RESUMO
Background Pulsed cyclophosphamide or mycophenolate mofetil for lupus nephritis has limited efficacy. We previously reported a case of mixed-class IV + V lupus nephritis successfully treated with cyclophosphamide and tacrolimus. This study assessed the efficacy and safety of multitarget therapy with cyclophosphamide and tacrolimus for the treatment of lupus nephritis. Methods In a prospective, single-arm, open label pilot study, we recruited 15 patients aged 18-64 years with active lupus nephritis who met the American College of Rheumatology criteria for a diagnosis of systemic lupus erythematosus (1997). The treatment protocol was a starting dose of prednisolone of 0.6-1.0 mg/kg/day for 2 weeks and then tapered to a maintenance dose, intravenous cyclophosphamide (500 mg biweekly for 3 months) and tacrolimus (3.0 mg/day). Tacrolimus was continued as maintenance therapy. Complete remission was defined as a spot urine protein/creatinine ratio of < 0.5 g/gCr with no active urine casts and a serum creatinine level that was either normal or within 30% of a previously abnormal baseline level. We retrospectively compared results for the study patients with those of 18 historical controls conventionally treated with cyclophosphamide and prednisolone. Results At baseline, the mean patient age was 41.5 ± 14.6 years (male:female ratio 2:13), urine protein/creatinine ratio 3.9 ± 2.3 g/gCr and serum creatinine 84.6 ± 34.6 µmol/L. Lupus nephritis classifications included classes IV ( n = 8), III + V ( n = 1), IV + V ( n = 5) and unclassified ( n = 1). Eleven patients completed the treatment protocol and four withdrew. At 6 months, 12 of 15 (80.0%) had achieved complete remission using intention-to-treat analysis, significantly more than historical controls (seven of 18 patients, 38.9%). A transient increase in serum creatinine and gastric symptoms occurred in three cases. One patient withdrew due to cytomegalovirus antigenemia and severe diabetes, and one patient died of thrombotic microangiopathy. Conclusions Multitarget therapy with cyclophosphamide and tacrolimus can be a therapeutic option for lupus nephritis. Clinical trials registration Combination therapy of tacrolimus and intravenous cyclophosphamide for remission induction of lupus nephritis, UMIN: 000004893, URL: https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type=summary&recptno=R000005830&language=E . Date of registration: 18 January 2011.
Assuntos
Ciclofosfamida/farmacologia , Nefrite Lúpica/tratamento farmacológico , Ácido Micofenólico/farmacologia , Tacrolimo/farmacologia , Administração Intravenosa , Adulto , Creatinina/sangue , Ciclofosfamida/administração & dosagem , Quimioterapia Combinada/métodos , Inibidores Enzimáticos/farmacologia , Feminino , Glucocorticoides/uso terapêutico , Humanos , Imunossupressores/farmacologia , Japão/epidemiologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/administração & dosagem , Projetos Piloto , Prednisolona/administração & dosagem , Prednisolona/uso terapêutico , Estudos Prospectivos , Indução de Remissão , Estudos Retrospectivos , Tacrolimo/administração & dosagem , Resultado do TratamentoRESUMO
Sepsis causes long-term disability, such as immune dysfunction, neuropsychological disorders, persistent inflammation, catabolism, and immunosuppression, leading to a high risk of death in survivors, although the contributing factors of mortality are unknown. The purpose of this experimental study in rats was to examine renal (rSNA) and splanchnic (sSNA) sympathetic nerve activity, as well as baroreflex sensitivity, in acute and chronic post-sepsis periods. The rats were divided into two groups: control group with naïve Wistar rats and sepsis group with 2-mL intravenous inoculation of Escherichia coli at 108 CFU/mL. Basal mean arterial pressure, heart rate, rSNA, sSNA, and baroreflex sensitivity were evaluated in all groups at the acute (6 h) and chronic periods (1 and 3 months). Basal rSNA and sSNA were significantly reduced in the surviving rats, as was their baroreflex sensitivity, for both pressor and hypotensive responses, and this effect lasted for up to 3 months. A single episode of sepsis in rats was enough to induce long-term alterations in renal and splanchnic sympathetic vasomotor nerve activity, representing a possible systemic event that needs to be elucidated. These findings showed that post-sepsis impairment of sympathetic vasomotor response may be one of the critical components in the inability of sepsis survivors to respond effectively to new etiological illness factors, thereby increasing their risk of post-sepsis morbidity.
