RESUMEN
STUDY DESIGN: This is a retrospective study. OBJECTIVES: The aim of this study is to examine the main features and short-term neurological outcomes associated with injuries to the spine due to diving into water in a Latin American country. SETTING: Salvador, Brazil. PATIENT SAMPLE: A total of 1324 subjects were admitted with spinal trauma between 1991 and 2006 (inclusive). Subjects aged between 14 and 65 years who sustained diving injuries corresponded to 10.6% (N=140) of the cases. OUTCOME MEASURES: Neurological status was determined by the Frankel Functional Scale (FFS) on admission and discharge. The FFS was secondarily converted to the American Spinal Injury Association impairment scale. METHODS: This study is a patient record database review that examines demographic and injury-related characteristics, details of hospital treatment and neurological status at the time of discharge. RESULTS: Males (N=129) outnumbered females (N=11) in a proportion of 12:1 (mean age: 28.62 years). The cervical spine region was the most affected area (92.1%) and 45% of the cases presented with tetraplegia. On admission, neurologically complete lesions accounted for 32.1% of the overall cases and 45.7% were neurologically intact. The mean length of stay (7.7 weeks) did not differ with regard to treatment option (P=0.83). During hospitalization, patients with incomplete neurological impairment had shorter lengths of stay and showed more neurological improvement than those with complete lesions (P=0.26 and 64.5 versus 2.2%, P<0.0001). CONCLUSION: Diving spine injuries have a high tetraplegia rate. Neurological recovery and shorter length of stay are associated with incomplete lesions.
Asunto(s)
Traumatismos en Atletas/epidemiología , Buceo/lesiones , Traumatismos de la Médula Espinal/epidemiología , Traumatismos de la Médula Espinal/terapia , Traumatismos Vertebrales/epidemiología , Traumatismos Vertebrales/terapia , Adolescente , Adulto , Anciano , Traumatismos en Atletas/terapia , Brasil/epidemiología , Evaluación de la Discapacidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cuadriplejía/epidemiología , Cuadriplejía/terapia , Radiografía , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Traumatismos Vertebrales/diagnóstico por imagen , Adulto JovenRESUMEN
Drug development in phytomedicine has been focused in the past on the discovery and analysis of new structures from natural products. The search aimed at the determination of the single "active principle" in plants, based on the assumption that a plant has one or a few ingredients which determine its therapeutic effects. But traditional systems of medicines like Ayurveda, traditional Chinese medicine or the European phytotherapy generally assume that a synergy of all ingredients of the plants will bring about the maximum of therapeutic efficacy. This approach has for long been impossible to investigate since adequate methods to standardize complex plant mixtures as well as to rationalize complex mode of actions were lacking. The introduction of high throughput technologies provides the opportunity to determine profiles of plants and to systematically explore the mode of action of combinatory drug regimes. The present review highlights the concept of synergy and gives examples of synergistic effects of plant constituents. It elaborates on how the high throughput technologies can be used in drug development from natural products with the aim of creating evidence-based plant medications in prevention and treatment of different diseases in the form of new single treatments or new combinatory drug regimes while exploiting synergy-effects.
Asunto(s)
Productos Biológicos/química , Productos Biológicos/uso terapéutico , Descubrimiento de Drogas , Animales , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Fitoterapia/métodos , Fitoterapia/tendencias , Transducción de Señal/efectos de los fármacosRESUMEN
Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.
Asunto(s)
Antineoplásicos/farmacología , Catequina/análogos & derivados , Proteínas Quinasas Activadas por Mitógenos , Músculo Liso Vascular/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Oncogénicas de Retroviridae/metabolismo , Transducción de Señal/efectos de los fármacos , Células 3T3 , Animales , Aorta , Becaplermina , Neoplasias Encefálicas , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Catequina/farmacología , Transformación Celular Neoplásica , Células Cultivadas , Glioblastoma , Humanos , Cinética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Proteínas Oncogénicas v-sis , Fosforilación , Fosfotirosina/metabolismo , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Endogámicas WKY , Proteínas Recombinantes/metabolismo , Proteínas Oncogénicas de Retroviridae/genética , Transducción de Señal/fisiología , Té , Transfección , Células Tumorales CultivadasRESUMEN
Full healing was achieved in seven consecutive patients whose wounds were either infected or colonised with methicillin-resistant Staphylococcus aureus. Antiseptics and antibiotics had previously failed to irradicate the clinical signs of infection.
