RESUMO
AIMS: Failure of right ventricular (RV) function worsens outcome in pulmonary hypertension (PH). The adaptation of RV contractility to afterload, the RV-pulmonary artery (PA) coupling, is defined by the ratio of RV end-systolic to PA elastances (Ees/Ea). Using pressure-volume loop (PV-L) technique we aimed to identify an Ees/Ea cut-off predictive for overall survival and to assess hemodynamic and morphologic conditions for adapted RV function in secondary PH due to heart failure with reduced ejection fraction (HFREF). METHODS AND RESULTS: This post hoc analysis is based on 112 patients of the prospective Magdeburger Resynchronization Responder Trial. All patients underwent right and left heart echocardiography and a baseline PV-L and RV catheter measurement. A subgroup of patients (n = 50) without a pre-implanted cardiac device underwent magnetic resonance imaging at baseline. The analysis revealed that 0.68 is an optimal Ees/Ea cut-off (area under the curve: 0.697, P < 0.001) predictive for overall survival (median follow up = 4.7 years, Ees/Ea ≥ 0.68 vs. <0.68, log-rank 8.9, P = 0.003). In patients with PH (n = 76, 68%) multivariate Cox regression demonstrated the independent prognostic value of RV-Ees/Ea in PH patients (hazard ratio 0.2, P < 0.038). Patients without PH (n = 36, 32%) and those with PH but RV-Ees/Ea ≥ 0.68 showed comparable RV-Ees/Ea ratios (0.88 vs. 0.9, P = 0.39), RV size/function, and survival. In contrast, secondary PH with RV-PA coupling ratio Ees/Ea < 0.68 corresponded extremely close to cut-off values that define RV dilatation/remodelling (RV end-diastolic volume >160 mL, RV-mass/volume-ratio ≤0.37 g/mL) and dysfunction (right ventricular ejection fraction <38%, tricuspid annular plane systolic excursion <16 mm, fractional area change <42%, and stroke-volume/end-systolic volume ratio <0.59) and is associated with a dramatically increased short and medium-term all-cause mortality. Independent predictors of prognostically unfavourable RV-PA coupling (Ees/Ea < 0.68) in secondary PH were a pre-existent dilated RV [end-diastolic volume >171 mL, odds ratio (OR) 0.96, P = 0.021], high pulsatile load (PA compliance <2.3 mL/mmHg, OR 8.6, P = 0.003), and advanced systolic left heart failure (left ventricular ejection fraction <30%, OR 1.23, P = 0.028). CONCLUSIONS: The RV-PA coupling ratio Ees/Ea predicts overall survival in PH due to HFREF and is mainly affected by pulsatile load, RV remodelling, and left ventricular dysfunction. Prognostically favourable coupling (RV-Ees/Ea ≥ 0.68) in PH was associated with preserved RV size/function and mid-term survival, comparable with HFREF without PH.
Assuntos
Insuficiência Cardíaca , Hipertensão Pulmonar , Disfunção Ventricular Direita , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/diagnóstico , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/etiologia , Prognóstico , Estudos Prospectivos , Volume Sistólico , Disfunção Ventricular Direita/diagnóstico , Disfunção Ventricular Direita/etiologia , Função Ventricular Esquerda , Função Ventricular DireitaRESUMO
BACKGROUND: A new zero-fluoroscopy technique for electrophysiology catheter navigation relying on intracardiac echocardiography (ICE) has been recently reported (Ice&ICE trial). We investigated potential differences in efficacy, safety or procedural performance between conventional fluoroscopy- and ICE-guided cryothermal ablation (CA) in symptomatic AVNRT patients. METHODS: Clinical and electrophysiological data of AVNRT patients included in the Ice&ICE trial (22 patients, 16 females; =zero-fluoroscopy group) were compared to those of consecutive AVNRT patients, who underwent fluoroscopy-guided CA (25 patients, 17 females; = fluoroscopy group) during the last 2 years in our institution. RESULTS: Slow pathway ablation or modulation was successful in all patients. Fluoroscopy time and radiation dose in the fluoroscopy group were 11.2 ± 9.0 min and 20.3 ± 16.2Gycm2, whereas no fluoroscopy was used in the opposite group (p < 0.001, respectively). EPS duration was not different between the groups (zero-fluoroscopy:101.6 ± 40.2 min, fluoroscopy:99.4 ± 37.2 min, p = n.s.). Catheter placement time was significantly shorter in the fluoroscopy group (2.2 ± 1.6 min vs. 12.0 ± 7.5 min, p < 0.05), whereas cryo-application duration (from the first cryo-mapping to the last CA) was significantly shorter in the zero-fluoroscopy group (27.5 ± 37.0 min vs. 38.1 ± 33.9 min, p < 0.05). Mean cryo-mapping and CA applications were numerically lower in the zero-fluoroscopy group (CM:7.5 ± 5.7 vs. 8.8 ± 6.2; CA:3.1 ± 1.7 vs. 3.2 ± 2.0, p = n.s.). No major adverse events occurred in both groups. After 15.0 ± 4.2 months, arrhythmia recurrence was not different between the groups (4.5% vs. 8.0%, p = n.s.). CONCLUSIONS: Zero-fluoroscopy ICE-guided EP catheter navigation shows comparable efficacy and safety to fluoroscopic guidance during CA in AVNRT patients. ICE visualization of catheters and endocardial structures within the triangle of Koch shortens the cryo-application duration, though time needed for catheter placement is longer, when compared with conventional fluoroscopic guidance, which results in similar mean EPS duration with both navigation techniques. TRIAL REGISTRATION: (German Clinical Trials Register ID: DRKS00011360 ; Registration Date 14.12.2016).