Assuntos
Barorreflexo , Sepse , Animais , Pressão Sanguínea , Modelos Animais de Doenças , Frequência Cardíaca , Rim , Ratos , Ratos Wistar , Sistema Nervoso SimpáticoRESUMO
The objective of this study was to elucidate the dynamic mechanism of infant tongue movement during sucking. We developed an integrated device with sensors for three-dimensional force measurements applied by the tongue to an artificial nipple. Three mini-size built-in cantilever sensors were installed in each of three sides of the regular hexagonal prism (nine sensors in total) inside the artificial nipple. Signals from the force sensors were amplified and displayed on a PC monitor via USB in real time. We conducted measurements using the system and confirmed that signals were outputted from all nine sensors. The output waveforms and force distributions showed that the force applied was larger at the nipple tip than at the nipple root and moved from the nipple root to the nipple tip.
Assuntos
Mamilos , Comportamento de Sucção , Ingestão de Alimentos , Humanos , Lactente , Monitorização Fisiológica , LínguaRESUMO
Sepsis causes long-term disability, such as immune dysfunction, neuropsychological disorders, persistent inflammation, catabolism, and immunosuppression, leading to a high risk of death in survivors, although the contributing factors of mortality are unknown. The purpose of this experimental study in rats was to examine renal (rSNA) and splanchnic (sSNA) sympathetic nerve activity, as well as baroreflex sensitivity, in acute and chronic post-sepsis periods. The rats were divided into two groups: control group with naïve Wistar rats and sepsis group with 2-mL intravenous inoculation of Escherichia coli at 108 CFU/mL. Basal mean arterial pressure, heart rate, rSNA, sSNA, and baroreflex sensitivity were evaluated in all groups at the acute (6 h) and chronic periods (1 and 3 months). Basal rSNA and sSNA were significantly reduced in the surviving rats, as was their baroreflex sensitivity, for both pressor and hypotensive responses, and this effect lasted for up to 3 months. A single episode of sepsis in rats was enough to induce long-term alterations in renal and splanchnic sympathetic vasomotor nerve activity, representing a possible systemic event that needs to be elucidated. These findings showed that post-sepsis impairment of sympathetic vasomotor response may be one of the critical components in the inability of sepsis survivors to respond effectively to new etiological illness factors, thereby increasing their risk of post-sepsis morbidity.
RESUMO
Receptor tyrosine kinases regulate cell behavior by activating specific signal transduction cascades. Epidermal growth factor (EGF) receptor tyrosine kinases include ErbB1, ErbB2, ErbB3 and ErbB4. ErbB4 is a tyrosine kinase receptor that binds neuregulins (NRG) and several other EGF family members. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis identified two isoforms of ErbB4 that differed in their cytoplasmic domain sequences. Specifically, RT-PCR using primers flanking the putative phosphatidyl inositol 3-kinase (PI3-K) binding site of ErbB4 generated two specific bands when human and mouse heart and kidney tissues were analysed. Cloning and sequencing of these RT-PCR products revealed that one of the ErbB4 isoforms (ErbB4 CYT-2) lacked a 16 amino acid sequence including a putative PI3-K binding site, that was present in the other isoform (ErbB4 CYT-1). RT-PCR analysis of mouse tissues suggested that the expression of ErbB4 CYT-1 and ErbB4 CYT-2 was tissue-specific. Heart, breast and abdominal aorta expressed predominantly ErbB4 CYT-1 whereas neural tissues and kidney expressed predominantly ErbB4 CYT-2. To ascertain whether the absence of the putative PI3-K binding site in ErbB4 CYT-2 also resulted in the loss of PI3-K activity, NIH3T3 cell lines overexpressing ErbB4 CYT-1 or ErbB4 CYT-2 were produced. NRG-1 bound to and stimulated equivalent tyrosine phosphorylation of both isoforms. However, unlike ErbB4 CYT-1, the ErbB4 CYT-2 isoform was unable to bind the p85 subunit of PI3-K and to stimulate PI3-K activity in these cells. Furthermore, tyrosine phosphorylation of p85 or association of PI3-K activity with phosphotyrosine was not induced in NRG-1 treated cells expressing ErbB4 CYT-2, indicating that this isoform was incapable of activating PI3-K even indirectly. It was concluded that a novel naturally occurring ErbB4 isoform exists with a deletion of the cytoplasmic domain sequence required for the activation of the PI3-K intracellular signal transduction pathway and that this is the only PI3-K binding site in ErbB4.
