RESUMEN
We previously isolated islet stellate cells (ISCs) from healthy Wistar rat islets. In the present study, we isolated "already primed by diabetic environment" ISCs from islets of Goto-Kakizaki rats, determined the gene profile of these cells, and assessed the effects of these ISCs on beta-cell function and survival. We detected gene expression of ISCs by digital gene expression. INS-1 cell proliferation, apoptosis, and insulin production were measured after being treated with ISCs supernatant (SN). We observed the similar expression pattern of ISCs and PSCs, but 1067 differentially expressed genes. Insulin production in INS-1 cells cultured with ISC-SN was significantly reduced. The 5-ethynyl-2'-deoxyuridine-positive INS-1 cells treated with ISC-SN were decreased. Propidium iodide- (PI-) positive INS-1 cells were 2.6-fold higher than those in control groups. Caspase-3 activity was increased. In conclusion, ISCs presented in fibrotic islet of GK rats might be special PSCs, which impaired beta-cell function and proliferation and increased beta-cell apoptosis.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Células Estrelladas Pancreáticas/metabolismo , Animales , Apoptosis/fisiología , Proliferación Celular/fisiología , Diabetes Mellitus Tipo 2/patología , Fibrosis/metabolismo , Fibrosis/patología , Células Secretoras de Insulina/patología , Células Estrelladas Pancreáticas/patología , RatasRESUMEN
BACKGROUND: Pancreatic stellate cells (PSCs) promote metastasis as well as local growth of pancreatic cancer. However, the factors mediating the effect of PSCs on pancreatic cancer cells have not been clearly identified. METHODS: We used a modified Boyden chamber assay as an in vitro model to investigate the role of PSCs in migration of Panc1 and UlaPaCa cells and to identify the underlying mechanisms. RESULTS: PSC supernatant (PSC-SN) dose-dependently induced the trans-migration of Panc1 and UlaPaCa cells, mainly via haptokinesis and haptotaxis, respectively. In contrast to poly-L-lysine or fibronectin, collagen I resembled PSC-SN with respect to its effect on cancer cell behaviours, including polarised morphology, facilitated adhesion, accelerated motility and stimulated trans-migration. Blocking antibodies against integrin α2/ß1 subunits significantly attenuated PSC-SN- or collagen I-promoted cell trans-migration and adhesion. Moreover, both PSC-SN and collagen I induced the formation of F-actin and focal adhesions in cells, which was consistent with the constantly enhanced phosphorylation of focal adhesion kinase (FAK, Tyr397). Inhibition of FAK function by an inhibitor or small interference RNAs significantly diminished the effect of PSC-SN or collagen I on haptotaxis/haptokinesis of pancreatic cancer cells. CONCLUSION: Collagen I is the major mediator for PSC-SN-induced haptokinesis of Panc1 and haptotaxis of UlaPaCa by activating FAK signalling via binding to integrin α2ß1.
Asunto(s)
Movimiento Celular/fisiología , Colágeno Tipo I/metabolismo , Hígado/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Células Estrelladas Pancreáticas/patología , Actinas/metabolismo , Adhesión Celular/fisiología , Línea Celular Tumoral , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Neoplasias Pancreáticas/enzimología , Células Estrelladas Pancreáticas/metabolismo , Fosforilación/fisiología , Transducción de SeñalRESUMEN
In type 2 diabetes mellitus, pancreatic stellate cells (PSCs) are present within and surrounding pancreatic islets and may cause progressive fibrosis and deterioration of pancreatic beta cell function. However, it is unknown whether pancreatic beta cells influence the biological behavior of PSCs. In the present study, we examined the impact of pancreatic beta cells on the proliferation, migration and extracellular matrix (ECM) production of PSCs. PSCs were treated with conditioned media from INS-1 cells (supernatant, SN). Although the proliferation of PSCs incubated with INS-1-SN was increased compared to control, INS-1-SN treatment induced matrix metalloproteinase-2 activity and reduced the production of ECM and TGF-ß1. In addition, PSCs treated with INS-1-SN reduced the secretion of cytokines that are known to mediate pancreatic beta cell death, such as FADD, Fas, IFN-γ, IL-1, TNF-α, and TRAIL. Our findings suggest that pancreatic beta cells may ameliorate islet fibrosis and the progression of islet dysfunction.