Assuntos
Cateterismo Cardíaco/métodos , Criocirurgia/métodos , Ecocardiografia/métodos , Procedimentos Endovasculares/métodos , Fluoroscopia/métodos , Cirurgia Assistida por Computador/métodos , Taquicardia por Reentrada no Nó Atrioventricular/cirurgia , Eletrocardiografia , Estudos de Viabilidade , Feminino , Humanos , Imageamento Tridimensional , Masculino , Pessoa de Meia-Idade , Pericárdio , Reprodutibilidade dos Testes , Estudos Retrospectivos , Taquicardia por Reentrada no Nó Atrioventricular/diagnóstico , Taquicardia por Reentrada no Nó Atrioventricular/fisiopatologia , Ultrassonografia de Intervenção/métodosRESUMO
INTRODUCTION: Stochastic damage of the ionizing radiation to both patients and medical staff is a drawback of fluoroscopic guidance during catheter ablation of cardiac arrhythmias. Therefore, emerging zero-fluoroscopy catheter-guidance techniques are of great interest. METHODS AND RESULTS: We investigated, in a prospective pilot study, the feasibility and safety of the cryothermal (CA) slow-pathway ablation in patients with symptomatic atrioventricular-nodal-re-entry-tachycardia (AVNRT) using solely intracardiac echocardiography (ICE) for endovascular and endocardial catheter visualization. Twenty-five consecutive patients (mean age 55.6 ± 12.0 years, 17 female) with ECG-documentation or symptoms suggesting AVNRT underwent an electrophysiology study (EPS) in our laboratory utilizing ICE for catheter navigation. Supraventricular tachycardia was inducible in 23 (92%) patients; AVNRT was confirmed by appropriate stimulation maneuvers in 20 (80%) patients. All EPS in the AVNRT subgroup could be accomplished without need for fluoroscopy, relying solely on ICE-guidance. CA guided by anatomical location and slow-pathway potentials was successful in all patients, median cryo-mappings = 6 (IQR:3-10), median cryo-ablations = 2 (IQR:1-3). Fluoroscopy was used to facilitate the trans-septal puncture and localization of the ablation substrate in the remaining 3 patients (one focal atrial tachycardia and two atrioventricular-re-entry-tachycardias). Mean EPS duration in the AVNRT subgroup was 99.8 ± 39.6 minutes, ICE guided catheter placement 11.9 ± 5.8 minutes, time needed for diagnostic evaluation 27.1 ± 10.8 minutes, and cryo-application duration 26.3 ± 30.8 minutes. CONCLUSIONS: ICE-guided zero-fluoroscopy CA in AVNRT patients is feasible and safe. Real-time visualization of the true endovascular borders and cardiac structures allow for safe catheter navigation during the ICE-guided EPS and might be an alternative to visualization technologies using geometry reconstructions.