Assuntos
Receptores ErbB/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Citoplasma/enzimologia , Ativação Enzimática , Humanos , Camundongos , Ligação Proteica , Isoformas de Proteínas/metabolismo , Receptor ErbB-4 , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Uptake of oxidized low density lipoprotein (Ox-LDL) and subsequent foam cell transformation have been implicated in early atherogenesis. Although multiple molecules, including class A and B scavenger receptors, have been identified as Ox-LDL receptors, additional receptors may also be involved in this process. Here, we provide evidence that lectin-like Ox-LDL receptor-1 (LOX-1), a novel Ox-LDL receptor initially identified in vascular endothelial cells, is also expressed in macrophages in humans and mice. Expression of LOX-1 can be induced after macrophage-like differentiation in vitro in human peripheral blood monocytes and the related cell line THP-1 cells. Furthermore, LOX-1 expression can also be detected in resident peritoneal macrophages, and can be upregulated by an inflammatory cytokine TNF-alpha. These results suggest that LOX-1 in macrophages may play an important role in Ox-LDL uptake and subsequent foam cell formation in this cell type.
Assuntos
Macrófagos/metabolismo , Receptores de LDL/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Northern Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Lectinas , Leucócitos Mononucleares , Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais , Camundongos , Monócitos , Receptores de LDL/genética , Receptores de LDL Oxidado , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Depuradores Classe E , Acetato de Tetradecanoilforbol/farmacologia , Regulação para CimaRESUMO
Lysophosphatidylcholine (lyso-PC) accumulates in tissues undergoing inflammation and atherosclerosis, where an infiltration of T cells is also seen. We found that lyso-PC increased IFN-gamma production and CD40L expression in CD4+ T cells stimulated with anti-CD3 Ab and recombinant CD80 molecules, whereas lyso-PC did not affect IL-2 and IL-4 production. These results suggest that lyso-PC, in combination with other stimuli, may regulate CD4+ T cell functions to propagate local inflammatory reactions and also imply a novel role played by a modified lipid in the selection of Th1/Th2 immune response as well as in the T cell mediated pathogenesis in atherosclerosis.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Glicoproteínas de Membrana/genética , Anticorpos/farmacologia , Antígeno B7-1/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40 , Linhagem Celular , Técnicas de Cocultura , Humanos , Interferon gama/biossíntese , Ativação Linfocitária , RNA Mensageiro/metabolismo , Proteínas Recombinantes/imunologiaRESUMO
Lysophosphatidylcholine (lyso-PC), a polar phospholipid increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to induce transcription of a variety of endothelial genes relevant to atherogenesis. Lyso-PC has been shown to activate c-jun N-terminal kinase (JNK) and activator protein 1 (AP-1) and thereby stimulate transcription of the c-jun gene. Here we provide evidence that lyso-PC can phosphorylate cyclic AMP responsive element binding protein (CREB) and thereby activate the jun2 12-O-tetradecanoylphorbol 13-acetate response element (jun2TRE) site of the c-jun promoter, which appears to be the major molecular mechanism involved in lyso-PC-induced c-jun gene expression in cultured bovine aortic endothelial cells (BAEC). Transient transfection of BAEC with a 1.6-kbp c-jun promoter and luciferase reporter fusion gene resulted in a 12.9-fold increase in luciferase activity by lyso-PC treatment. Serial deletion mutation in c-jun promoter and luciferase reporter gene assay revealed that the 5' promoter region between nucleotide numbers -268 and -127, which contains a jun2TRE binding sequence, was most crucial for