Asunto(s)
Comunicación Celular , Matriz Extracelular/metabolismo , Células Secretoras de Insulina/metabolismo , Células Estrelladas Pancreáticas/metabolismo , Animales , Línea Celular , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Citocinas/biosíntesis , Diabetes Mellitus Tipo 2/metabolismo , RatasRESUMEN
BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) is a multi-faceted protein-modulating cell-cell and cell-matrix interactions. In cancer, SPARC can be not only associated with a highly aggressive phenotype, but also acts as a tumour suppressor. The aim of this study was to characterise the function of SPARC and its modulation by fibroblast growth factor receptor (FGFR) 1 isoforms in pancreatic ductal adenocarcinoma (PDAC). METHODS AND RESULTS: Exogenous SPARC inhibited growth, movement, and migration. ShRNA inhibition of endogenous SPARC in ASPC-1 and PANC-1 cells resulted in increased anchorage-dependent and -independent growth, transwell migration, and xenograft growth as well as increased mitogenic efficacy of fibroblast growth factor (FGF) 1 and FGF2. Endogenous SPARC expression in PANC-1 cells was increased in FGFR1-IIIb over-expressing cells, but decreased in FGFR1-IIIc over-expressing cells. The up-regulation of endogenous SPARC was abrogated by the p38-mitogen-activated protein kinase inhibitor SB203580. SPARC was detectable in conditioned medium of pancreatic stellate cells (PSCs), but not PDAC cells. Conditioned medium of PDAC cells reduced endogenous SPARC expression of PSCs. CONCLUSION: Endogenous SPARC inhibits the malignant phenotype of PDAC cells and may, therefore, act as a tumour suppressor in PDAC. Endogenous SPARC expression can be modulated by FGFR1-III isoform expression. In addition, PDAC cells may inhibit endogenous SPARC expression in surrounding PSCs by paracrine actions.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas de Neoplasias/fisiología , Osteonectina/fisiología , Neoplasias Pancreáticas/patología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/fisiología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , División Celular/fisiología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Movimiento Celular/fisiología , Medios de Cultivo Condicionados/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Osteonectina/biosíntesis , Osteonectina/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Comunicación Paracrina , Fenotipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Piridinas/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes de Fusión/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
The failure of malignant cells to undergo apoptosis is a major obstacle in cancer therapy, and thus identifying the underlining molecules involved therein is imperative for improving patient survival. An important mechanism of drug resistance is cell adhesion-mediated drug resistance (CAM-DR). In this study we identify a novel switch by which glioblastoma multiforme (GBM) cells alter the mode of CAM-DR. In the absence of a microenvironmental cue provided by components of the extracellular matrix (ECM), GBM cells are able to employ an alternative, but equally effective, mode of CAM-DR by forming spheres via cell-cell interactions. Intriguingly, when inhibiting cell-cell interactions in the absence of ECM components, either by low cell density or by inhibition of gap junctions (intercellular connexin tunnels) through chemical inhibition with carbenoxyolone or co-incubation with the connexin-mimicking Gap 27 Cx37,43 peptide, GBM cells were sensitized to tumor necrosis factor-related apoptosis-inducing ligand- and CD95-induced apoptosis. By demonstrating that GBM cells can alternate from one form of CAM-DR (cell-substrate tethering) to another (homocellular cell-cell adhesion) and that inhibition of both forms is necessary for apoptosis sensitization, our findings not only have important implications for novel approaches to restore defective apoptosis programs, but also reveal a novel role of gap junctions in GBM.
