RESUMO
BACKGROUND: : The postprandial state after a high-fat meal favors endothelial dysfunction and contributes to the development of atherosclerosis. Little is known about the course of circulating microparticles (MPs) and endothelial progenitor cells (EPCs) after the consumption of a high-fat meal. Both are important for the maintenance and function of endothelial cells. METHODS: : Ten healthy males consumed a meal with French fries and hot pork sausage. In a crossover design (4 weeks, 1 meal per week) they coingested a drink (mineral water, coke, red wine, liquor). Before and 1 and 2 hours after the meal, blood samples were drawn and endothelial function (expressed as reactive hyperemia index) was measured by a peripheral arterial tone technology. Number of EPCs, total MPs, and endothelial-derived MPs were measured using flow cytometry. RESULTS: : Reactive hyperemia index decreased by about 5% in those tests drinking mineral water, and by about 20% in the coke group, but remained unaffected in the red wine and liquor group. The number of EPCs were not significantly affected. The number of total and endothelial-derived MPs increased after a single meal, most in the coke group (increase by about 62%), and less in the red wine group (by about 5%). DISCUSSION: : A single high-fat meal deteriorates endothelial function, associated with a significant increase in circulating MPs. These changes were modified by the drink coindigested to the meal. The postprandial state was getting worse when a cola was consumed, but less hazardous when red wine was consumed.
Assuntos
Consumo de Bebidas Alcoólicas , Micropartículas Derivadas de Células/metabolismo , Gorduras na Dieta/administração & dosagem , Endotélio Vascular/fisiologia , Vinho , Adulto , Pressão Sanguínea , Contagem de Células , Estudos Cross-Over , Células Endoteliais , Frequência Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Prandial/fisiologia , Células-TroncoRESUMO
Previous studies suggest that endothelial progenitor cells (EPCs) contribute to vascular repair processes. In contrast, circulating microparticles (MPs) are reported to be part of a process that is damaging to vascular cells. Numerous studies suggest that the "balance" between EPCs and MPs is important for the integrity of vascular cells and preservation of endothelial function. In the present study, we assess the impact of acetylsalicylate (ASA) - which is, beside statins and physical exercise, a third basic column in the preventive therapy of coronary artery disease (CAD) - on EPCs and MPs in patients with CAD. We investigated the effect of treatment (8 weeks) with ASA (100 mg/d) on endothelial function (flow-mediated vasodilation, FMD), number of circulating EPCs, and endothelial- and platelet-derived microparticles (eMP, pMP) in 15 male patients (age 59.5 ± 12.3 years) with CAD but nonsignificant stenosis. The number of pMPs and eMPs decreased by 62.7% (p < 0.05) and 28.4% (p < 0.05), respectively. The number of circulating EPCs (VEGFR2(+)CD34(+)), expressed as of circulating polymorphonuclear leukocytes, remained unchanged. Despite the reduced number of pMPs and eMPs in response to the ASA therapy, the FMD responses and the maximal dilator effects of nitroglycerin were unaffected. In a control experiment, patients (n = 6) treated with the selective COX-2 inhibitor etoricoxib (90 mg/day) for 8 weeks showed no changes in the number of pMPs, eMPs, and EPCs and in FMD. We report on a novel effect of ASA treatment on the number of circulating endothelial- and platelet-derived microparticles in patients with cardiovascular disease. The mechanism remains elusive, and appears not to be associated with the COX-2 pathway.
