine induced transient PLIN2 expression in hMSCs that could result in an accumulation of lipid droplets and overexpression of PLIN1 and PLIN4, providing information of possible interest for olanzapine-induced weight gain.
Assuntos
Adipócitos/efeitos dos fármacos , Antipsicóticos/farmacologia , Benzodiazepinas/farmacologia , Proteínas de Transporte/metabolismo , Gotículas Lipídicas/metabolismo , Fosfoproteínas/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Olanzapina , Perilipina-1 , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
Angiotensin II (Ang II) has been implicated in the development of abdominal aortic aneurysm (AAA). In vascular smooth muscle cells (VSMC), Ang II activates epidermal growth factor receptor (EGFR) mediating growth promotion. We hypothesized that inhibition of EGFR prevents Ang II-dependent AAA. C57BL/6 mice were co-treated with Ang II and ß-aminopropionitrile (BAPN) to induce AAA with or without treatment with EGFR inhibitor, erlotinib. Without erlotinib, 64.3% of mice were dead due to aortic rupture. All surviving mice had AAA associated with EGFR activation. Erlotinib-treated mice did not die and developed far fewer AAA. The maximum diameters of abdominal aortas were significantly shorter with erlotinib treatment. In contrast, both erlotinib-treated and non-treated mice developed hypertension. The erlotinib treatment of abdominal aorta was associated with lack of EGFR activation, endoplasmic reticulum (ER) stress, oxidative stress, interleukin-6 induction and matrix deposition. EGFR activation in AAA was also observed in humans. In conclusion, EGFR inhibition appears to protect mice from AAA formation induced by Ang II plus BAPN. The mechanism seems to involve suppression of vascular EGFR and ER stress.
Assuntos
Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/prevenção & controle , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Aminopropionitrilo , Angiotensina II , Animais , Aorta Abdominal/enzimologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/enzimologia , Ruptura Aórtica/enzimologia , Ruptura Aórtica/prevenção & controle , Células Cultivadas , Citoproteção , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Matriz Extracelular/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Integrin-linked kinase predominantly localizes at focal adhesions to regulate actin cytoskeletal dynamics, including cell migration and matrix remodelling. Although recent studies have suggested both physiological and pathophysiological roles of integrin-linked kinase in the cardiovascular and renal system, its involvement in hypertensive organ dysfunctions, such as those that occur in kidney, has not been investigated. In the present issue of Clinical Science, Alique and co-workers have demonstrated that angiotensin II-induced renal inflammatory responses were attenuated in mice with conditional deficiency of integrin-linked kinase, which were associated with suppression of nuclear factor κB activation and reactive oxygen species generation but not hypertension. The significance, potential mechanisms and future direction are presented and discussed in this Commentary.
Assuntos
Angiotensina II/farmacologia , Nefrite/enzimologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Feminino , HumanosRESUMO
Although AngII (angiotensin II) and its receptor AT1R (AngII type 1 receptor) have been implicated in AAA (abdominal aortic aneurysm) formation, the proximal signalling events primarily responsible for AAA formation remain uncertain. Caveolae are cholesterol-rich membrane microdomains that serve as a signalling platform to facilitate the temporal and spatial localization of signal transduction events, including those stimulated by AngII. Cav1 (caveolin 1)-enriched caveolae in vascular smooth muscle cells mediate ADAM17 (a disintegrin and metalloproteinase 17)-dependent EGFR (epidermal growth factor receptor) transactivation, which is linked to vascular remodelling induced by AngII. In the present study, we have tested our hypothesis that Cav1 plays a critical role for the development of AAA at least in part via its specific alteration of AngII signalling within caveolae. Cav1-/- mice and the control wild-type mice were co-infused with AngII and ß-aminopropionitrile to induce AAA. We found that Cav1-/- mice with the co-infusion did not develop AAA compared with control mice in spite of hypertension. We found an increased expression of ADAM17 and enhanced phosphorylation of EGFR in AAA. These events were markedly attenuated in Cav1-/- aortas with the co-infusion. Furthermore, aortas from Cav1-/- mice with the co-infusion showed less endoplasmic reticulum stress, oxidative stress and inflammatory responses compared with aortas from control mice. Cav1 silencing in cultured vascular smooth muscle cells prevented AngII-induced ADAM17 induction and activation. In conclusion, Cav1 appears to play a critical role in the formation of AAA and associated endoplasmic reticulum/oxidative stress, presumably through the regulation of caveolae compartmentalized signals induced by AngII.