Asunto(s)
Neoplasias Encefálicas/patología , Adhesión Celular/genética , Resistencia a Antineoplásicos , Glioblastoma/patología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proteínas de la Matriz Extracelular/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Transducción de SeñalRESUMEN
Oxidized low density lipoproteins (oxLDL), macrophages and T-lymphocytes are present in atherosclerotic lesions. We and others have shown that oxLDL is cytotoxic for macrophages, endothelial, smooth muscle and activated T-lymphocytes and induce apoptosis. Here we demonstrate that (i) oxidized LDL (oxLDL), oxidized VLDL (oxVLDL) and hydrogen peroxide (H2O2) induce apoptosis in human T-lymphocytes and (ii) mitogen-activated protein kinases are involved in this process. Apoptosis was monitored by immunofluorescence microscopy and flow cytometry for annexin V binding, Apo 2.7 expression, the TUNEL reaction and caspase 3 activity. In the presence of oxLDL (100 microg/ml), oxVLDL (50 microg/ml) and H2O2 (5 mM), the fraction of apoptotic cells increased within 6 hours to more than 70%. Preincubation of lymphocytes with the MAPKK inhibitor PD-98059 and the p38MAPK inhibitor SB-203580 almost completely abolished these effects. Furthermore, oxLDL and H2O2 but not native LDL strongly enhanced phosphorylation of JNK, p38MAPK and p42/44MAPK. The results suggest that in the resting lymphocyte apoptosis triggered by oxidized lipoproteins and oxidative stress depends on the activation of p44/42MAPK and p38MAPK cascades.
Asunto(s)
Apoptosis/efectos de los fármacos , Lipoproteínas LDL/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Linfocitos T/metabolismo , Anexina A5/metabolismo , Caspasa 3/análisis , Caspasa 3/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Oxidación-Reducción , Estrés Oxidativo , Piridinas/farmacología , Factores de TiempoRESUMEN
BACKGROUND: A broad spectrum of hepatobiliary disorders are found in patients with inflammatory bowel diseases. The aim of the present work was to study interactions between gut and liver in experimental rat models of colitis and small bowel inflammation. MATERIALS AND METHODS: Colitis was induced either by trinitrobenzene sulphonic acid or dextran sodium sulphate. Small-bowel inflammation was induced by indomethacin. Bile acid secretion, bile acid pool, and cholesterol 7-alpha hydroxylase were studied. Cholesterol 7-alpha hydroxylase protein expression was analysed in the microsomal liver fraction. As portal mediators released form the inflamed gut we measured lipopolysaccharide, tumour necrosis factor-alpha and interleukin-1beta in portal serum. The hepatic inflammatory response was evaluated by binding activity of nuclear factor-kappaB, activator protein-1 and alpha-2-macroglobulin. RESULTS: Increased bile acid secretion, total bile acid content in gut and liver (bile acid pool size), and hepatic cholesterol 7-alpha hydroxylase protein and mRNA levels were found in the two colitis models associated with only a minor hepatic acute phase and cytokine response. In contrast, during indomethacin-induced small-bowel inflammation bile acid secretion, pool size, and cholesterol 7-alpha hydroxylase decreased in parallel to a strong hepatic cytokine and acute phase response. CONCLUSIONS: Colitis without portal cytokine release and acute phase reaction shows an induction of bile acid secretion, pool size, and cholesterol 7-alpha hydroxylase. In contrast, intestinal inflammation after indomethacin treatment is associated with an acute phase response and a repression of bile acid synthesis.