Asunto(s)
Miel , Resistencia a la Meticilina , Infecciones Estafilocócicas/terapia , Staphylococcus aureus , Infección de Heridas/terapia , Adolescente , Adulto , Anciano , Alginatos/uso terapéutico , Vendajes , Alemania , Ácido Glucurónico/uso terapéutico , Ácidos Hexurónicos/uso terapéutico , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Cuidados de la Piel/métodos , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Factores de Tiempo , Resultado del Tratamiento , Cicatrización de Heridas , Infección de Heridas/diagnóstico , Infección de Heridas/microbiologíaRESUMEN
Tobacco use is causally associated with head and neck squamous cell cancer (HNSCC). Here, we present the results of a case-control study that investigated the effects that the genetic variants of the cytochrome (CYP)1A1, CYP1B1, glutathione-S-transferase (GST)M1, GSTT1, and GSTP1 genes have on modifying the risk of smoking-related HNSCC. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. CYP1B1 codon 432 polymorphism was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of CYP1B1 polymorphism was significantly higher (P < 0.001) in the group of smoking cases when compared with smoking controls. Additionally, an odds ratio (OR) of 4.53 (2.62-7.98) was discovered when investigating smoking and nonsmoking cases for the susceptible genotype CYP1B1*2/*2, when compared with the presence of the genotype wild type. In combination with polymorphic variants of the GST genes, a synergistic-effect OR was observed. The calculated OR for the combined genotype CYP1B1*2/*2 and GSTM1*2/*2 was 12.8 (4.09-49.7). The calculated OR for the combined genotype was 13.4 (2.92-97.7) for CYP1B1*2/*2 and GSTT1*2/*2, and 24.1 (9.36-70.5) for the combination of CYP1B1*2/*2 and GSTT1-expressors. The impact of the polymorphic variants of the CYP1B1 gene on HNSCC risk is reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with susceptible genotype CYP1B1*2/*2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1 wild type. Combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. The results of the present study indicate that polymorphic variants of CYP1B1 relate significantly to the individual susceptibility of smokers to HNSCC.
Asunto(s)
Alelos , Hidrocarburo de Aril Hidroxilasas , Carcinoma de Células Escamosas/genética , Sistema Enzimático del Citocromo P-450/genética , Genes p53/genética , Neoplasias de Cabeza y Cuello/genética , Mutación , Carcinoma de Células Escamosas/enzimología , Estudios de Casos y Controles , Codón , Citocromo P-450 CYP1B1 , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Gutatión-S-Transferasa pi , Glutatión Transferasa/genética , Neoplasias de Cabeza y Cuello/enzimología , Humanos , Isoenzimas/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Factores de Riesgo , Fumar/efectos adversos , Fumar/genéticaRESUMEN
Vascular smooth muscle cell hypertrophy and proliferation may participate in the pathophysiology of cardiovascular disease. The analysis of changes in gene expression in vascular smooth muscle cells is crucial to the understanding of the molecular biology of cardiovascular disease. An effective method for analysis of gene expression is the differential display approach. Applying the differential display approach, we identified a gp130RB13-6-related gene in vascular smooth muscle cells following stimulation with platelet-derived growth factor-BB and angiotensin II. It is well known that gp130RB13-6 is a phosphodiesterase/nucleotide pyrophosphatase. Northern blotting and reverse transcriptase-polymerase chain reaction analysis revealed a dramatic down-regulation of the gp130RB13-6-related mRNA after six hours of stimulation of the cells with both agonists. Recently, gp130RB13-6 was identified as a rat neural differentiation and tumor cell surface plasma membrane glycoprotein. These findings demonstrate that the expression of gp130RB13-6 mRNA in vascular smooth muscle cells is remarkably regulated by growth factors and therefore may play an important role in the regulation of vascular smooth muscle cell growth.