Assuntos
Cateterismo Cardíaco , Criocirurgia , Ecocardiografia Doppler em Cores , Ecocardiografia Doppler de Pulso , Taquicardia por Reentrada no Nó Atrioventricular/cirurgia , Taquicardia Supraventricular/cirurgia , Ultrassonografia de Intervenção/métodos , Potenciais de Ação , Adulto , Idoso , Cateterismo Cardíaco/efeitos adversos , Estimulação Cardíaca Artificial , Criocirurgia/efeitos adversos , Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Estudos de Viabilidade , Feminino , Frequência Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Projetos Piloto , Valor Preditivo dos Testes , Estudos Prospectivos , Taquicardia por Reentrada no Nó Atrioventricular/diagnóstico por imagem , Taquicardia por Reentrada no Nó Atrioventricular/fisiopatologia , Taquicardia Supraventricular/diagnóstico por imagem , Taquicardia Supraventricular/fisiopatologia , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: Interventional closure of the left atrial appendage (LAA) in patients with non-valvular atrial fibrillation, high thromboembolic and bleeding risk or bleeding history is an alternative therapeutic strategy to oral anticoagulation. It is not known if the exclusion of the LAA from the blood circulation affects the left atrial volume (LAV) and consequently its prognostic value or the circulatory performance of the heart in humans. METHODS: We aimed to prospectively assess potential changes in baseline LAV, left ventricular ejection fraction (LVEF), NT-proBNP-level and the covered distance in the 6-min walk-test 6 weeks and 6 months after LAA closure with the WATCHMAN™ device. We used serial 3-dimensional transthoracic and transesophageal echocardiography to assess LAV, residual interatrial shunt and device performance in 58 consecutive patients with successful LAA closure. RESULTS: Accurate 3D-echocardiographic data for LAV measurements were evaluable for 51 (91%) patients. Maximum LAV (LAVmax) at baseline was 102.8 ± 30.8 ml and increased significantly to 107.7 ± 32.8 ml after 6 weeks (p < 0.01) and 113.5 ± 34.2 ml after 6 months (p < 0.01). Minimal LAV (LAVmin) increased from 76.9 ± 29.5 ml at baseline to 81.8 ± 30.2 ml after 45 days (p < 0.01) and 82.1 ± 33.3 ml after 6 months (p < 0.01). Similarly, their indexes to BSA (LAVImax and LAVImin) increased significantly, as well. Patients without a residual left-to-right interatrial shunt showed a significantly higher increase in LAVmax or LAVmin. Baseline LVEF, NT-proBNP-level or the distance covered at the 6-min walk test did not significantly change 6 weeks or 6 months after LAA closure. CONCLUSIONS: LAVmax and LAVmin increase significantly after interventional LAA closure. LA enlargement does not correlate with clinical progression of heart failure. Persistent left-to-right interatrial shunt counteracts the LA enlargement. A reduced LA compliance after exclusion of the LAA from the blood circulation with consecutive increase in LA pressure may be a potential cause of LA enlargement and warrants further investigation. TRIAL REGISTRATION: German Clinical Trials Register ID: DRKS00010768 ; Registration Date 07.07.2016.
Assuntos
Apêndice Atrial/fisiopatologia , Fibrilação Atrial/terapia , Remodelamento Atrial , Cateterismo Cardíaco/instrumentação , Hemodinâmica , Idoso , Idoso de 80 Anos ou mais , Apêndice Atrial/diagnóstico por imagem , Fibrilação Atrial/diagnóstico por imagem , Fibrilação Atrial/fisiopatologia , Biomarcadores/sangue , Cateterismo Cardíaco/efeitos adversos , Ecocardiografia Tridimensional , Ecocardiografia Transesofagiana , Tolerância ao Exercício , Feminino , Humanos , Masculino , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Estudos Prospectivos , Recuperação de Função Fisiológica , Volume Sistólico , Fatores de Tempo , Resultado do Tratamento , Função Ventricular Esquerda , Teste de CaminhadaRESUMO
OBJECTIVE: Hypoxia plays a pivotal role in development and progression of restenosis after vascular injury. Under hypoxic conditions the hypoxia-inducible factors (HIFs) are the most important transcription factors for the adaption to reduced oxygen supply. Therefore the aim of the study was to investigate the effect of a local HIF-inhibition and overexpression on atherosclerotic plaque development in a murine vascular injury model. METHODS AND RESULTS: After wire-induced vascular injury in ApoE-/- mice a transient, local inhibition of HIF as well as an overexpression approach of the different HIF-subunits (HIF-1α, HIF-2α) by adenoviral infection was performed. The local inhibition of the HIF-pathway using a dominant-negative mutant dramatically reduced the extent of neointima formation. The diminished plaque size was associated with decreased expression of the well-known HIF-target genes vascular endothelial growth factor-A (VEGF-A) and its receptors Flt-1 and Flk-1. In contrast, the local overexpression of HIF-1α and HIF-2α further increased the plaque size after wire-induced vascular injury. CONCLUSIONS: Local HIF-inhibition decreases and HIF-α overexpression increases the injury induced neointima formation. These findings provide new insight into the pathogenesis of atherosclerosis and may lead to new therapeutic options for the treatment of in stent restenosis.