Asunto(s)
Ácidos y Sales Biliares/biosíntesis , Colitis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Ácido Trinitrobencenosulfónico/farmacología , Animales , Colitis/inducido químicamente , Sulfato de Dextran/farmacología , Indometacina/farmacología , Enfermedades Inflamatorias del Intestino/inducido químicamente , Masculino , Modelos Biológicos , FN-kappa B/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción AP-1/metabolismoRESUMEN
OBJECTIVES AND METHODS: In idiopathic pulmonary fibrosis (IPF), proliferation of fibroblasts and increased matrix deposition result in pulmonary damage and respiratory insufficiency. We cultured human fibroblasts from lung biopsies of healthy adults and of three patients with IPF (histologically usual interstital pneumonitis, UIP) in order to compare proliferation ([(3)H]thymidine incorporation, cell count) and matrix protein expression (immune fluorescence, quantification of fibronectin synthesis using time-resolved immune fluorescence) of normal and UIP fibroblasts in response to various growth factors. FINDINGS: The growth factors platelet-derived growth factor-BB (PDGF), epidermal growth factor (EGF), insulin growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), tumor necrosis factor alpha (TNFalpha), Transforming growth factor-beta (TGFbeta(1)), and fibroblast growth factor-2 (FGF-2) stimulate proliferation of normal lung fibroblasts significantly more than proliferation of UIP fibroblasts. Immunofluorescence reveals extensive expression of collagen I, collagen III, and fibronectin induced by serum, TGFbeta(1), and TNFalpha. This expression is more pronounced in UIP fibroblasts than in normal fibroblasts. Quantification of fibronectin synthesis reveals an enhanced fibronectin synthesis by UIP fibroblasts in response to PDGF, EGF, IGF-1, IGF-2, TNFalpha, TGFbeta(1), and FGF-2). CONCLUSIONS: Fibroblasts from normal and UIP lungs differ in their response to growth factors: Whereas normal fibroblasts show a predominantly proliferative response, UIP fibroblasts show an enhanced synthetic activity. Different fibroblast responses may contribute to progressive pulmonary fibrosis in patients with UIP.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/fisiología , Sustancias de Crecimiento/farmacología , Fibrosis Pulmonar/fisiopatología , Actinas/metabolismo , Fibroblastos/efectos de los fármacos , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Fibrosis Pulmonar/patologíaRESUMEN
In recent years, numerous studies have provided novel insights into the pathomechanisms of pancreatic fibrogenesis. This includes in particular the identification and characterization of the pancreatic stellate cells (PSCs) and their role in the synthesis of extracellular matrix (ECM) proteins. It has become clear that pancreatic stellate cell activation is regulated by a complex network of growth factors and cytokines and results in increased expression and release of collagens I and II, fibronectin and other components of ECM. Among the cytokines involved in PSC activation and other fundamental mechanisms of pancreatic fibrosis, transforming growth factor beta (TGFbeta) is of particular relevance. TGFbeta stimulates PSC activation and induces transcription of ECM proteins mainly via activation of the Smad proteins which regulate gene expression through functional interaction with co-operating partner proteins such as the zinc finger transcription factor Sp1. Recent progress in understanding of the biochemical and molecular mechanisms of pancreatic fibrosis, is reviewed here.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Páncreas/metabolismo , Páncreas/patología , Enfermedades Pancreáticas/metabolismo , Enfermedades Pancreáticas/patología , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibrosis , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Pancreatitis/metabolismo , Pancreatitis/patología , Proteínas Represoras , Proteínas Smad , Transactivadores/metabolismo , Factores de Transcripción , Regulación hacia ArribaRESUMEN
Biological markers in nasal secretions provide valuable information on nasal pathophysiology. However, published data on biomarker concentrations in nasal fluids are remarkably inconsistent, and the bias due to different sampling techniques, has not yet been systematically evaluated. Concentrations of various protein were repeatedly determined in nasal secretions of 16 healthy volunteers. The proteins were detected by using: 1) alpha2-macroglobulin as a marker for plasma contamination; 2) lactoferrin as a marker for glandular secretion; 3) lactate dehydrogenase as a marker for tissue injury; and 4) interleukin (IL)-1beta, IL-8, tumour necrosis factor-alpha, and eosinophil cationic protein and tryptase as indicators for tissue inflammation. A total of four different sampling methods, including nasal lavage (NL) and a new polyurethane foam sampler technique (PFST) were employed. Analyte concentrations in NL were approximately 10-times lower than in specimens obtained by PFST. Due to the unpredictable dilution during NL, various analytes were below the detection limit of the high sensitivity assays employed. With PFST, concentrations below the detection limit rarely occurred. The specimens did not significantly differ regarding plasma contamination, glandular secretion or tissue injury. The considerable variability of reported analyte concentrations in nasal secretions mainly results from different sampling techniques. To collect nasal secretions, samplers are considered superior to nasal lavage techniques.