Assuntos
Aspirina/farmacologia , Plaquetas/metabolismo , Micropartículas Derivadas de Células/efeitos dos fármacos , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/prevenção & controle , Endotélio Vascular/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Doença da Artéria Coronariana/patologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Etoricoxib , Humanos , Masculino , Pessoa de Meia-Idade , Piridinas/farmacologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/patologia , Sulfonas/farmacologia , Vasodilatação/efeitos dos fármacosRESUMO
BACKGROUND: The anti-infective agent Taurolidine (TRD) has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. METHODS: Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3) were incubated with TRD (100 µM, 250 µM and 1000 µM). Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent-microarray platform to identify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. RESULTS: TRD 250 µM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3) as well as genes involved in the ER stress response (PPP1R15A), in ubiquitination (TRAF6) and mitochondrial apoptotic pathways (PMAIP1). CONCLUSIONS: This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Taurina/análogos & derivados , Tiadiazinas/farmacologia , Antineoplásicos/farmacologia , Apoptose , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Sobrevivência Celular , Citometria de Fluxo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Taurina/farmacologia , Transcrição GênicaRESUMO
BACKGROUND: Bone-marrow derived endothelial progenitor cells (CD34+ and VEGFR2+ KDR+ EPC) and endothelial-derived microparticles (CD 31+Annexin V+, EMP; indicator for endothelial apoptosis) were examined in the peripheral blood of 35 male, clinically stable patients with 3-vessel coronary artery disease (CAD). The patients were divided in 2 groups, those with preserved or normal function (n = 17; EF 65 +/- 6%) and those with reduced left ventricular (LV) function (n = 18; EF 36 +/- 11%). METHODS AND RESULTS: The number of circulating EPCs was decreased by 25% (P = .07) and the number of EMPs was increased by 109 % (P < .05) in patients with LV dysfunction compared with those with normal or preserved LV function. EPCs were positively correlated (r = 0.24 for patients with LV dysfunction and r = 0.28 for patients with preserved LV function) with endothelial function as assessed by flow-mediated vasodilatation. In contrast, EMPs were inversely correlated (r = -0.42 for patients with LV dysfunction and r = -0.49 for patients with preserved LV function). CONCLUSIONS: CAD patients with significant LV dysfunction show an increased index of endothelial cell damage. This decrease (or lack of compensatory elevation) of EPCs may result in a reduced potential for repair and thus contribute at least in part to the pathogenesis of endothelial dysfunction.
Assuntos
Anexina A5/sangue , Células Endoteliais , Endotélio Vascular/fisiopatologia , Isquemia Miocárdica/sangue , Isquemia Miocárdica/fisiopatologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/sangue , Disfunção Ventricular Esquerda/fisiopatologia , Idoso , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/fisiopatologia , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Células-Tronco , Volume Sistólico , Ultrassonografia , Disfunção Ventricular Esquerda/sangue , Disfunção Ventricular Esquerda/diagnóstico por imagemRESUMO
The treatment of choice for esophageal cancer is considered surgical resection, but a median survival of around 20 months after treatment is still discouraging. The value of adjuvant or neoadjuvant radiation or chemotherapy is limited and to date, benefits have only been described for certain tumor stages. Therefore, new therapeutic options are required. As alternative chemotherapeutics, we tested the antibiotic taurolidine (TRD) on KYSE 270 human esophageal carcinoma cells alone and in combination with rhTRAIL (TNF related apoptosis-inducing ligand). Viability, apoptosis and necrosis were visualized by TUNEL assay and quantitated by FACS analysis. Gene expression was analysed by RNA microarray. The most effective concentration of TRD as single substance (250 micromol/l) induced apoptosis to a maximum of 40% after 12-h dose dependently, leaving 4% viable cells after 48 h; by comparison, rhTRAIL did not have a significant effect. The combination of both substances doubled the effect of TRD alone. Gene expression profiling revealed that TRD downregulated endogenous TRAIL, TNFRSF1A, TRADD, TNFRSF1B, TNFRSF21, FADD, as well as MAP2K4, JAK2 and Bcl2, Bcl2l1, APAF1 and caspase-3. TNFRSF25, cytochrome-c, caspase-1, -8, -9, JUN, GADD45A and NFKBIA were upregulated. TRAIL reduced endogenous TRAIL, Bcl2l1 and caspase-1 expression. BIRC2, BIRC3, TNFAIP3, and NFKBIA were upregulated. The combined substances upregulated endogenous TRAIL, NFKBIA and JUN, whereas DFFA and TRAF3 were downregulated compared to TRD as single substance. We conclude that TRD overcomes TRAIL resistance in KYSE 270 cells. Synergistic effects are dependent on the same and on distinct apoptotic pathways which, jointly triggered, result in an amplified response. Several apoptotic pathways, including the TNF-receptor associated and the mitochondrial pathway, were differentially regulated by the substances on gene expression level. Additionally transcription factors seem to be influenced, NFKB in particular. Endogenous TRAIL expression is increased by the combination of substances, whereas it is reduced by each single substance. Taking into consideration that the non-toxic TRD was able to reduce rhTRAIL toxicity and dose, combined therapy with TRD and rhTRAIL may offer new options for treatment in esophageal cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/patologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Taurina/análogos & derivados , Tiadiazinas/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Neoplasias Esofágicas/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas de Neoplasias/metabolismo , Taurina/farmacologiaRESUMO