Assuntos
Apolipoproteínas E/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Artéria Femoral/lesões , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Neointima/prevenção & controle , Placa Aterosclerótica/prevenção & controle , Adenoviridae , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Reestenose Coronária , Modelos Animais de Doenças , Endotélio Vascular/lesões , Artéria Femoral/patologia , Vetores Genéticos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Masculino , Camundongos , Camundongos Knockout , Placa Aterosclerótica/etiologia , Transdução de Sinais , Transdução Genética , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Macrophages are often associated to pathophysiological processes and were found at hypoxic areas. However, cell adaption greatly depends on hypoxia-inducible factors (HIF). Activation of these transcription factors is induced by heterodimerization of an α-(HIF-1α, -2α, -3α) and HIF-1ß subunit. The main regulatory pathway is represented by α-subunit stability. Beside, little is known about the exact mechanisms of fine-tuning in Hif-regulation. The present study characterizes the hypoxia-induced regulation of HIF-1α and -2α in human macrophages. The hypoxic increase of both subunits is initially mediated by protein stabilization. Sustained hypoxia caused a distinct regulation of HIF-1α and -2α. The striking increase of HIF-2α protein expression was contrasted by a dramatic decrease of HIF-1α. The long-term downregulation of HIF-1α is due to downregulation of its mRNA. This decrease was accompanied by increased expression of ahif, a natural cis-antisense transcript of HIF-1α. The ahif-transcript was strongly inducible by hypoxia and rapidly degraded under reoxygenation. Using an adenoviral overexpression and siRNA silencing approach revealed that the targeted regulation of ahif is mediated by the HIF-system itself. Furthermore it could be shown that ahif indeed is able to modulate the hypoxic expression of HIF-1α and influences the expression of the HIF-target gene Enolase-2. Taken together, this study characterizes a new regulation process of the HIF-transcription factor-system in human macrophages under hypoxia. For the first time evidence is provided that ahif is regulated by the HIF-system and influences HIF-1α expression in primary human macrophages.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular/genética , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/imunologia , Músculo Liso Vascular/citologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Transcrição GênicaRESUMO
MiRNAs are a class of endogenous tiny RNAs that act as inhibitors of translation or promote RNA degradation by duplex-formation within the 3'-UTR of target mRNAs. They play an important role during a wide range of cellular processes by fine-tuning of gene expression. The differentiation of monocytes to macrophages plays a pivotal role in physiological as well as pathophysiological processes such as atherosclerosis. Monocytes which can be found in well-oxygenated blood migrate into areas with a high inflammation, such as the atherosclerotic plaque. There, they differentiate into macrophages. Interestingly, macrophages were found mainly at hypoxic sites of the plaque. Key regulators for the adaptation to hypoxia are the hypoxia-inducible factors (Hif). Therefore the aim of the present study was to investigate the regulation of the Hif-system by miRNAs during the process of monocyte differentiation. The present study shows that during the differentiation of monocytes into macrophages a dramatically change in the expression pattern of Hif-1α and Hif-2α took place. This was associated with a downregulation of microRNAs encoded by the miR-17-92 cluster. An in silico analysis of the 3'-UTR of Hif-α subunits for binding sites of miRNAs was performed using different miRNA databases in concert with a secondary structure prediction algorithm. This analysis revealed that both 3'-UTRs contain binding sites for miRNAs of the miR-17-92 cluster. Transfection of HeLa cells with miR-17 and miR-20a led to an inhibition of Hif-1α and -2α mRNA and protein expression and a lowered Hif DNA binding activity. Using a Luciferase-Reporter assay, it could be shown, that both Hif-α subunits are targeted by miR-17 and miR-20a. Furthermore, miR-overexpression in primary human macrophages demonstrates the important role of this microRNA-mediated regulation of the Hif-system for adaption of macrophages to hypoxia. In conclusion, the present study shows that the Hif-system is activated during monocyte-to-macrophage differentiation. This activation is in part mediated by a miRNA-dependent mechanism, which seems to be crucial for the adaption of macrophages to hypoxia.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/metabolismo , MicroRNAs/genética , Monócitos/metabolismo , Regiões 5' não Traduzidas/genética , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Western Blotting , Diferenciação Celular/genética , Hipóxia Celular/genética , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Macrófagos/citologia , MicroRNAs/metabolismo , Modelos Genéticos , Monócitos/citologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.