Asunto(s)
Biomarcadores/análisis , Líquido del Lavado Nasal/química , Mucosa Nasal/metabolismo , Ribonucleasas , Adolescente , Adulto , Análisis de Varianza , Proteínas Sanguíneas/análisis , Citocinas/análisis , Proteínas en los Gránulos del Eosinófilo , Humanos , L-Lactato Deshidrogenasa/análisis , Lactoferrina/análisis , Persona de Mediana Edad , Serina Endopeptidasas/análisis , Estadísticas no Paramétricas , Triptasas , alfa-Macroglobulinas/análisisRESUMEN
BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important pathophysiological role in ischaemic renal failure and drug-induced renal injury such as cyclosporin A (CsA)- and tacrolimus-associated nephrotoxicity. In contrast, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) seem to accelerate renal regeneration after ischaemic and drug-induced renal injury. This study aimed to investigate the influence of HGF and EGF on ET-1 synthesis in cultured human umbilical vein endothelial cells (HUVEC) and renal artery endothelial cells (RAEC). In addition, we have investigated whether mycophenolic acid (MPA), a new immunosuppressive drug, which in contrast to CsA and tacrolimus lacks nephrotoxic side effects, modulates ET-1 synthesis in endothelial cells. METHODS: ET-1 release was measured with a specific enzyme-linked immunosorbent assay. ET-1 mRNA expression was investigated by reverse transcription polymerase chain reaction. RESULTS: HGF and EGF (0.001-10 nM) exerted a significant concentration-dependent inhibitory effect on ET-1 release by HUVEC and RAEC (minimum 56.1+/-4.3% of control, n=6, mean+/-SE). The suppressive effect of HGF and EGF on ET-1 synthesis was dose-dependently antagonized by the tyrosine kinase inhibitors tyrphostin AG1478, lavendustin A and methyl 2,5-dihydroxycinnamate. Incubation of HUVEC and RAEC with MPA (2.5, 10, 25, and 50 microg/ml) for 3-5 h induced a significant reduction of ET-1 mRNA expression. After 48 h incubation with MPA (1-50 microg/ml) a significant decrease of ET-1 release and DNA content per culture well was observed, whereas ET-1 release referred to the DNA content in the corresponding culture well did not differ significantly from controls. CONCLUSIONS: The present findings demonstrate that HGF and EGF reduce ET-1 synthesis in endothelial cells via their receptor tyrosine kinase activity and suggest that the renoprotective effects of HGF and EGF might be linked to their inhibitory action on ET-1 synthesis. This study also provides evidence that, in contrast to CsA and tacrolimus, MPA does not stimulate ET-1 synthesis. This might explain the clinical observation that renal function often improves when CsA or tacrolimus is replaced by mycophenolate mofetil.