Assuntos
Angiotensina II/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Caveolina 1/metabolismo , Regulação da Expressão Gênica , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Proteínas ADAM/metabolismo , Proteína ADAM17 , Adenoviridae/metabolismo , Animais , Células Cultivadas , Inativação Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Imuno-Histoquímica , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos de Músculo Liso/citologia , Estresse Oxidativo , Regiões Promotoras Genéticas , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Sistema Renina-Angiotensina , Transdução de SinaisRESUMO
BACKGROUND: Oxidized phosphatidylcholines (oxPC) and lysophosphatidylcholine (lysoPC) generated during the formation of oxidized low-density lipoprotein (oxLDL) are involved in atherosclerotic lesion development. We investigated the time course-changes in phosphatidylcholine (PC) molecular species during oxidation of LDL to determine how those atherogenic PCs are produced. METHODS: Human and rabbit LDLs were pretreated with or without a selective platelet-activating factor acetylhydrolase (PAF-AH) inhibitor. LDL was oxidized by incubation with copper sulfate, and PC profiles were analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: When human LDL was oxidized, the peak areas for polyunsaturated fatty acid (PUFA)-containing PC species dramatically decreased after a short lag period, concomitantly lysoPC species increased sharply. Although a variety of oxPC species containing oxidized fatty acyl groups or cleaved acyl chains are formed during LDL oxidation, only a few oxPC products accumulated in oxLDL: 1-palmitoyl-2-(9-oxo-nonanoyl) PC and long-chain oxPC with two double bonds. Pretreatment of LDL with the PAF-AH inhibitor greatly reduced lysoPC production while it had no effect on lipid peroxidation reactions and oxPC profiles. Rabbit LDL, which has a different composition of PC molecular species and needs a longer time to reach achieve full oxidation than human LDL, also accumulated lysoPC during oxidation. The increase in lysoPC in rabbit oxLDL was suppressed by pretreatment with the PAF-AH inhibitor. The major oxPC species formed in rabbit oxLDL were almost the same as human oxLDL. CONCLUSIONS: These results suggest that lysoPC species are the major products and PAF-AH activity is crucial for lysoPC generation during oxidation of LDL. The oxPC species accumulated are limited when LDL is oxidized with copper ion in vitro.
Assuntos
Lipoproteínas LDL/química , Fosfatidilcolinas/química , 1-Alquil-2-acetilglicerofosfocolina Esterase/antagonistas & inibidores , 1-Alquil-2-acetilglicerofosfocolina Esterase/química , Animais , Apolipoproteínas B/química , Sulfato de Cobre/química , Humanos , Cinética , Oxidantes/química , Oxirredução , Coelhos , Inibidores de Serina Proteinase/química , Sulfonas/química , Espectrometria de Massas em TandemRESUMO
Periodontitis is characterized by chronic gingival tissue inflammation, and inflammatory mediators such as IL-8 and prostaglandin E(2) (PGE(2)) are associated with disease progression. Previously we showed that oxidatively modified low-density lipoprotein (oxLDL) was present in gingival crevicular fluid. In this study, the role of oxLDL in the gingival epithelial cell inflammatory response was further investigated using Ca9-22 cells and primary human oral keratinocytes (HOK). Treatment of Ca9-22 cells and HOK with oxLDL induced an up-regulation of IL-8 and the PGE(2)-producing enzymes, cyclooxygenase-2 and microsomal PGE(2) synthase-1. These responses induced by oxLDL were significantly suppressed by a nuclear factor-kappa B (NF-κB) inhibitor. However, unlike the result in macrophages, oxLDL did not lead to an increase in CD36 expression in these two cells. These results suggest that oxLDL elicits gingival epithelial cell inflammatory responses through an activation of the NF-κB pathway. These data suggest a mechanistic link between periodontal disease and lipid metabolism-related disorders, including atherosclerosis.