Assuntos
Colesterol/metabolismo , Lipoproteínas LDL/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Fenótipo , Animais , Aorta Abdominal/citologia , Aorta Abdominal/metabolismo , Comunicação Celular/fisiologia , Células Cultivadas , LDL-Colesterol/metabolismo , Técnicas de Cocultura , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Macrófagos/citologia , Microscopia de Fluorescência , Músculo Liso Vascular/citologia , Ratos , Ratos WistarRESUMO
Atherosclerotic plaques are characterized by hypoxic even anoxic areas and by high concentrations of oxidized lipoproteins. Moreover, unstable plaques attract a high number of macrophages despite the proapoptotic background within these plaques. Recently, it was shown that these macrophages are positive for Hif-1α. This subunit is a part of hypoxia-inducible factor 1 (Hif-1), a key transcriptional factor under hypoxia. Till date, it is not understood whether the Hif-system (consisting of Hif-1, Hif-2 and Hif-3) is involved in protection of macrophages under these proatherogenic conditions. The present study delineates that oxLDL causes fundamental changes in the regulation of the Hif-system in primary human macrophages. First, both oxLDL and hypoxia mediate accumulation of Hif-1α protein. Second, treatment with a combination of oxLDL and hypoxia is acting in an additive manner on Hif-1α protein content. Third, oxLDL alone does not increase Hif-2α protein, but abolishes the hypoxic induction of Hif-2α completely. OxLDL treatment alone was not toxic for macrophages under neither normoxia nor hypoxia. But, inhibition of Hif-pathway by adenoviral expression of a dominant-negative mutant combined with oxLDL treatment independently of the oxygen tension leads to apoptosis, as determined by caspase-3 activation and induction of DNA fragmentation. Furthermore, this inhibition also mediates the opening of the mitochondrial permeability transition pore. In conclusion, the present data show that Hif-1α regulation is essential for survival of oxLDL-treated macrophages independent of the oxygen tension. Therefore, this newly characterized mechanism might also have an important influence for the vulnerability of atherosclerotic plaques.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Oxigênio/fisiologia , Transdução de Sinais/fisiologia , Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Caspase 3/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genéticaRESUMO
Hypoxia-inducible factors (HIF) are transcription factors responding to reduced oxygen levels and are of utmost importance for regulation of a widespread of cellular processes, e.g., angiogenesis. In contrast to HIF-1α/HIF-2α, the relevance of HIF-3α for the regulation of the HIF pathway in human vascular cells is largely unknown. HIF-3α mRNA increases under hypoxia in endothelial and vascular smooth muscle cells. Analysis of HIF-3α isoforms revealed a cell type-specific pattern, but only one isoform, HIF-3α2, is hypoxia-inducible. Reporter gene assays of the appropriate promoter localized a 31-bp fragment, mediating this hypoxic regulation. The contribution of HIF-1/2 and NFκB to the HIF-3α induction was verified. Functional studies focused on overexpression of HIF-3α isoforms, which decrease the hypoxia-mediated expression of VEGFA and Enolase2. These data support the notion of a hypoxia-induced inhibitory function of HIF-3α and demonstrate for the first time the existence of this negative regulation of HIF-signaling in vascular cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Proteínas Reguladoras de Apoptose , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Células Cultivadas , Regulação para Baixo/genética , Células Endoteliais/fisiologia , Perfilação da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Especificidade de Órgãos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras , Elementos de Resposta/genética , Transdução de Sinais/genética , TransfecçãoRESUMO
Estrogens are suggested to play a role in the development and progression of proliferative diseases such as breast cancer. Like other steroid hormone receptors, the estrogen receptor-alpha (ERalpha) is a substrate of protein kinases, and phosphorylation has profound effects on its function and activity. Given the importance of DNA-dependent protein kinase (DNA-PK) for DNA repair, cell cycle progression, and survival, we hypothesized that it modulates ERalpha signaling. Here we show that, upon estrogen stimulation, DNA-PK forms a complex with ERalpha in a breast cancer cell line (MELN). DNA-PK phosphorylates ERalpha at Ser-118. Phosphorylation resulted in stabilization of ERalpha protein as inhibition of DNA-PK resulted in its proteasomal degradation. Activation of DNA-PK by double-strand breaks or its inhibition by siRNA technology demonstrated that estrogen-induced ERalpha activation and cell cycle progression is, at least, partially dependent on DNA-PK.
Assuntos
Proteína Quinase Ativada por DNA/metabolismo , Receptor alfa de Estrogênio/metabolismo , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Sítios de Ligação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células COS , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Cromonas/farmacologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Receptor alfa de Estrogênio/genética , Estrogênios/metabolismo , Estrogênios/farmacologia , Humanos , Immunoblotting , Imunoprecipitação , Autoantígeno Ku , Morfolinas/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Ubiquitina/metabolismoRESUMO
The cellular response to DNA double-strand break (DSB) occurs through an integrated sensing and signalling network that maintains genomic stability. Oestrogen (E2), among its many functions, is known to have a positive effect on global genomic DNA repair; however, the mechanism by which it functions is unclear. A central enzyme involved in DNA DSB repair in mammalian cells is the DNA-dependent protein kinase (DNA-PK). Here, we show that E2 enhances DNA-PK catalytic subunit (DNA-PKcs) promoter activity with subsequent transcriptional and translational upregulation of DNA-PKcs in a breast cancer cell line. We identify two potential E2 receptor-alpha (ERalpha)-binding sites in a region upstream from the DNA-PKcs initiation site. By using small interfering RNA and the specific E2 receptor antagonist ICI 182,780, we demonstrate that ERalpha knockdown reduces E2-induced upregulation of DNA-PKcs expression and activity in breast carcinoma cells. E2-induced DNA-PK transactivation results in an increased ability of the cells to repair DNA DSB. This previously unknown mechanism of DNA-PK regulation sheds new light on tumour biology and reveals new possibilities for the prevention and therapy of E2-sensitive proliferative diseases.