Glucagon like peptide-2 (GLP-2) exerts intestinotrophic actions, but the underlying mechanisms are still a matter of debate. Recent studies demonstrated the expression of the GLP-2 receptor on fibroblasts located in the subepithelial tissue, where it might induce the release of growth factors such as keratinocyte growth factor (KGF) or vascular endothelial growth factor (VEGF). Therefore, in the present studies we sought to elucidate the downstream mechanisms involved in improved intestinal adaptation by GLP-2. Human colonic fibroblasts (CCD-18Co), human colonic cancer cells (Caco-2 cells) and rat ileum IEC-18 cells were used. GLP-2 receptor mRNA expression was determined using real time RT-PCR. Conditioned media from CCD-18Co cells were obtained following incubation with GLP-2 (50-250 nM) for 24 h. Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay, and wound healing was determined with an established migration-assay. Transforming Growth Factor beta (TGF-beta), VEGF and KGF mRNA levels were determined by RT-PCR. Protein levels of VEGF and TGF-beta in CCD-18Co cells following GLP-2 stimulation were determined using ELISA. Neutralizing TGF-beta and VEGF-A antibodies were utilized to assess the role of TGF-beta and VEGF-A in the process of wound healing. GLP-2 receptor expression was detected in CCD-18Co cells. Conditioned media from CCD-18Co cells dose-dependently induced proliferation in Caco-2 cells, but not in IEC-18 cells. Conditioned media also enhanced cell migration in IEC-18 cells (P<0.01), while migration was even inhibited in Caco-2 cells (P<0.0012). GLP-2 significantly stimulated mRNA expression of VEGF and TGF-beta, but not of KGF in CCD-18Co. The migratory effects of GLP-2 were completely abolished in the presence of TGF-beta and VEGF-A antibodies. GLP-2 exerts differential effects on the epithelium of the small intestine and the colon. Thus, in small intestinal cells GLP-2 stimulates wound repair, whereas no such effects were observed in colonic cells. The mechanisms underlying GLP-2 induced intestinal wound repair seem to involve the secretion of VEGF and, subsequently, TGF-beta from subepithelial fibroblasts, whereas KGF appeared to be less important.
Assuntos
Colo/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Peptídeo 2 Semelhante ao Glucagon/metabolismo , Íleo/metabolismo , Comunicação Parácrina , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Monoclonais , Células CACO-2 , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Colo/efeitos dos fármacos , Meios de Cultivo Condicionados/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Peptídeo 2 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 2 , Humanos , Íleo/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Receptores de Glucagon/genética , Receptores de Glucagon/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , CicatrizaçãoRESUMO
BACKGROUND: Cytochrome P450 (CYP) enzyme 2J2, an epoxygenase predominantly expressed in the heart, metabolizes arachidonic acid to biologically active eicosanoids. One of the CYP2J2 products, 11, 12-epoxyeicosatrienoic acid, has several vasoprotective effects. The CYP2J2-G-50T-promotor polymorphism decreases gene expression and is associated with coronary artery disease. This association supports the vascular protective role of CYP-derived eicosanoids in cardiovascular disease. In the present study, we investigated the influence of this polymorphism on survived myocardial infarction in two study groups of patients with on average high cardiovascular risk profile. METHODS: The CYP2J2 polymorphism was genotyped in two groups of patients that were collected with the same method of clinical data collection. Data from 512 patients with sleep apnoea (group: OSA) and on average high cardiovascular risk profile and from another 488 patients who were admitted for coronary angiography (CAR-group) were evaluated for a potential correlation of the CYP2J2 polymorphism G-50T and a history of myocardial infarction. The G-50T polymorphism of the CYP2J2 gene was genotyped by allele specific restriction and light cycler analysis. RESULTS: The T-allele of the polymorphism was found in 111 (11.1%; CAR-group: N = 65, 13.3%; OSA: N = 46, 9.0%). 146 patients had a history of myocardial infarction (CAR: N = 120, 24.6%; OSA: N = 26, 5.1%). Cardiovascular risk factors were equally distributed between the different genotypes of the CYP2J2 G-50T polymorphism. In the total group of 1000 individuals, carriers of the T-allele had significantly more myocardial infarctions compared to carriers of the wild type (T/T or G/T: 21.6%; G/G: 13.7%; p = 0.026, odds ratio 1.73, 95%-CI [1.06-2.83]). In the multivariate logistic regression analysis the odds ratio for a history of myocardial infarction in carriers of the T-allele was 1.611, 95%-CI [0.957-2.731] but this trend was not significant (p = 0.073). CONCLUSION: In presence of other risk factors, the CYP2J2 G-50T failed to show a significant role in the development of myocardial infarction. However, since our result is close to the border of significance, this question should be clarified in larger, prospective studies in the future.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Predisposição Genética para Doença , Infarto do Miocárdio/genética , Miocárdio/enzimologia , Idoso , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/metabolismo , Análise Mutacional de DNA , Feminino , Alemanha , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/epidemiologia , Razão de Chances , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