Asunto(s)
Músculo Liso Vascular/enzimología , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/genética , ARN Mensajero/genética , Angiotensina II/farmacología , Animales , Secuencia de Bases , División Celular/genética , División Celular/fisiología , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Fosfodiesterasa I , Factor de Crecimiento Derivado de Plaquetas/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Transducción de SeñalRESUMEN
To examine the limitations of reverse transcription (RT)-PCR for the detection of circulating tumor cells in blood, we established a RT-PCR for carcinoembryonic antigen (CEA). Whole blood (10(7) nucleated cells) was mixed with cells from the colon cancer cell line LS174T (concentrations ranging from 0 to 10(6) cells). RT-PCR was performed to detect CEA mRNA in blood under various conditions. Healthy blood donors (n = 24) were examined by the established method for detecting CEA mRNA in blood. We were able to show that there is a detection limit for RT-PCR of 10 tumor cells in total and of 1 tumor cell in 10(5) nucleated cells. To obtain these results, a high number of PCR cycles (first PCR, 30 cycles; nested PCR, 45 cycles) was required. Under these PCR conditions, we found a positive PCR signal in 33% of healthy blood donors (n = 8). To overcome this problem, we reduced the nested PCR to 35 cycles. At that point, none of the controls showed a positive signal for CEA, and there was a subsequent decrease of the detection limit to 1 tumor cell in 10(2)-10(3) nucleated cells, lower than the detection limit of an immunocytological examination (1 tumor cell in 10(4) nucleated cells). When the amplification was performed with the tumor cells only and with no nucleated blood cells present, under exactly the same conditions, there was still a detection limit of 1 tumor cell in 106 nucleated cells. Our data clearly show that there is a severe loss of expected sensitivity of RT-PCR if it is performed in blood or nucleated blood cells. We conclude that PCR for CEA mRNA expression is not more sensitive than immunocytology and is, furthermore, plagued by the problem of a high percentage of false positive results.
Asunto(s)
Antígeno Carcinoembrionario/sangre , Células Neoplásicas Circulantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Antígeno Carcinoembrionario/biosíntesis , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Humanos , Masculino , ARN Mensajero/sangre , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
Angiotensin II is involved in blood pressure regulation, cell growth and angioneogenesis. The angiotensin receptors which mediate the intracellular effects of angiotensin II are expressed in numerous tissues and cell types. We studied the expression of angiotensin II receptors in cultured human skin fibroblasts derived from a skin biopsy. Angiotensin II binding characteristics were analyzed by radioligand binding assays. The DNA synthesis was assessed by [H]thymidine incorporation assays. Intracellular calcium concentrations were measured by fura-2 spectrofluorometry, and mRNA expression levels were analyzed by northern blot technology. Two distinct angiotensin receptors were detectable on human skin fibroblasts: the AT1 receptor with Kd = 1.0 +/- 0.7 nmol/l and Bmax = 17.9 +/- 0.9 fmol/mg protein, and an angiotensin(1-7) binding site with Kd = 26 +/- 6.6 nmol/l and Bmax = 80.4 +/- 3.5 fmol/mg protein, as shown by competition binding assays using selective angiotensin II receptor antagonists and the heptapeptide angiotensin(1-7). The angiotensin AT1 receptor mRNA was substantially expressed in human skin fibroblasts and was subjected to homologous downregulation. In human skin fibroblasts angiotensin II caused a profound increase in intracellular calcium which was blocked by angiotensin AT1 receptor antagonists such as Exp-3174. Furthermore, both angiotension II and angiotensin(1-7) led to increased DNA synthesis in human skin fibroblasts. In conclusion, cultured human skin fibroblasts express angiotensin AT1 receptors and a putatively new angiotensin receptor activated by angiotensin(1-7), both coupled to signaling pathways involved in DNA synthesis.