lyso-PC-induced transcription. The 5' promoter region between -76 and -27, which contains an AP-1 site, also affected lyso-PC-induced transcription of the c-jun gene. Point mutation in the jun2TRE site reduced lyso-PC-induced transcription of the c-jun promoter-luciferase fusion gene by a 70.3% decrease in c-jun promoter activity. Electrophoretic mobility shift assays showed increased binding of (32)P-labeled oligonucleotides with jun2TRE in nuclear extracts isolated from lyso-PC-treated BAEC, which was abolished or supershifted by anti-CREB antibody. Immunoblotting with anti-phosphorylated CREB antibody showed rapid phosphorylation of this protein after lyso-PC treatment. These results indicate that lyso-PC phosphorylates CREB, which was then bound to the jun2TRE site of the c-jun promoter and activated transcription. Activation of jun2TRE may play a key role in the transcriptional activation of c-jun as well as other endothelial genes depending upon these transcription factors.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Genes jun , Lisofosfatidilcolinas/metabolismo , Regiões Promotoras Genéticas , Animais , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Fosforilação , Transcrição Gênica/efeitos dos fármacosRESUMO
Lysophosphatidylcholine (lyso-PC) is a major phospholipid component of atherogenic lipoproteins. Lyso-PC has been shown to differentially upregulate the adhesion molecules, such as VCAM-1 and ICAM-1, as well as smooth muscle growth factors, such as PDGF-A, B chains and HB-EGF gene expression in various cultured endothelial cells. In this paper, we demonstrate increased expression of cell- and matrix-associated forms of PDGF-B protein elicited by lyso-PC and further characterized potential signal transduction mechanisms responsible for lyso-PC-induced human umbilical vein endothelial cell. Cycloheximide inhibited PDGF-B but not ICAM-1 mRNA induction by lyso-PC, suggesting the dependence on de novo protein synthesis for PDGF-B, but not ICAM-1. A protein kinase C (PKC) inhibitor did not block lyso-PC-induced increases in PDGF-B or ICAM-1 mRNA. The elevated level of cAMP blocked both PDGF-B and ICAM-1 upregulation by lyso-PC. However cAMP-elevating agents did not suppress ICAM-1 upregulation by PMA. Taken together, PDGF-B and ICAM-1 gene induction by lyso-PC may involve different signaling mechanisms; however, both appear to be independent of PMA-regulatable PKC activation but are suppressed by increased levels of intracellular cAMP.
Assuntos
Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Lisofosfatidilcolinas/farmacologia , Fator de Crescimento Derivado de Plaquetas/biossíntese , Northern Blotting , Western Blotting , Células Cultivadas , HumanosRESUMO
A critical event in the early stages of atherosclerosis is the focal accumulation of lipid-laden foam cells derived from macrophages. In various cholesterol-fed animal models of atherosclerosis, localized attachment of circulating monocytes to arterial endothelial cells appeared to precede the formation of foam cells. It is suggested that monocyte recruitment into early lesions depends on the endothelial adhesiveness for monocytes and lymphocytes. In vivo and in vitro experiments have identified molecules, such as ICAM-1, VCAM-1, and P-selectin, that can support the adhesion of monocytes and lymphocytes. Moreover, oxidized LDL, lysophosphatidyl-choline, and oxidized fatty acids induce the expression not only of these adhesion molecules but also of scavenger receptors, such as CD-36, SR-A, and LOX-1. Recently, we isolated and characterized the novel receptors for oxidized LDL, namely, LOX-1 and SR-PSOX. Expression of LOX-1 is found on endothelial cells, smooth muscle cells, and macrophages, whereas SR-PSOX is expressed on macrophages. In this paper the significance of oxidized LDL and its receptors, LOX-1 and SR-PSOX, in terms of atherogenesis is discussed.