BACKGROUND: The increased sympathetic nervous activity in patients with obstructive sleep apnea (OSA) is largely responsible for the high prevalence of arterial hypertension, and it is suggested to adversely affect triglyceride and high-density lipoprotein (HDL) cholesterol levels in these patients. The functionally relevant polymorphisms of the beta2-adrenergic receptor (Arg-47Cys/Arg16Gly and Gln27Glu) have been shown to exert modifying effects on these risk factors in previous studies, but results are inconsistent. METHODS: We investigated a group of 429 patients (55 +/- 10.7 years; 361 men, 68 women) with moderate to severe obstructive sleep apnea (apnea/hypopnea index (AHI) 29.1 +/- 23.1/h) and, on average, a high cardiovascular risk profile (body mass index 31.1 +/- 5.6, with hypertension in 60.1%, dyslipidemia in 49.2%, and diabetes in 17.2% of patients). We typed the beta2-adrenergic receptor polymorphisms and investigated the five most frequent haplotypes for their modifying effects on OSA-induced changes in blood pressure, heart rate, and lipid levels. The prevalence of cardiovascular risk factors and coronary heart disease (n = 55, 12.8%) and survived myocardial infarction (n = 27, 6.3%) were compared between the genotypes and haplotypes. RESULTS: Multivariate linear/logistic regressions revealed a significant and independent (from BMI, age, sex, presence of diabetes, use of antidiabetic, lipid-lowering, and antihypertensive medication) influence of AHI on daytime systolic and diastolic blood pressure, heart rate, prevalence of hypertension, and triglyceride and HDL levels. The beta2-adrenergic receptor genotypes and haplotypes showed no modifying effects on these relationships or on the prevalence of dyslipidemia, diabetes, and coronary heart disease, yet, for all three polymorphisms, heterozygous carriers had a significantly lower relative risk for myocardial infarction (Arg-47Cys: n = 195, odds ratio (OR) = 0.32, P = 0.012; Arg16Gly: n = 197, OR = 0.39, P = 0.031; Gln27Glu: OR = 0.37, P = 0.023). Carriers of the most frequent haplotype (n = 113) (haplotype 1; heterozygous for all three polymorphisms) showed a five-fold lower prevalence of survived myocardial infarction (OR = 0.21, P = 0.023). CONCLUSION: Our study showed no significant modifying effect of the functionally relevant beta2-adrenergic receptor polymorphisms on OSA-induced blood pressure, heart rate, or lipid changes. Nevertheless, heterozygosity of these polymorphisms is associated with a lower prevalence of survived myocardial infarction in this group with, on average, a high cardiovascular risk profile.
Assuntos
Infarto do Miocárdio/complicações , Infarto do Miocárdio/genética , Polimorfismo Genético , Receptores Adrenérgicos beta 2/genética , Apneia Obstrutiva do Sono/complicações , Apneia Obstrutiva do Sono/genética , Idoso , Pressão Sanguínea , Estudos de Coortes , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Infarto do Miocárdio/diagnóstico , Fatores de Risco , Apneia Obstrutiva do Sono/diagnósticoRESUMO
BACKGROUND: Cyclo-oxygenase (COX)-2 isoform appears to be a major source of the vasodilator, prostacyclin. Both prostacyclin and nitric oxide may contribute to the regulation of vascular tone and endothelial-mediated homeostasis. OBJECTIVE: To evaluate the effects of acute COX-2 inhibition on endothelial function in the forearm circulation of patients with essential hypertension. METHODS: Forty patients with essential hypertension as the only risk factor were studied. Forearm blood flow was measured by venous occlusion phlethysmography. Vascular responses were measured after intra-arterial infusion of the endothelium-dependent agonist acetylcholine (15, 30, or 40 microg/min) and the endothelium-independent agonist nitroprusside (1.6, 3.2, or 4.0 microg/min). Patients were allocated randomly to groups to receive the non-selective COX-inhibitor, acetylsalicylate-lysin (500 mg intravenously), or the selective COX-2 inhibitor, parecoxib, an injectable prodrug of valdecoxib (20 mg intravenously). The infusion procedure was repeated 30 min later. RESULTS: Acetylcholine increased concentration-dependent forearm blood flow. This increase was significantly augmented by acetylsalicylate-lysin, but significantly diminished by parecoxib. Neither acetylsalicylate-lysin nor parecoxib modified the vasodilator responses to nitroprusside. CONCLUSION: COX-2 inhibition with the prodrug, parecoxib, diminishes the acetylcholine-induced vasodilatation in the forearm circulation of patients with essential hypertension. It is speculated that the production of vasodilator prostaglandins may be important in pathological states with endothelial dysfunction, at least in patients with essential hypertension.