Assuntos
Periodontite Crônica/enzimologia , Ciclo-Oxigenase 2/metabolismo , Gengiva/enzimologia , Oxirredutases Intramoleculares/metabolismo , Lipoproteínas LDL/metabolismo , Antígenos CD36/biossíntese , Linhagem Celular , Periodontite Crônica/induzido quimicamente , Dinoprostona/biossíntese , Gengiva/efeitos dos fármacos , Humanos , Interleucina-8/biossíntese , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Lipoproteínas LDL/farmacologia , Microssomos/enzimologia , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/enzimologia , NF-kappa B/metabolismo , Prostaglandina-E Sintases , Regulação para CimaRESUMO
Apolipoprotein E (apoE) is one of the major transporters of cholesterol in the body and is essential for maintaining various neural functions in the brain. Given that hypercholesterolemia is a risk factor in Alzheimer's disease (AD), it has been suggested that altered cholesterol metabolism may be involved in the development of the pathogenesis, including neural degeneration, commonly observed in AD patients. Neurotrophic factors and their receptors, which are known to regulate various neural functions, are also known to be altered in various neurodegenerative diseases. We therefore hypothesized that cholesterol metabolism may itself influence the neurotrophin system within the brain. We decided to investigate this possibility by modulating the amount of dietary cholesterol given to apoE-knockout (apoE-KO) and wild-type (WT) mice, and examining the mRNA expression of various neurotrophin ligands and receptors in their hippocampal formations. Groups of eight-week-old apoE-KO and WT mice were fed a diet containing either "high" (HCD) or "normal" (ND) levels of cholesterol for a period of 12 weeks. We found that high dietary cholesterol intake elevated BDNF mRNA expression in both apoE-KO and WT mice and TrkB mRNA expression in apoE-KO animals. On the other hand, NGF and TrkA mRNA levels remained unchanged irrespective of both diet and mouse type. These findings indicate that altered cholesterol metabolism induced by HCD ingestion combined with apoE deficiency can elicit a differential response in the various neurotrophin ligand/receptor systems in the mouse hippocampus. Whether such changes can lead to neural degeneration, and the mechanisms that may be involved in this, awaits further research.
Assuntos
Apolipoproteínas E/deficiência , Fator Neurotrófico Derivado do Encéfalo , Colesterol na Dieta , Hipocampo/metabolismo , Receptor trkB , Doença de Alzheimer/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Colesterol na Dieta/efeitos adversos , Colesterol na Dieta/metabolismo , Humanos , Hipercolesterolemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Crescimento Neural/efeitos dos fármacos , Fator de Crescimento Neural/metabolismo , RNA Mensageiro/metabolismo , Receptor trkA/efeitos dos fármacos , Receptor trkA/metabolismo , Receptor trkB/efeitos dos fármacos , Receptor trkB/metabolismoRESUMO
Oxidative modification of lipoproteins is implicated in the occurrence and development of atherosclerotic lesions. Earlier studies have elucidated on the mechanisms of foam cell formation and lipid accumulation in these lesions, which is mediated by scavenger receptor-mediated endocytosis of oxidized low-density lipoprotein (oxLDL). Mounting clinical evidence has supported the involvement of oxLDL in cardiovascular diseases. High-density lipoprotein (HDL) is known as anti-atherogenic; however, recent studies have shown circulating oxidized HDL (oxHDL) is related to cardiovascular diseases. A modified structure of oxLDL, which was increased in the plasma of patients with acute myocardial infarction, was characterized. It had two unique features: (1) a fraction of oxLDL accompanied oxHDL, and (2) apoA1 was heavily modified, while modification of apoB, and the accumulation of oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) was less pronounced. When LDL and HDL were present at the same time, oxidized lipoproteins actively interacted with each other, and oxPC and lysoPC were transferred to another lipoprotein particle and enzymatically metabolized rapidly. This brief review provides a novel view on the dynamics of oxLDL and oxHDL in circulation.
RESUMO
The continuous formation and accumulation of oxidized lipids (e.g., lipid hydroperoxides (LOOH)) which are present even in plasma lipoproteins of healthy subjects, are ultimately considered to be linked to various diseases. Because lipid peroxidation mechanisms (i.e., radical, singlet oxygen, and enzymatic oxidation) can be suppressed by certain proper antioxidants (e.g., radical oxidation is efficiently suppressed by tocopherol), in order to suppress lipid peroxidation successfully, the determination of the peroxidation mechanism involved in the formation of LOOH is deemed crucial. In this study, to determine the peroxidation mechanisms of plasma lipoproteins of healthy subjects, we develop novel analytical methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PC 16:0/18:2;OOH) and cholesteryl linoleate hydroperoxide (CE 18:2;OOH) isomers. Using the newly developed methods, these PC 16:0/18:2;OOH and CE 18:2;OOH isomers in the low-density lipoprotein (LDL) and high-density lipoprotein (HDL) of healthy subjects are analyzed. Consequently, it is found that predominant PC 16:0/18:2;OOH and CE 18:2;OOH isomers in LDL and HDL are PC 16:0/18:2;9OOH, PC 16:0/18:2;13OOH, CE 18:2;9OOH, and CE 18:2;13OOH, which means that PC and CE in LDL and HDL are mainly oxidized by radical and/or enzymatic oxidation. In conclusion, the insights about the oxidation mechanisms shown in this study would be useful for a more effective suppression of oxidative stress in the human organism.