Assuntos
Proteína Quinase Ativada por DNA/genética , Receptor alfa de Estrogênio/genética , Ativação Transcricional , Animais , Sítios de Ligação , Células COS , Domínio Catalítico , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteína Quinase Ativada por DNA/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Fulvestranto , Humanos , Modelos Biológicos , RNA Interferente Pequeno/metabolismo , Radiação IonizanteRESUMO
The Ser/Thr-protein kinase Pim-1 has been discovered as a novel transducer of survival- and cell cycle promoting signals in the hematopoietic cell system. Although its significance for proliferation of vascular smooth muscle cells (VSMC) in vitro and neointima formation in vivo has been suggested recently, the mechanism has barely been characterized. This study aimed to foster the understanding of Pim-1 expression and regulation in murine VSMC in response to factors typically present within the atherosclerotic plaque. While oxidative stress, VEGF-A165 and angiotensin II did not have any effect on Pim-1 expression, VSMC strongly increased (3-fold) Pim-1 mRNA upon stimulation with PDGF(bb), followed by its protein upregulation. Half life of Pim-1 RNA and protein were determined to be 25 min and 6 h, respectively. PDGF(bb) induced a strong, 10-fold increase in BrdU-uptake, a marker of proliferation. This was effectively blocked by either Pim-1-specific inhibitor quercetagetin or adenovirally introduced Pim-1 shRNA. We further identified the signaling pathways linking PDGF(bb) to Pim-1 in VSMC: as expected, we determined transcriptional stimulation of Pim-1 via Janus-activated kinase (Jak), but also an additional pathway involving protein kinase C (PKC) and the mitogen-activated protein kinase Mek1/2. Blockade of Akt signaling did, however, not interfere with Pim-1 upregulation, suggesting an independence of either survival system. PDGF(bb)-induced proliferation of VSMC is partly attributed to transcriptionally upregulated Pim-1 and was assigned to distinct cell signaling. Our findings help to understand the fundamental processes of vasculoproliferative diseases thus opening avenues for its prevention and treatment.
Assuntos
Proliferação de Células , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Animais , Células Cultivadas , Regulação da Expressão Gênica , Janus Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismoRESUMO
OBJECTIVES: Therapeutic augmentation of collateral artery growth (ie, arteriogenesis) is of particular clinical interest for improving blood flow in vascular occlusive disease. Quantification of collateralization in small animal models is difficult, however, and the commonly used technique of laser Doppler perfusion imaging (LDPI) has always been criticized. Therefore, a new method, termed indocyanine green angiography (ICGA), was established for in vivo imaging of arteriogenesis in mice and compared with LDPI. METHOD: Using the accepted model of ligation of the left femoral artery of 45 C57BL6 mice, we determined arteriogenesis both by LDPI and ICGA, which were applied before and periodically after ligation of the left femoral artery (each group n = 7). Collateral artery growth within the hind limb was additionally verified by histologic workup. RESULTS: Determination of flow by ICGA, as represented by maximal pixel intensity (ratio of left/right hind limb) demonstrated a drop from 0.97 +/- 0.06 before ligation to 0.11 +/- 0.12 directly after ligation, which recovered to 0.48 +/- 0.22 after 1 week, to 0.65 +/- 0.11 after 2 weeks, and to 0.59 +/- 0.22 after 3 weeks (n = 7, P < .05). Similarly, flow determined as the perfusion index (slope of pixel intensity, ratio left/right) dropped from 1.18 +/- 0.4 before ligation to 0.02 +/- 0.03 immediately after ligation but recovered to 0.08 +/- 0.01 after 1 week, to 0.17 +/- 0.01 after 2 weeks, and to 0.17 +/- 0.06 after 3 weeks (n = 7, P < .05). Quantification by LDPI demonstrated a drop from 1.06 +/- 0.06 (left/right ratio) before ligation to 0.37 +/- 0.03 immediately after ligation. In contrast to ICGA, perfusion recuperated completely within 1 week to 1.01 +/- 0.14 and tended to be even higher in the ligated than in the unligated hind limb after 2 (1.09 +/- 0.25) and 3 weeks (1.20 +/- 0.29), pointing towards limitations of this technique. Histologic analysis confirmed the significant increase in the number of collaterals. The intraindividual ratio increased from 1.0 +/- 0.05 before ligation to 1.35 +/- 0.10 at 2 weeks and 1.41 +/- 0.08 at 3 weeks after ligation (P < .05). CONCLUSION: Our data demonstrate that ICGA represents a potent tool for the quantification of collateral flow in small animal models. The current standard of LDPI seems to rather represent blood movements within the superficial skin but not of the entire hind limb.