Asunto(s)
Angiotensina II/metabolismo , Receptores de Angiotensina/fisiología , Unión Competitiva , Calcio/metabolismo , División Celular , Células Cultivadas , Fibroblastos/citología , Humanos , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Receptores de Angiotensina/clasificación , Transducción de Señal , Piel/citologíaRESUMEN
Accumulating evidence suggests that plasma levels of the plasminogen activator inhibitor 1 (PAI-1) may modulate the risk of coronary artery disease. The regulation of PAI-1 levels underlies not only environmental but also genetic influences. The 4G/5G polymorphism of the PAI-1 gene has recently gained additional relevance as a possible cardiovascular risk factor, as the 4G allele may be associated with enhanced expression of the PAI-1 gene. This retrospective cohort study examined the effect of the PAI-1 4G/5G genotype on longevity among 205 subjects aged 80 years and older. Such studies in larger cohorts have recently become available along with new methods for the rapid and easy determination of gene polymorphisms. We utilized a light-cycler assisted method which is a fast and flexible method of analyzing the PAI-1 4G/5G polymorphism on the gene level. In these 205 persons the 4G/5G allele was found in 96 persons (47%), the 4G/4G variant in 62 (30%), and the 5G/5G allele in 47 (23%). These data are similar to the allele distribution described in other large cohorts not restricted to old age. Thus the results of this study are not suggestive of an important contribution of the PAI-1 genotype on total mortality.
Asunto(s)
Longevidad/genética , Inhibidor 1 de Activador Plasminogénico/genética , Polimorfismo Genético/genética , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Fluorescencia , Frecuencia de los Genes/genética , Genotipo , Humanos , Masculino , Fenotipo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , TemperaturaRESUMEN
alpha1-Antitrypsin (AAT) deficiency is a common inherited cause of emphysema and cirrhotic liver disease. Current laboratory diagnosis of Pi (proteinase inhibitor) status by protein analysis depends on the availability of blood samples and has a limited accuracy. Single-strand conformational polymorphism (SSCP) analysis and direct DNA sequencing can be performed from blood cells or from tissue samples, but it is a time-consuming procedure not suitable for screening purposes. We used a Light-Cycler assisted PCR approach to identify the PiZ mutation and to determine hetero- and homozygous carrier status from whole blood and from paraffin-embedded archival tissue specimens. The results were compared to those obtained by standard PCR amplification followed by SSCP and direct DNA sequencing. Light-Cycler assisted PCR identified heterozygous PiZ mutations in 16 samples, a homozygous PiZ status in three cases, and wild-type PiM in five control samples. In all cases the results were confirmed by SSCP and direct DNA sequencing. Light-Cycler assisted PCR has a high detection rate for the PiZ mutation. It can be performed from blood or from fixed archival tissues, requires only small amounts of DNA, and allows a rapid diagnosis on a high output level.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , ADN/análisis , Cartilla de ADN/química , Genotipo , Humanos , Hígado/patología , Fenotipo , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: In the present study we investigated whether low density lipoprotein is able to enhance the growth promoting effects of thrombin in vascular smooth muscle cells. METHODS: DNA synthesis was examined by measurement of the [3H]thymidine incorporation into the cell DNA. Cell count was measured with a Neubauer cell box. Thrombin receptor mRNA was determined by Northern blotting. Ca2+ was measured by the fura 2-method. RESULTS: Thrombin (5 nmol/l), thrombin receptor activating protein (3 mumol/l) and low density lipoprotein (33 nmol/l) induce a 652 +/- 80%, 593 +/- 80% and a 316 +/- 60% increase in [3H]thymidine incorporation into DNA (mean +/- SD, n = 3), respectively. A coincubation of thrombin or thrombin receptor activating protein with low density lipoprotein led to a 1245 +/- 160% or 1200 +/- 40% increase of DNA synthesis (mean +/- SD, n = 3). Thus, coincubation of low density lipoprotein and thrombin causes a synergistic rather than an additive mitogenic effect on smooth muscle cells. Thrombin and low density