Assuntos
Aspirina/análogos & derivados , Inibidores de Ciclo-Oxigenase/efeitos adversos , Endotélio Vascular/enzimologia , Hipertensão/tratamento farmacológico , Isoenzimas/antagonistas & inibidores , Isoxazóis/efeitos adversos , Lisina/análogos & derivados , Vasodilatação/efeitos dos fármacos , Acetilcolina/administração & dosagem , Adulto , Idoso , Aspirina/administração & dosagem , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Feminino , Antebraço/irrigação sanguínea , Humanos , Hipertensão/fisiopatologia , Lisina/administração & dosagem , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Prostaglandina-Endoperóxido Sintases , Fluxo Sanguíneo Regional/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/administração & dosagemRESUMO
Circulating microparticles (MPs) are recently discussed as "biologically active", participating in the pathology of diseases rather than being a marker of damaging processes. It was the purpose of the present study to investigate the effects of MPs, as isolated from the blood of healthy volunteers, on the induction of apoptosis and necrosis in cultured KYSE-270 esophageal and ASPC1 pancreas carcinoma cells. MPs were obtained from the blood of 20 healthy volunteers (11 women; mean age 33.3 years). Viability, apoptosis, and necrosis were determined by flow cytometry using Annexin V/propidium iodide and tetramethylrhodamine ethyl ester perchlorate (TMRE)/propidium iodide for staining. Incubation of KYSE and ASPC1 carcinoma cells with MPs (1-20.000/µl) for 48 h reduced significantly viability of the cells, and induced apoptosis, but not necrosis. This apoptotic effect was significant at a concentration of ≥1.000 MPs/µl in both cell types. Pre-treatment of MPs with either the global caspase inhibitor ZVAD-FMK or Annexin V which blocks phosphatidyl serine in the outer membrane of MPs with high affinity, almost abolished MP-induced apoptosis. A specific enzyme assay as well Western blot analysis confirmed the presence (activity, protein) of the apoptotic enzyme caspase-3 in MPs. Incubation of carcinoma cells with MPs (20.000/µl) resulted in an increase in caspase-3 protein in carcinoma cells; this increase could be prevented by pre-treatment of MPs with Annexin V. It is suggested that MPs induce concentration-dependent apoptosis in KSYE esophageal and ASPC1 pancreas carcinoma cells in vitro by transferring caspases into target cells. This process probably requires a target cell-MP interaction, and membrane-bound anionic phosphatidyl serine may be involved.