Assuntos
Arteriopatias Oclusivas/fisiopatologia , Circulação Colateral , Angiofluoresceinografia , Corantes Fluorescentes/administração & dosagem , Verde de Indocianina/administração & dosagem , Fluxometria por Laser-Doppler , Músculo Esquelético/irrigação sanguínea , Animais , Arteriopatias Oclusivas/diagnóstico por imagem , Arteriopatias Oclusivas/patologia , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Artéria Femoral/cirurgia , Imuno-Histoquímica , Injeções , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Fluxo Sanguíneo Regional , Fatores de Tempo , UltrassonografiaRESUMO
Functional tricuspid regurgitation secondary to mitral valve disease can not be attributed to the dilatation of the tricuspid annulus alone. Furthermore, geometrical changes of the right ventricle lead to tethering of the tricuspid valve leaflets and thereby to an incomplete leaflet coaptation. With this pathologic entity, conventional isolated tricuspid valve annuloplasty will presumably result in significant residual tricuspid regurgitation. The surgical goal should be the reduction of tricuspid annulus dilatation and annihilation of tethering forces on the tricuspid leaflets. In combination with conventional tricuspid valve annuloplasty, right ventricular reduction surgery, as demonstrated, may be effective in reaching these goals and hereby avoiding residual tricuspid regurgitation in this patient population.
Assuntos
Cardiomiopatia Dilatada/cirurgia , Insuficiência da Valva Mitral/cirurgia , Insuficiência da Valva Tricúspide/cirurgia , Disfunção Ventricular Direita/cirurgia , Idoso de 80 Anos ou mais , Ponte Cardiopulmonar , Ecocardiografia , Feminino , Implante de Prótese de Valva Cardíaca , Ventrículos do Coração/cirurgia , Humanos , Valva Mitral/cirurgia , Músculos Papilares/cirurgia , Complicações Pós-Operatórias/diagnóstico , Recidiva , Suturas , Valva Tricúspide/cirurgiaRESUMO
BACKGROUND: In cases of in-stent restenosis, intracoronary radiotherapy with beta-emitters and gamma-emitters has been shown to reduce the risk of repeat restenosis. The present randomised, placebo-controlled study addresses the question of whether intracoronary radiotherapy applied by the easy-to-handle Rhenium liquid-filled angioplasty balloon system is also able to reduce the angiographic re-restenosis rate in stents. METHODS AND RESULTS: At our center, from May 2000 to December 2003, 165 patients (mean age 64+/-10, median 65 years; 127 men, 38 women) with symptomatic in-stent restenosis underwent either intracoronary brachytherapy or sham procedure. Index clinical and angiographic parameters were largely comparable in both groups. Radiation therapy was performed with a standard percutaneous transluminal coronary angioplasty (PTCA) balloon catheter inflated with liquid Rhenium in the redilated in-stent restenosis for 240-890, mean 384+/-125 s with low pressure (3 atm) in order to reach 30 Gy at 0.5 mm depth of the vessel wall. In 82 patients, intracoronary radiotherapy was carried out without complications, but one of the 83 patients who underwent sham procedure suffered small myocardial infarction. During follow-up, stent thrombosis with subsequent non-Q-wave myocardial infarction occurred in one patient in each group (6 days and 8 months after the procedure, respectively). At 6 months after the index procedure, repeat angiography was performed in 156 of the 164 patients with successful procedure (rate 95%): restenosis (stenosis >50% in diameter) or reocclusion was observed in only 19 of 78 (=24%) patients of the radiation but in 31 of 78 (=40%) patients of the sham procedure group (P=0.04). Event-free survival (free of death, myocardial infarction, target vessel revascularization) at 1 year was 87% for patients being radiated and 74% for patients having undergone sham procedure (P=0.05). CONCLUSIONS: Intracoronary radiation therapy with the liquid-filled beta-emitting Rhenium balloon is not only easy to perform, safe, and comparably inexpensive but also an effective option to prevent repeat restenosis and the need for target vessel revascularization in cases of in-stent restenosis.