lipoprotein induced a 22 +/- 8.4% and a 29% +/- 6% increase in cell number, respectively. Simultaneous treatment of vascular smooth muscle cells with thrombin and low density lipoprotein caused a 63 +/- 14% increase in cell number (mean +/- SD, n = 3). To further elucidate the underlying mechanism, we studied the effect of low density lipoprotein on the expression of thrombin receptor mRNA. Low density lipoprotein caused a 2.5-fold increase of thrombin receptor mRNA within 24 h, as assessed by Northern analysis. Preincubation of cells for 24 h with 33 nmol/l low density lipoprotein resulted in an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration from 538 +/- 54 to 923 +/- 75 nmol/l (mean +/- SD, n = 4). CONCLUSION: In summary, low density lipoprotein may enhance the mitogenic effect of thrombin probably by an up-regulation of thrombin receptor gene expression in vascular smooth muscle cells or by an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration.
Asunto(s)
Lipoproteínas LDL/farmacología , Músculo Liso Vascular/efectos de los fármacos , Trombina/farmacología , Animales , Aorta Torácica , Northern Blotting , Calcio/análisis , Calcio/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Líquido Intracelular/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , ARN/análisis , Ratas , Ratas Endogámicas WKY , Estadísticas no ParamétricasRESUMEN
OBJECTIVES: NO production has been attributed to play a major role in cardiac diseases such as cardiac hypertrophy and cardiac remodeling after myocardial infarction which display significant gender-based differences. Therefore we assessed the effect of 17 beta-estradiol (E2) on estrogen receptor (ER) alpha and beta and endothelial and inducible NO synthase in neonatal and adult rat cardiomyocytes. METHODS: The presence of ER alpha and ER beta was demonstrated by immunofluorescence and western blot analysis as well as the expression pattern of inducible NO synthase (iNOS) and endothelial NOS (eNOS) in isolated cardiomyocytes from neonatal and adult rats. Furthermore, regulation of myocardial iNOS and eNOS expression by estrogen was evaluated in the myocardium from ovariectomized or sham-operated adult Wistar-Kyoto rats. RESULTS: Incubation with E2 led to translocalization of the ER into the nucleus and increased receptor protein expression. E2 stimulated expression of iNOS and eNOS in both neonatal and adult cardiac myocytes. Coincubation with the pure anti-estrogen ICI 182,780 inhibited upregulation of ER and NOS expression. In ovariectomized rats myocardial iNOS and eNOS protein levels were significantly lower compared to sham-operated female animals. CONCLUSION: Taken together, these results show that E2 stimulates the expression of iNOS/eNOS in neonatal and adult cardiomyocytes in-vivo and in-vitro. These novel findings provide a potential mechanism of how estrogen may modulate NOS expression and NO formation in the myocardium.
Asunto(s)
Estradiol/farmacología , Miocardio/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , Western Blotting , Células Cultivadas , Inducción Enzimática/efectos de los fármacos , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Fulvestrant , Miocardio/química , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Ovariectomía , Ratas , Ratas Endogámicas WKY , Receptores de Estrógenos/análisis , Receptores de Estrógenos/metabolismo , Estimulación QuímicaRESUMEN
Low-density lipoprotein (LDL) is thought to be involved in the growth of various cell types including human endothelial cells. Nevertheless, little is known about the signal transduction mechanisms underlying the growth-promoting effects of LDL in endothelial cells. Furthermore, the question whether native LDL participates in the described effects remains unanswered. Here, we show that native LDL induces a dose-dependent elevation in free intracellular Ca(2+)-concentration ([Ca2+]i) as well as a rapid and prolonged increase in intracellular pH (pHi) in human umbilical arterial endothelial cells (HUAEC). Native LDL induces a dose-dependent increase of early growth response gene-1 (egr-1) mRNA expression. The effect is maximal 30 min after addition of LDL to the culture medium. Moreover, native LDL causes an increase in DNA-synthesis and cell proliferation. In addition, the effect of acidic fibroblast growth factor (aFGF) on HUAEC proliferation was enhanced by native LDL.