Assuntos
Apoptose , Caspases/metabolismo , Micropartículas Derivadas de Células/metabolismo , Neoplasias Esofágicas/patologia , Neoplasias Pancreáticas/patologia , Fosfatidilserinas/metabolismo , Western Blotting , Neoplasias Esofágicas/sangue , Feminino , Humanos , Necrose , Neoplasias Pancreáticas/sangue , Células Tumorais CultivadasRESUMO
BACKGROUND: Immunoadsorption (IA) is used in patients with chronic inflammatory dilative cardiomyopathy (iDCM) to remove cardiotoxic autoantibodies, and to improve myocardial function. We examined the effects of IA on endothelial function, circulating endothelial progenitor cells, and circulating microparticles, including endothelial-derived microparticles, in patients with chronic iDCM. METHODS: Thirteen patients (10 males, 3 females, mean age 52.3 years) with advanced congestive heart failure (NYHA III and IV) secondary due to chronic iDCM (with signs of myocardial inflammation in biopsies, but without persistence of virus genome), and reduced left ventricular ejection fraction (EF < 35%) underwent IA. Blood samples were drawn before an IA course of 5 days, and 6 months after IA. Blood levels of endothelial progenitor cells (EPCs as defined as VEGFR2(+)CD34(+) cells), of microparticles (MPs as defined as Annexin V(+) particles with a diameter between 0.1 and 1 µm), and of endothelial-derived MPs (eMPs as defined as CD31(+bright) CD42b(-)/Annexin V(+) particles) were analyzed by flow cytometry. Endothelial function (expressed as reactive hyperemia index (RHI)) and arterial stiffness were assessed by PAT-technology (peripheral arterial tone) using fingertips. RESULTS: Left ventricular systolic function (EF%) improved on average at 6 months from 26.3 ± 4.8 to 37.9 ± 9.6% (mean ± SEM; p < 0.05). The LV end-diastolic diameter reduced after 6 months from 68.4 ± 8.2 to 61.6 ± 7.9 mm; p < 0.05). Endothelial function improved from 1.53 ± 0.09 to 1.80 ± 0.12 (p < 0.05). The arterial stiffness index remained unchanged. Number of total MPs decreased on average by 36.8% (p < 0.05), the number of eMPs by 39.6% (p < 0.05), respectively. The level of circulating EPC remained unchanged (EPC/PMNC 0.26 ± 0.07 vs. 0.27 ± 0.05 , p = n.s.). CONCLUSIONS: IA treatment improves endothelial function in patients with chronic iDCM. This effect is associated with a significant drop in circulating microparticles. The causal relationship between circulating microparticles and endothelial function is discussed.
Assuntos
Cardiomiopatia Dilatada/fisiopatologia , Cardiomiopatia Dilatada/terapia , Endotélio Vascular/fisiopatologia , Técnicas de Imunoadsorção , Inflamação/fisiopatologia , Inflamação/terapia , Adulto , Cardiomiopatia Dilatada/complicações , Contagem de Células , Micropartículas Derivadas de Células , Feminino , Humanos , Inflamação/complicações , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Células-TroncoRESUMO
BACKGROUND: Immunoadsorption (IA) is used in patients with inflammatory dilative cardiomyopathy (iDCM) to remove cardiotoxic autoantibodies, and to improve myocardial function. Even if IA is used only once, the level of anti-cardiac antibodies remains low over time. Changes in cell-mediated immunity after IA may contribute to this observation. The aim of this study is to investigate the effect of IA on cell-mediated immunity especially on regulatory and early activated T cells. METHODS: Ten patients (50.1 ± 9.3 years) with chronic iDCM (with signs of myocardial inflammation in biopsies, but without persistence of virus genome), reduced left ventricular ejection fraction (EF < 35%) underwent IA. Blood samples were drawn before and after an IA course of 5 days, and 1, 3, and 6 months after IA. Leukocyte subpopulations were quantified by FACS analysis. RESULTS: Left ventricular systolic function improved on average at 6 months from 25.6 ± 4.9 to 37.3 ± 10.1% (p < 0.05). The left ventricular end-diastolic diameter was reduced after 6 months (63.3 ± 3.1 vs. 57.1 ± 4.1 mm; p < 0.05). IA therapy led to an increase of regulatory T cells (CD4(+), CD25(+) and CD127(low)) over time (up to 6 months). This was associated with a decrease of activated T cells (CD4(+)/CD69(+) and CD8(+)/CD69(+) cells) and CD28(+) T cells (co-stimulatory cells), which was detectable for at least 6 months after IA. CONCLUSION: It is suggested that changes in cell-mediated immunity contribute to the beneficial effect of IA on myocardial function, and on maintenance of reduced blood levels of cardiotoxic autoantibodies.