Asunto(s)
Endotelina-1/biosíntesis , Endotelio Vascular/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Inmunosupresores/farmacología , Ácido Micofenólico/farmacología , Células Cultivadas , Endotelina-1/antagonistas & inhibidores , Endotelina-1/genética , Endotelio Vascular/citología , Humanos , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Arteria Renal , Venas UmbilicalesRESUMEN
Cumulating evidence suggests that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of chronic liver injury and fibrogenesis. We investigated the effects of oxidized low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis of cultured human and rat hepatic stellate cells (HSC). As shown on protein and mRNA levels, oxLDL dose-dependently stimulated the synthesis of collagen types I and III and fibronectin of cultured HSC. The effect was biphasic, with a maximum between 5 and 25 microg/mL oxLDL (c-fibronectin concentration in HSC supernatants increased 3.9-fold; collagen type I increased 4-fold). Higher oxLDL concentrations were cytotoxic. LDL modified with malondialdehyde (MDA) was not toxic, but stimulated extracellular matrix synthesis as well. As demonstrated by immunofluorescence microscopy (double staining of CD36 and iso-alpha-smooth muscle actin [iso-alpha-sm actin]), immunoblot, and reverse-transcription polymerase chain reaction (RT-PCR), respectively, cultured human HSC express the oxLDL receptor, CD36 (glycoprotein IIIb). Colocalization of CD36 and iso-alpha-sm actin on sinusoidal lining cells was further demonstrated using sections of human fibrotic liver. Preincubation of cultured human HSC with the monoclonal antibody, OKM5, known to block CD36-mediated oxLDL uptake, resulted in a reduction of the oxLDL-stimulated collagen type I synthesis by 56%. In summary, our results demonstrate that low concentrations of modified lipoproteins (oxLDL and MDA-LDL) represent fibrogenic mediators that bind to CD36 and stimulate matrix synthesis of HSC.
Asunto(s)
Antígenos CD36/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Lipoproteínas LDL/toxicidad , Hígado/metabolismo , Northern Blotting , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Lipoproteínas LDL/metabolismo , Hígado/citología , Cirrosis Hepática/etiologíaRESUMEN
Endothelin-1 (ET-1) is a potent vasoconstrictive peptide exerting its effects predominantly by paracrine and autocrine mechanisms. ET-1 acts as a mitogen and co-mitogen on vascular smooth muscle cells, and accumulating evidence suggests that ET-1 is involved in the pathogenesis of atherosclerosis. Deposition of low density lipoproteins (LDL) in the vessel wall is known to play a crucial role in the development of atherosclerotic lesions. In the present study, we have investigated the effect of native LDL (nLDL) and oxidatively modified LDL (oxLDL) on ET-1 synthesis and endothelin receptor expression in cultured human coronary artery smooth muscle cells and human monocyte-derived macrophages. Native LDL and oxLDL induced a significant stimulation of ET-1 release and ET-1 mRNA expression in human coronary artery smooth muscle cells and monocyte-derived macrophages. Antibodies against the scavenger receptor CD36 significantly reduced the oxLDL-induced stimulation of ET-1 synthesis. The antioxidants trolox and probucol did not significantly inhibit the LDL-induced rise of ET-1 release. Endothelin B receptor expression was up-regulated in both cell types after incubation with nLDL and oxLDL. In coronary smooth muscle cells, endothelin A receptor expression was slightly increased by LDL, whereas endothelin A receptor was not detectable in monocyte-derived macrophages. Coronary artery smooth muscle cells secreted a more than 150-fold higher amount of immunoreactive ET-1 into the cell culture medium than monocyte-derived macrophages. In summary, the present data, demonstrating a LDL-induced up-regulation of the endothelin system in coronary smooth muscle cells and in monocyte-derived macrophages, provide further support for a pathophysiological role of endothelin in coronary atherosclerosis and suggest that ET-1 might be involved in mediating the atherogenic effects of LDL.
Asunto(s)
Vasos Coronarios/metabolismo , Endotelina-1/biosíntesis , Lipoproteínas LDL/farmacología , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de Endotelina/metabolismo , Células Cultivadas , Vasos Coronarios/citología , Medio de Cultivo Libre de Suero , Endotelina-1/genética , Humanos , L-Lactato Deshidrogenasa/metabolismo , Lipoproteínas HDL/farmacología , Lipoproteínas LDL/química , Lipoproteínas LDL/aislamiento & purificación , Músculo Liso Vascular/citología , Oxidación-Reducción , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor de Endotelina B , Receptores de Endotelina/genética , Regulación hacia ArribaRESUMEN
The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.
Asunto(s)
Matriz Extracelular/metabolismo , Páncreas/citología , Páncreas/metabolismo , Animales , División Celular/fisiología , Células Cultivadas/efectos de los fármacos , Ceruletida , Sustancias de Crecimiento/farmacología , Masculino , Páncreas/patología , Páncreas/fisiología , Pancreatitis/inducido químicamente , Pancreatitis/metabolismo , Pancreatitis/patología , Fenotipo , Ratas , Ratas Wistar , Valores de ReferenciaRESUMEN
Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.