Asunto(s)
Proteínas de Unión al ADN/genética , Endotelio Vascular/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Inmediatas-Precoces , Lipoproteínas LDL/farmacología , Factores de Transcripción/genética , Calcio/metabolismo , División Celular , Células Cultivadas , ADN/biosíntesis , Proteína 1 de la Respuesta de Crecimiento Precoz , Endotelio Vascular/citología , Factor 1 de Crecimiento de Fibroblastos/farmacología , Genes Inmediatos-Precoces/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Peroxidación de Lípido , ARN Mensajero/biosíntesis , Arterias UmbilicalesRESUMEN
Gangliosides appear to regulate proliferation of different cell types. In the present study, we investigated the effects of gangliosides GM1, GM2 and GM3 on platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) growth. In addition, we examined the effects of gangliosides on the PDGF-BB-dependent signalling transduction pathway in rat aortic VSMC. GM2 and GM1 inhibit the PDGF-BB-dependent receptor tyrosine autophosphorylation, stimulation of the PLC-gamma 1, increase of inositol-1,4,5-trisphosphate (InsP3), elevation in cytosolic free Ca2+ ([Ca2+]i), expression of the immediate early growth response gene c-fos and cell proliferation with the following rank order of potency GM2 > GM1. Although GM3 did not influence the PDGF-BB-dependent receptor autophosphorylation and PLC-gamma 1 activation, it effectively inhibited the PDGF-BB-dependent InsP3 formation, [Ca2+]i and cell growth. Binding studies with 125I-PDGF-BB on VSMC in the presence and absence of 10 to 50 microM of each ganglioside revealed that GM1 and GM2 effectively inhibited the specific binding of PDGF-BB with an IC50 value of 20 microM for GM2 and 30 microM for GM1. GM3 had no significant effect on the specific 125I-PDGF-BB binding. These observations suggest that GM1 and GM2 may interact with PDGF-BB or its receptor resulting in a prevention of its binding. GM3 was able to suppress the PDGF-BB-dependent increase of InsP3 and [Ca2+]i downstream of the PDGF-BB-dependent receptor autophosphorylation and PLC-gamma 1 activity.
Asunto(s)
Gangliósido G(M1)/farmacología , Gangliósido G(M2)/farmacología , Gangliósido G(M3)/farmacología , Músculo Liso Vascular/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Aorta/citología , Becaplermina , Calcio/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Fosfatos de Inositol/análisis , Isoenzimas/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Fosfolipasa C gamma , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/análisis , Ratas , Ratas Endogámicas WKY , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismoRESUMEN
The activation of mitogen-activated protein (MAP) kinase and increase in intracellular free calcium concentration ([Ca2+]i) are discussed in reference to activation of different protein kinases and growth of vascular smooth muscle cells (VSMCs). The aim of the present study was to investigate the role of angiotensin (Ang) II-induced increase in [Ca2+]i for activation of 44-kD/42-kD MAP kinase (p44mapk/p42mapk) and DNA synthesis in VSMCs. Experiments were performed by chelation of [Ca2+]i by the intracellular chelator 1,2-bis-(o-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). Ca2+ was measured by the fura 2 method. MAP kinase activation was determined by the Western blotting method. DNA synthesis was determined by measurement of [3H]thymidine incorporation into the cell DNA. Treatment of VSMCs with 20 micromol/L MAPTAM for 30 minutes resulted in a complete abolishment of the maximal Ang II-induced increase at 10 seconds. Ang II phosphorylated the p44mapk/p42mapk in a time-dependent manner, showing a maximum at 3 minutes. In MAPTAM-treated cells, the maximal phosphorylation of MAP kinase isoforms was shifted to 5 minutes, and dephosphorylation was delayed compared with untreated cells. In concordance with this finding, the induction of the MAP kinase phosphatase-1 was markedly impaired in MAPTAM-treated cells. Ang II induced a 2.3-fold increase in [3H]thymidine incorporation into DNA synthesis in untreated cells. This effect was not reduced in MAPTAM-treated cells. Treatment of the cells with PD 98059 (10 micromol/L), a MAP kinase kinase inhibitor, caused 85% inhibition of the Ang II-induced activation of MAP kinases but did not inhibit the Ang II-induced DNA synthesis. In conclusion, the Ang II-induced stimulation of the MAP kinase is a Ca2+-dependent process. Furthermore, blockade of the Ang II-induced stimulation of the early intracellular events, such as increase in [Ca2+]i or phosphorylation of the MAP kinase, is not accompanied by an inhibition of the Ang II-induced DNA synthesis.