Assuntos
Angioplastia Coronária com Balão/métodos , Reestenose Coronária/radioterapia , Rênio/uso terapêutico , Idoso , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Radioisótopos , Resultado do TratamentoRESUMO
This is the first report of a cardiac manifestation of a primary hyperoxaluria type II (PH II) with the hemodynamic characteristics of a severe restrictive cardiomyopathy. PH II is a rare inherited metabolic disease characterized by a deficiency of D-glycerate dehydrogenase, which has also glyoxylate reductase activity. This defect causes an accumulation of hydroxypyruvate the precursor of oxalate. The renal excretion of oxalate is impaired causing a deposition of oxalate mainly in the kidneys. To date, less than fifty cases have been reported. Systemic oxalosis in PH II is an occasional finding; thus far, myocardial oxalosis due to PH II has never been reported. Described is the case of a 41 year old male with renal failure and severe neuropathy of unknown cause, who underwent endomyocardial biopsy under the suspicion of cardiac amyloidosis. Echocardiography and cardiac catheterization showed a severe restrictive cardiomyopathy; endomyocardial biopsy established the diagnosis of oxalosis. Plasma oxalate levels were markedly increased, therefore a liver biopsy was performed. Immunoreactivity for D-glycerate dehydrogenase/ glyoxylate reductase was absent and activity of the enzyme was < 5% of normal. In summary, these findings established the diagnosis of a restrictive cardiomyopathy due to PH II.
Assuntos
Cardiomiopatia Restritiva/diagnóstico , Cardiomiopatia Restritiva/etiologia , Hiperoxalúria Primária/complicações , Hiperoxalúria Primária/diagnóstico , Adulto , Biomarcadores/sangue , Cardiomiopatia Restritiva/fisiopatologia , Humanos , Hiperoxalúria Primária/sangue , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Ácido Oxálico/sangue , Volume Sistólico , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/fisiopatologia , Pressão VentricularRESUMO
Although caveolin-1 is not expressed in cardiomyocytes, this protein is assumed to act as a key regulator in the development of cardiomyopathy. In view of recent discordant findings we aimed to elucidate the cardiac phenotype of independently generated caveolin-1 knockout mice (cav-1(-/-)) and to unveil causative mechanisms. Invasive hemodynamic measurements of cav-1(-/-) show a severely reduced systolic and diastolic heart function. Additionally, genetic ablation of caveolin-1 leads to a striking biventricular hypertrophy and to a sustained eNOS-hyperactivation yielding increased systemic NO levels. Furthermore, a diminished ATP content and reduced levels of cyclic AMP in hearts of knockout animals were measured. Taken together, these results indicate that genetic disruption of caveolin-1 is sufficient to induce a severe biventricular hypertrophy with signs of systolic and diastolic heart failure. Collectively, our findings suggest a causative role of a sustained nitrosative stress in the development of the pronounced cardiac impairment.
Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Caveolina 1/deficiência , Caveolina 1/genética , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/genética , Cardiomiopatia Hipertrófica/enzimologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/fisiopatologia , AMP Cíclico/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Direita/enzimologia , Hipertrofia Ventricular Direita/genética , Hipertrofia Ventricular Direita/fisiopatologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Índice de Gravidade de DoençaRESUMO
Angiotensin-converting enzyme (ACE) inhibitors interfere with several key events of vascular inflammation resulting in impressive reductions in coronary vascular events. However, in human arteries ACE inhibitors block the production of angiotensin II (AngII) incompletely because of the involvement of alternative pathways in local AngII formation. Therefore, our study concentrated on the presumed modulation by ACE inhibition of local AngII-mediated inflammatory actions by a mechanism independent of blockage of AngII formation. We analyzed the effect of the ACE inhibitor ramiprilat on AngII-dependent cell adhesion molecule (CAM) expression and adhesion of monocytic THP-1 cells to endothelial cells. AngII induced upregulation of P-selectin, VCAM-1 and ICAM-1 on endothelial cells via activation of AT 1, which was correlated with enhanced THP-1 adhesion in flow chamber assays. Both enhanced adhesion and adhesion molecule expression were significantly reduced by pretreatment with ramiprilat. Ramiprilat reduced AT 1 expression on endothelial cells and decreased the AngII-induced p65 translocation into the nucleus. Diminished AT 1 expression and adhesion molecule expression in response to ramiprilat treatment were partially reversed after incubation with a bradykinin 2 receptor antagonist, suggesting that elevated bradykinin levels under ACE inhibition may be involved in the beneficial effect of ACE inhibitors. Thus, modulation of the local AngII system by ramiprilat may at least in part contribute to the benefits of ACE inhibition in the treatment of atherosclerotic diseases.