Assuntos
Arteriosclerose/fisiopatologia , Quimiocinas CXC , Lipoproteínas LDL/fisiologia , Proteínas de Membrana , Animais , Arteriosclerose/sangue , Quimiocina CXCL16 , Colesterol/sangue , Humanos , Receptores Imunológicos/fisiologia , Receptores de LDL/fisiologia , Receptores de LDL Oxidado , Receptores Depuradores , Receptores Depuradores Classe ERESUMO
The effect of pretreatment with metallothionein (MT) inducers (bismuth nitrate or zinc chloride) on clastogenicity of anticancer drugs was investigated. Bismuth nitrate (50 mumol/kg) or zinc chloride (400 mumol/kg) was administered s.c. to mice once a day for two days prior to treatment with 3.3 mumol/kg of cis-diamminedichloroplatinum(II) (cis-DDP), 3.4 mumol/kg of adriamycin (ADR), 72 mumol/kg of cyclophosphamide (CPA) or 0.41 mumol/kg of L-phenylalanine mustard (L-PAM). The frequency of occurrence of erythrocytes with micronuclei in bone marrow was increased by each anticancer drug at 24 h after treatment. Micronucleus formation was significantly prevented by pretreatment with either bismuth nitrate or zinc chloride. MT concentration in bone marrow cells of mice at the time of treatment with anticancer drugs increased to 2- and 3.5-fold by pretreatment with bismuth nitrate and zinc chloride, respectively. These results indicate that MT induction in bone marrow cells effectively prevents micronucleus induction of anticancer drugs.
Assuntos
Antineoplásicos/toxicidade , Metalotioneína/biossíntese , Mutagênicos/toxicidade , Animais , Bismuto/farmacologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Cloretos/farmacologia , Cisplatino/toxicidade , Ciclofosfamida/toxicidade , Doxorrubicina/toxicidade , Masculino , Melfalan/toxicidade , Camundongos , Camundongos Endogâmicos ICR , Testes para Micronúcleos , Nitratos/farmacologia , Compostos de Zinco/farmacologiaRESUMO
A 48-year-old male developed acute respiratory failure owing to Legionnaires' disease (LD). Antibiotic treatment including erythromycin and rifampicin was not effective, thus transbronchial lung biopsy was performed. The histological examination of the lung showed intra-alveolar fibrosis. Corticosteroid therapy was begun and he responded well with definite clinical improvement. Bronchoalveolar lavage (BAL) was performed three times in the following year. The first BAL showed an increase of lymphocytes which then decreased significantly in the following studies. This case report thus demonstrates the importance of lung biopsy of protracted LD and the usefulness of BAL in the assessment of corticosteroid therapy.
Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Doença dos Legionários/complicações , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/patologia , Contagem de Células , Humanos , Linfocitose/patologia , Masculino , Pessoa de Meia-Idade , Prednisolona/uso terapêutico , Fibrose Pulmonar/tratamento farmacológico , Fatores de TempoRESUMO
Lysophosphatidylcholine (lyso-PC), a polar phospholipid product increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to differentially induce functional intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 and mRNA for platelet-derived growth factor (PDGF)-A and -B chains and heparin-binding epidermal growth factor-like growth factor in various cultured endothelial cells. In this study, we have demonstrated increased expression of cell- and matrix-associated forms of PDGF-B chain (PDGF-B) protein elicited by lyso-PC and further characterized potential signal transduction mechanisms responsible for lyso-PC-induced gene expression, focusing on PDGF-B and ICAM-1 genes in cultured human umbilical vein endothelial cell models. Cycloheximide almost completely inhibited PDGF-B but not ICAM-1 mRNA induction elicited by lyso-PC, suggesting that dependence on de novo protein synthesis for PDGF-B is different from that for ICAM-1. Prolonged exposure to phorbol myristate acetate (PMA), which depletes protein kinase C (PKC), or staurosporine, a PKC inhibitor, did not block lyso-PC-induced increases in PDGF-B or ICAM-1 mRNA. Forskolin and dibutyryl cAMP, which elevate intracellular cAMP levels, blocked both PDGF-B and ICAM-1 upregulation elicited by lyso-PC; however, these cAMP-elevating agents did not suppress ICAM-1 upregulation by PMA. Taken together, PDGF-B and ICAM-1 gene induction by lyso-PC may involve different signaling mechanisms; however, both appear to be independent of PMA-regulatable PKC activation but are suppressed by increased levels of intracellular cAMP.