Asunto(s)
Lipoproteínas LDL/análisis , Apolipoproteína B-100 , Apolipoproteínas B , Células Cultivadas , Fraccionamiento Químico , Sulfato de Cobre , Vasos Coronarios , Electroforesis en Gel de Agar , Electroforesis Capilar/métodos , Fluorescencia , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/metabolismo , Músculo Liso Vascular/metabolismo , Oxidación-ReducciónRESUMEN
The role of nitric oxide (NO) in the pathophysiology of inflammatory bowel diseases (IBD) is controversially discussed. The aim of the present study was to investigate the role of NO inhibition in the acute phase of rat 2,4,6-trinitrobenzenesulphonic acid (TNB)-colitis. To inhibit NO synthesis we used aminoguanidine (AG) as a selective inhibitor of inducible nitric oxide synthase (iNOS). TNB-colitis was induced in rats with and without pretreatment with AG (200 mg kg-1 body weight in the drinking water). The severity of colitis was observed over a period of 7 days. On days 1 and 2, AG reduced concentrations of plasma nitrate and nitrite as well as of portal 6-keto-prostaglandin 1alpha. AG pretreatment increased colonic damage and inflammatory response, assessed by colonic myeloperoxidase and serum lactate dehydrogenase activity, macroscopic damage score, tumour necrosis factor-alpha concentration in stool and colonic glutathione content. The AG-treated group showed a higher and prolonged nuclear factor kappaB (NF-kappaB)/Rel binding activity in the colon. We conclude that NOS inhibition by AG is not beneficial in acute intestinal inflammation. With regard to appropriate therapeutic strategies, NF-kappaB/Rel activation might be a more suitable target.
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Colitis/enzimología , Colitis/patología , Colon/efectos de los fármacos , Colon/patología , Guanidinas/farmacología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Ácido Trinitrobencenosulfónico , Enfermedad Aguda , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Colon/metabolismo , Hígado/metabolismo , Masculino , FN-kappa B/metabolismo , Infiltración Neutrófila/efectos de los fármacos , Óxido Nítrico/metabolismo , Prostaglandinas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción ReIB , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The bcl-2 proto-oncogene is overexpressed in a variety of human cancers and plays an important role in programmed cell death. Recent reports implied that the 3'-untranslated region (3'UTR) functions effectively in the regulation of gene expression. Here, we attempt to assay the ability of triplex forming oligonucleotides (TFOs) to inhibit expression of a target gene in vivo and to examine the potential of the 3'UTR of the bcl-2 proto-oncogene in the regulation of bcl-2 gene expression. To do this, we have developed a novel cellular system that involves transfection of a Doxycyclin inducible expression plasmid containing the bcl-2 ORF and the 3'UTR together with a TFO targeted to the 3'UTR of the bcl-2 proto-oncogene. Phosphorothioate-modified TFO targeted to the 3'UTR of the bcl-2 gene significantly downregulated the expression of the bcl-2 gene in HeLa cells as demonstrated by western blotting. Our results indicate that blocking the functions of the 3'UTR using the TFO can downregulate the expression of the targeted gene, and suggest that triplex strategy is a promising approach for oligonucleotide-based gene therapy. In addition, triplex-based sequence targeting may provide a useful tool for studying the regulation of gene expression.
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Regiones no Traducidas 3'/química , Oligonucleótidos/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Secuencia de Bases , Western Blotting , ADN Recombinante , Regulación hacia Abajo , Fluoresceína-5-Isotiocianato , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Plásmidos/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-bcl-2/genética , Tionucleótidos/química , Tionucleótidos/genética , Tionucleótidos/metabolismo , TransfecciónRESUMEN
Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.