Assuntos
Cardiomiopatia Dilatada/imunologia , Imunidade Celular , Ativação Linfocitária/imunologia , Proteína Estafilocócica A/imunologia , Linfócitos T/imunologia , Adulto , Autoanticorpos , Feminino , Humanos , Técnicas de Imunoadsorção , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Hypertensive urgency/emergency occurs frequently, yet no prospective data on common secondary causes, including sleep apnea (SA), renal artery stenosis (RAS), and hyperaldosteronism, are available. METHODS: Patients presenting to the emergency room for over 1 year with systolic blood pressure > or =180 mmHg and/or diastolic blood pressure > or =100 mmHg and typical symptoms were included. RAS was diagnosed by direct duplex/Doppler ultrasound of the renal artery, resistance index, and imaging. The aldosterone/renin ratio (ARR) was determined from morning blood samples taken with the patients supine after > or =2 h of rest. A positive ARR (>50) was followed by saline infusion to exclude primary hyperaldosteronism. SA was evaluated by nasal breathing flow screening; when positive [apnea/hypopnea index (AHI) >5/h], complete polysomnography was performed. RESULTS: Of 161 patients (age, 66.0 +/- 13.1 years; BMI, 28.6 +/- 5.1 kg), 131 had previously identified hypertension (duration, 12.7 +/- 11.5 years; 1.9 +/- 1.5 antihypertensive medications). SA was found in 114 (70.8%) patients [18% mild (AHI: 5-15/h), 26.8% moderate (15.1-30/h), and 24.2% severe (>30/h)]. Aldosterone levels exceeded 160 pg/ml in 22 of 23 patients with hyperaldosteronism; 4 had primary and 12 had secondary hyperaldosteronism. Thirteen (8.1%) patients had RAS. Three secondary causes were found in 1 patient (0.6%), > or =2 in 25 (15.5%), and > or =1 in 124 patients (77.0%). Of 150 detected secondary causes, only 5 were recognized previously. CONCLUSIONS: Secondary causes of hypertension are common and predominantly unrecognized in patients with hypertensive urgency/emergency. Co-prevalence of secondary causes occurs in about 15% and should be considered before therapeutic intervention.
Assuntos
Serviço Hospitalar de Emergência/estatística & dados numéricos , Hiperaldosteronismo/epidemiologia , Hipertensão/epidemiologia , Obstrução da Artéria Renal/epidemiologia , Síndromes da Apneia do Sono/epidemiologia , Idoso , Causalidade , Comorbidade , Reações Falso-Positivas , Feminino , Alemanha/epidemiologia , Humanos , Hiperaldosteronismo/diagnóstico , Hipertensão/diagnóstico , Masculino , Prevalência , Obstrução da Artéria Renal/diagnóstico , Medição de Risco , Fatores de Risco , Síndromes da Apneia do Sono/diagnósticoRESUMO
BACKGROUND: Taurolidine (TRD) represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines. MATERIALS AND METHODS: Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas) were incubated with increasing concentrations of TRD (100 microM, 250 microM and 1000 microM) for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining). Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC) and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death. RESULTS: All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines--suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines. CONCLUSION: This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.
Assuntos
Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Taurina/análogos & derivados , Tiadiazinas/farmacologia , Acetilcisteína/farmacologia , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutationa/antagonistas & inibidores , Humanos , Espécies Reativas de Oxigênio , Taurina/farmacologiaRESUMO
BACKGROUND: Clopidogrel hydrogensulfate is a thienopyridine acting as an important antiplatelet agent alone or in combination with acetyl salicylic acid to prevent cardiovascular complications. A different clopidogrel salt, clopidogrel besylate, was approved in Germany as a "new drug" in May 2008. Only one study with 46 healthy men compared the plasma concentrations of both clopidogrel formulas. In our crossover study we measured the antiplatelet effect of clopidogrel hydrogensulfate (CHS, clopidogrel bisulfate) and clopidogrel besylate (CB) using two techniques, whole blood impedance aggregometry and flow cytometry in healthy subjects. METHODS: Twenty-one healthy volunteers (14 male, 7 female, mean age 36.3 years) were treated either with CHS or CB (300 mg loading, followed by 75 mg/day) and after a wash-out period of at least 21 days, the participants were switched to the other clopidogrel salt in a crossover design. Blood samples were drawn before and 2, 4 and 48 h after the initial dose was taken. Flow cytometry measurements of CD62P (P-selectin) expression were done at baseline and 48 h thereafter with three different ADP concentrations (5, 15, 50 micromol/L ADP). Whole blood impedance aggregometry testing (Chrono-log Model 590) was performed at baseline and after 2, 4 and 48 h with two ADP concentrations (5 and 20 micromol/L ADP). RESULTS: Using flow cytometry, the mean inhibitory effect of clopidogrel on the CD62P expression was similar and no significant differences were noted in subjects treated with either of the clopidogrel formulas for hydrogensulfate or besylate salt (5 micromol/L ADP: 8.12 +/- 5.53 CHS vs. 6.48 +/- 5.01 CB; 15 micromol/L ADP: 9.33 +/- 6.44 CHS vs. 8.99 +/- 8.27 CB; 50 micromol/L ADP: 11.17 +/- 6.81 CHS vs. 9.52 +/- 6.17 CB). It is important to note that clopidogrel CB shows similar and conspicuously high interindividual variability as was reported earlier on CHS. We observed both possibilities, subjects responding less to the hydrogensulfate salt, but better to the besylate salt, and vice versa. Using aggregometry, both salt formulas achieved similar inhibitory effects regarding initial platelet function (2 h/5 micromol/L ADP: CHS 4.5 +/- 3.66 Omega; CB 3.89 +/- 3.81 Omega and 4 h/5 micromol/L ADP: CHS 5.78 +/- 3.51 Omega; CB 4.89 +/- 4.03 Omega) as well as during the maintenance phase (48 h/5 micromol/L ADP: CHS 2.86 +/- 2.92 Omega; CB 3.43 +/- 3.06 Omega). Once again the aggregometry results for CB showed a similarly high interindividual variability as also holds true for CHS. Some subjects had a better antiplatelet effect with clopidogrel besylate and vice versa with clopidogrel hydrogensulfate. CONCLUSION: The crossover study using whole blood aggregometry and flow cytometry shows no overall significant difference in the antiplatelet effect of clopidogrel hydrogensulfate as compared to clopidogrel besylate. However, it is important to note that besides high interindividual there is also high intraindividual variability between the two different clopidogrel formulas. We observed both: subjects responding less to besylate salt, but better to hydrogensulfate salt, and vice versa.