Asunto(s)
Angiotensina II/farmacología , Aorta/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , ADN/biosíntesis , Músculo Liso Vascular/metabolismo , Transducción de Señal/efectos de los fármacos , Vasoconstrictores/farmacología , Animales , Aorta/citología , Calcio/metabolismo , Células Cultivadas , Músculo Liso Vascular/citología , Ratas , Ratas Endogámicas WKYRESUMEN
In the present study we describe the intracellular pathways for the transmission of growth signals by the potent vasoconstricting eicosanoids prostaglandin H2 and thromboxane A2 in smooth muscle cells from rat aorta. Carbocyclic thromboxane A2 and U46619 are stable thromboxane A2 mimetics acting at the common thromboxane A2/prostaglandin H2 receptor. Carbocyclic thromboxane A2 (10(-6) mol/L) induced an approximately 2.5-fold increase in [Ca2+]i above the basal value at 25 seconds. Maximal stimulation of the 42-kD mitogen-activated protein kinase isoform by both thromboxane A2 mimetics occurred at 5 minutes. Both thromboxane A2 mimetics at a concentration of 10(-6) mol/L induced the expression of c-fos and early growth response gene-1 (egr-1) mRNA, with a maximum at 30 minutes. Carbocyclic thromboxane A2 (10(-6) mol/L) induced a 3.3-fold increase in [3H]thymidine incorporation into cell DNA above the basal value and produced a 3.5-fold elevation of platelet-derived growth factor-BB-dependent [3H]thymidine incorporation into cell DNA. Similar effects of U46619 (10(-6) to 10(-5) mol/L) alone did in combination with platelet-derived growth factor-BB on cell DNA synthesis were obtained. The thromboxane A2/prostaglandin H2 receptor antagonist SQ29548 (10(-6) mol/L) completely suppressed the mitogenic effect of both thromboxane A2 mimetics (10(-6) mol/L). Pertussis toxin (10 to 100 ng/mL) did not influence the mitogenic effects of the thromboxane A2 mimetics. Carbocyclic thromboxane A2 (10(-6) mol/L) and platelet-derived growth factor-BB (20 ng/mL) per ser caused a 44% and 100% increase in cell number, respectively. In the presence of carbocyclic thromboxane A2 (10(-6) mol/L), platelet-derived growth factor-BB induced a 152% increase in cell number. Similar results were obtained with U46619 alone or in combination with platelet-derived growth factor-BB.