Assuntos
AMP Cíclico/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Lisofosfatidilcolinas/farmacologia , Fator de Crescimento Derivado de Plaquetas/genética , Proteína Quinase C/fisiologia , Células Cultivadas , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Fator de Crescimento Derivado de Plaquetas/biossíntese , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Ativação TranscricionalRESUMO
Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a mitogen and chemotactic factor, binds to two receptor tyrosine kinases, erbB1 and erbB4. Now we demonstrate that HB-EGF also binds to a novel 140 kDa receptor on MDA-MB 453 cells. Purification of this receptor showed it to be identical to N-arginine dibasic convertase (NRDc), a metalloendopeptidase of the M16 family. Binding to cell surface NRDc and NRDc in solution was highly specific for HB-EGF among EGF family members. When overexpressed in cells, NRDc enhanced their migration in response to HB-EGF but not to EGF. Conversely, inhibition of endogenous NRDc expression in cells by antisense morpholino oligonucleotides inhibited HB-EGF-induced cell migration. Anti-erbB1 neutralizing antibodies completely abrogated the ability of NRDc to enhance HB-EGF-dependent migration, demonstrating that this NRDc activity was dependent on erbB1 signaling. Although NRDc is a metalloproteinase, enzymatic activity was not required for HB-EGF binding or enhancement of cell migration; neither did NRDc cleave HB-EGF. Together, these results suggest that NRDc is a novel specific receptor for HB-EGF that modulates HB-EGF-induced cell migration via erbB1.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Metaloendopeptidases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Neoplasias da Mama , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Feminino , Células HeLa , Heparina/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Metaloendopeptidases/genética , Camundongos , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Especificidade por Substrato , Transfecção , Células Tumorais CultivadasRESUMO
The accumulation of monocyte/macrophages and T lymphocytes in arterial intima is a hallmark of early atherogenesis. To investigate the temporal relationships between endothelial expression of adhesion molecules (eg, P-selectin and vascular cell adhesion molecule-1 [VCAM-1]) and intimal accumulation of macrophages and T lymphocytes, immunostaining was performed by using serial frozen sections from intercostal branch points of thoracic aortas of New Zealand White rabbits that had been fed a 0.3% cholesterol diet. After 1 week of cholesterol feeding, neither macrophages nor T lymphocytes were detected, although endothelial expression of P-selectin and VCAM-1 was observed. After 3 weeks, macrophages were detectable in 75% and T lymphocytes were present in 25% of the rabbits. Expression of P-selectin and VCAM-1 was sustained until 10 weeks. Infiltration of T lymphocytes was restricted in areas in which macrophages were accumulated and did not appear to precede macrophage infiltration. E-selectin expression was not detectable before accumulation of mononuclear leukocytes; however, very few endothelial cells covering foam cell lesions expressed E-selectin after 6 weeks. Similar results were obtained in Watanabe heritable hyperlipidemic rabbits aged 1, 2, and 3 months. Taken together, localized expression of P-selectin and VCAM-1 may play a key role in the initial recruitment of macrophages and T lymphocytes in early atherogenesis.
Assuntos
Aorta/metabolismo , Aorta/patologia , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Selectina-P/metabolismo , Túnica Íntima/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Colesterol na Dieta/farmacologia , Selectina E/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotoxinas/farmacologia , Macrófagos/patologia , Masculino , Coelhos , Linfócitos T/patologiaRESUMO
Lysophosphatidylcholine (lyso-PC), a biologically active phospholipid, appears to modulate various endothelial cell functions through tyrosine kinase-dependent signaling pathways. In cultured bovine aortic endothelial cells (BAEC), we have found that a 130 kDa protein (p130) was rapidly tyrosine phosphorylated within 2 min and sustained for, at least, 1 hr in response to 10 mumol/L of lyso-PC but not to phorbol myristate acetate (PMA). Prolonged preexposure to PMA did not affect lyso-PC-induced p130 tyrosine phosphorylation, suggesting that mechanisms independent of protein kinase C may be involved. Fractionation of the cell lysates revealed that p130 was detectable in the membrane fraction but not in the cytosolic fraction. Immunoprecipitation followed by immunoblotting of lyso-PC-treated BAEC identified p130 as bovine PECAM-1. Tyrosine phosphorylation of PECAM-1 appears to be one of the earliest events elicited by lyso-PC, and may play a role in lyso-PC-induced modulation of endothelial functions.