Assuntos
Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Ticlopidina/análogos & derivados , Adulto , Clopidogrel , Estudos Cross-Over , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ticlopidina/administração & dosagem , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Disseminated soft tissue sarcoma still represents a therapeutic dilemma because effective cytostatics are missing. Therefore we tested TRAIL and Tarolidine (TRD), two substances with apoptogenic properties on human fibrosarcoma (HT1080). METHODS: Viability, apoptosis and necrosis were visualized by TUNEL-Assay and quantitated by FACS analysis (Propidiumiodide/AnnexinV staining). Gene expression was analysed by RNA-Microarray and the results validated for selected genes by rtPCR. Protein level changes were documented by Western Blot analysis. NFKB activity was analysed by ELISA and proliferation assays (BrdU) were performed. RESULTS AND DISCUSSION: The single substances TRAIL and TRD induced apoptotic cell death and decreased proliferation in HT1080 cells significantly. Gene expression of several genes related to apoptotic pathways (TRAIL: ARHGDIA, NFKBIA, TNFAIP3; TRD: HSPA1A/B, NFKBIA, GADD45A, SGK, JUN, MAP3K14) was changed. The combination of TRD and TRAIL significantly increased apoptotic cell death compared to the single substances and lead to expression changes in a variety of genes (HSPA1A/B, NFKBIA, PPP1R15A, GADD45A, AXL, SGK, DUSP1, JUN, IRF1, MYC, BAG5, BIRC3). NFKB activity assay revealed an antipodal regulation of the several subunits of NFKB by TRD and TRD+TRAIL compared to TRAIL alone. CONCLUSION: TRD and TRAIL are effective to induce apoptosis and decrease proliferation in human fibrosarcoma. A variety of genes seems to be involved, pointing to the NFKB pathway as key regulator in TRD/TRAIL-mediated apoptosis.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Fibrossarcoma/tratamento farmacológico , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Taurina/análogos & derivados , Tiadiazinas/farmacologia , Apoptose/genética , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , NF-kappa B/biossíntese , NF-kappa B/genética , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Taurina/administração & dosagem , Taurina/farmacologia , Tiadiazinas/administração & dosagemRESUMO
OBJECTIVE: Endothelial progenitor cells (EPCs) may have an important role in vascular homeostasis and repair. METHODS: We examined the level of circulating EPCs in pre- (n = 22; mean age 28.7 years), and postmenopausal healthy females without (n = 30; mean age 61.6 years) or under current hormone replacement therapy (HRT) (n = 19; mean age 59.8 years). RESULTS: Premenopausal females had the highest level of circulating EPCs (0.147 +/- 0.076 per thousand of polymorphnuclear cells). The level of EPCs was lowest in postmenopausal females (0.094 +/- 0.058 per thousand), and increased significantly with HRT on average by 25.5%. In addition, the proliferative capacity of circulating EPCs was assessed under cell culture conditions. This capacity was significantly increased in EPCs isolated from postmenopausal subjects under current HRT as compared to corresponding samples obtained from postmenopausal females without HRT. CONCLUSIONS: This observation is in line with the hypothesis that the hormonal status in females modulates the cardiovascular risk and that circulating EPCs could be involved in this phenomenon.