Asunto(s)
Aorta/metabolismo , Músculo Liso Vascular/metabolismo , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Receptores de Tromboxanos/metabolismo , Tromboxano A2/análogos & derivados , Vasoconstrictores/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Animales , Becaplermina , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Replicación del ADN/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Endogámicas WKY , Transducción de Señal , Tromboxano A2/farmacologíaRESUMEN
In the present study, we describe possible mechanisms by which hypercholesterolemia may contribute to the development of cardiovascular diseases. Treatment of rat aortic smooth muscle cells for 20 hours with cholesterol-rich liposomes (500 micrograms/mL cholesterol, 100 micrograms/mL low-density lipoprotein) resulted in a 76 +/- 12% increase in total cholesterol content. The effects of cholesterol enrichment were examined by determination of changes in cell membrane fluidity. Fluidity of the cholesterol-enriched cell membranes was decreased at all temperatures between 15 degrees C and 40 degrees C. Changes in membrane fluidity in whole cell membranes represented changes in fluidity of microsomal membranes isolated by Percoll gradient ultracentrifugation. The basal [Ca2+]i and the maximal platelet-derived growth factor (PDGF)-BB-induced [Ca2+]i was elevated by 30% and 90% in cholesterol-enriched cells, respectively. In contrast, the resting pH, and the PDGF-BB-induced stimulation of the Na+/H+ exchange were not affected in cholesterol-enriched cells. The effect of PDGF-BB on [3H]thymidine incorporation in cholesterol-enriched cells was elevated by 40% in comparison with untreated cells. Our findings show that cellular cholesterol may be involved in the development of vascular diseases via modulation of the PDGF-induced increase in [Ca2+]i and DNA synthesis in vascular smooth muscle cells.
Asunto(s)
Calcio/metabolismo , Colesterol/farmacología , ADN/biosíntesis , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Becaplermina , LDL-Colesterol/farmacología , Sinergismo Farmacológico , Concentración de Iones de Hidrógeno/efectos de los fármacos , Liposomas/farmacología , Fluidez de la Membrana/efectos de los fármacos , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismo , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Endogámicas WKYRESUMEN
In the present study, we examined whether changes in the membrane fluidity of vascular smooth muscle cells (VSMCs) alter their DNA synthesis. For this purpose, the membrane fluidity of the cells was modulated after treatment of VSMCs with 1,2-dioleoyl phosphatidylcholine (PC). Treatment of VSMCs with 1,2-dioleoyl PC-rich medium containing 10% heat-inactivated human serum and 3 mg/ ml 1,2-dioleoyl PC for 24 h resulted in an increase in VSMC membrane fluidity at all temperatures from 15 degrees to 40 degrees C as well as a 51% inhibition of DNA synthesis, compared with untreated cells. Remarkably, enrichment of VSMCs with 1,2-dioleoyl PC/cholesterol-rich medium containing 10% human serum, 3 mg/ml 1,2-dioleoyl PC and 2 mg/ml cholesterol restored both membrane fluidity and DNA synthesis to the levels of untreated cells. The present findings show an inverse association between increased membrane fluidity and cellular DNA synthesis.
Asunto(s)
ADN/biosíntesis , Fluidez de la Membrana/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Fosfatidilcolinas/farmacología , Animales , Aorta Torácica/citología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Células Cultivadas , Colesterol/farmacología , Medios de Cultivo , Humanos , Fluidez de la Membrana/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Ratas , Ratas Endogámicas WKY , TermodinámicaRESUMEN
Mechanical factors are thought to play an important role in the induction of myocardial hypertrophy. Yet, it is not known whether active contraction induces genes that probably represent initial steps in the hypertrophic response in the adult myocardium--and if so, whether the mechanical or the electrical component of the twitch governs this response. We therefore investigated whether electrical stimulation of contraction was able to induce the immediate-early genes (IEGs) egr-1 and c-fos in adult rat cardiomyocytes. Cyclical contraction led to an increase in egr-1 and c-fos mRNA levels within 30 min. Full inhibition of contraction during electrostimulation by the Ca(2+)-desensitizer 2,3-butanedione monoxime (BDM) totally blocked this IEG-response without altering membrane potential. These data suggest that in adult myocardium, the mechanical rather than the electrical activity is responsible for the IEG-response during active twitch.