Assuntos
Endotélio Vascular/metabolismo , Lisofosfatidilcolinas/farmacologia , Fosfotirosina/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Animais , Aorta , Bovinos , Membrana Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Immunoblotting , Técnicas de Imunoadsorção , Fosforilação , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Accumulation of substantial numbers of activated T lymphocytes, as well as monocyte/macrophages, in focal areas of arterial intima appears to be a hallmark of atherogenesis. Our previous report demonstrated that lysophosphatidylcholine (lyso-PC), a polar phospholipid component that is increased in atherogenic lipoproteins and atherosclerotic lesions, can upregulate the expression of heparin-binding epidermal growth factor-like growth factor and the interleukin (IL)-2 receptor in cultured human peripheral T lymphocytes. In this study, we show that lyso-PC can also enhance interferon gamma (IFN-gamma) secretion and gene expression in human T lymphocytes. Lyso-PC-induced upregulation of IFN-gamma depended on the presence of IL-2, IL-12, or phytohemagglutinin in culture media and was similarly observed in both CD4+ and CD8+ subsets. Actinomycin D chase by Northern blotting showed that lyso-PC significantly prolonged IFN-gamma mRNA half-lives in human T cells. Transient transfection of IFN-gamma promoter-reporter gene construct in the human T-cell line Jurkat cells demonstrated that lyso-PC stimulated the transcription of IFN-gamma promoter-driven luciferase gene. Analyses of serial deletion mutations of IFN-gamma promoter revealed that the lyso-PC-responsive element is located between base pairs - 102 and -78 of the transcription initiation site of the IFN-gamma gene. Enhanced expression of IFN-gamma in T lymphocytes by lyso-PC may play a crucial role in atherogenesis.
Assuntos
Citocinas/farmacologia , Interferon gama/fisiologia , Lisofosfatidilcolinas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Expressão Gênica , Humanos , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-12/farmacologia , Interleucina-2/farmacologia , Fito-Hemaglutininas/farmacologia , Processamento Pós-Transcricional do RNA , Taxa Secretória/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transcrição GênicaRESUMO
Atherosclerotic lesions contain substantial numbers of activated T lymphocytes in addition to monocytes/macrophages. T cell-derived cytokines and growth factors may play a role in atherogenesis; however, stimuli responsible for T-cell activation in atherogenesis have not been fully elucidated. In this study, we provide evidence that lysophosphatidylcholine (lyso-PC), a polar phospholipid component increased in atherogenic lipoproteins and atherosclerotic lesions, can upregulate gene expression and secretion of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in cultured T lymphocytes isolated from human peripheral blood. Effects of lyso-PC on T lymphocytes appear to be selective and specific, since lyso-PC also increases interleukin (IL)-2 receptor expression but does not affect mRNA levels for IL-2 or IL-4. Lyso-PC-induced upregulation of HB-EGF and IL-2 receptor mRNA in peripheral T cells is mostly dependent on exogenous IL-2 in conditioned medium. The effect of lyso-PC on HB-EGF induction was more potent in CD4+ cells than in CD8+ cells, although lyso-PC increases IL-2 receptor expression dramatically in both CD4+ cells and CD8+ cells. Lyso-PC similarly increased HB-EGF expression in Jurkat cells, a cell line for human CD4+ T lymphocytes. These results in vitro suggest that lyso-PC may be an important stimulus for T cells in atherogenesis in vivo to upregulate HB-EGF and that T cell-derived smooth muscle growth factors may modulate atherosclerotic progression.