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Endotelina-1/metabolismo , Endotelio Vascular/metabolismo , Factor de Crecimiento de Hepatocito/genética , Lipoproteínas LDL/farmacología , Células Cultivadas , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/fisiopatología , Vasos Coronarios/citología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Proteínas Proto-Oncogénicas c-met/genética , ARN Mensajero/análisisRESUMEN
Progression to androgen independence remains the main problem that impacts on survival and quality of life in prostate cancer patients. We have investigated the potency of tributyrin, an orally available prodrug of butyrate, to induce growth arrest, differentiation and apoptosis in LNCaP, PC-3 and TSU-PR1 human prostate cancer cell lines. Cells were treated with 0.1 to 5 mM tributyrin or sodium butyrate. Growth inhibition, cell cycle arrest and apoptosis induction was assessed using standard methods. Both agents induced a more differentiated, fibroblast-like phenotype in androgen-sensitive as well as androgen-resistant cell lines. Expression of prostate-specific antigen was increased in LNCaP cells by tributyrin as a indicator of differentiation. The IC(50) for sodium butyrate was 2.5 mM in PC-3 and TSU-PR1 cells. LNCaP cells exhibited <50% growth inhibition at 5 mM sodium butyrate. However, the IC(50) for tributyrin was 0.8 mM in PC-3 cells, 1.2 mM in TSU-PR1 cells and 3.1 mM in LNCaP cells. Flow cytometry revealed a strong G1-arrest after exposure to tributyrin or sodium butyrate. Both agents resulted in a strong increase of apoptosis rates compared with mock-treated cells. Overall, tributyrin had a 2.5- to 3-fold growth inhibitory and apoptosis-inducing potency compared with equimolar concentrations of sodium butyrate. Our results demonstrate that tributyrin is more potent than butyrate in regard to cell growth inhibition and apoptosis induction at pharmacologically relevant concentrations. Hence, tributyrin may be a promising candidate for clinical protocols in prostate cancer.
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Andrógenos/farmacología , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Triglicéridos/toxicidad , Butiratos/toxicidad , División Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos , Citometría de Flujo , Humanos , Masculino , Neoplasias de la Próstata , Células Tumorales CultivadasRESUMEN
BACKGROUND/AIMS: A variety of hepatobiliary abnormalities has been described in patients with inflammatory bowel diseases. However, the pathophysiological mechanisms leading to these liver alterations are poorly understood. The aim of the present study was to investigate parameters of liver function in a trinitrobenzenesulfonic acid (TNB)-induced rat colitis model. METHODS: Glucose output, bile acid secretion, bile acid uptake, and the cytochrome P-450 metabolic capacity during TNB-colitis were studied in the perfused liver model. Furthermore, hepatic bile acid- and glycogen content was measured. To evaluate the inflammatory response in the colon and liver, NF-kappaB/Rel was quantified by electrophoretic mobility shift assays. As an NF-kappaB/Rel regulated gene the inducible NO-synthase (NOS2) was evaluated by Western blot analysis. As possible mediators released from the inflamed colon into the portal vein, endotoxin and the stable metabolite of prostaglandin I2 (6-keto-prostaglandin-F1alpha) were determined. RESULTS: Glucose output, bile acid secretion, bile acid uptake, and cytochrome P-450 metabolic capacity decreased on the first and second day of TNB-colitis. Hepatic bile acid content increased at day 14 of colitis. Glycogen content was reduced, most likely due to an inadequate chow intake of these animals. A low level of portal endotoxin was detectable during the first 2 days of colitis. In addition, 6-keto-prostaglandin-F1alpha was clearly increased in portal blood. NF-kappaB/Rel binding activity and inducible NOS2 were strongly positive in the colon during colitis. Although low levels of portal endotoxin were measured during the first 2 days of colitis, no significant NF-kappaB/Rel activity and NOS2 induction were detected in the liver. CONCLUSION: Our results indicate that during the acute state of the TNB-colitis, bile acid secretion and cytochrome P-450 function are disturbed in the absence of distinct inflammatory changes in the liver.