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BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.
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Canales Iónicos Sensibles al Ácido/biosíntesis , Canales Iónicos Sensibles al Ácido/genética , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/metabolismo , Animales , Células Cultivadas , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Preparación de Corazón Aislado/métodos , Masculino , Ratones , Ratones Noqueados , Isquemia Miocárdica/terapia , Daño por Reperfusión Miocárdica/terapia , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Polimorfismo de Nucleótido Simple/fisiología , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Venenos de Araña/farmacologíaRESUMEN
AIMS: Metformin may have clinical benefits in dialysis patients; however, its safety in this population is unknown. This systematic review evaluated the safety of metformin in dialysis patients. METHODS: MEDLINE, Embase, CENTRAL, PsycINFO and the Cochrane Library were searched for randomised controlled trials and observational studies evaluating metformin use in dialysis patients. Three authors reviewed the studies and extracted data. The primary outcomes were mortality, occurrence of lactic acidosis and myocardial infarction (MI) in patients taking metformin during dialysis treatment for ≥12 months (long term). Risk of bias was assessed using Risk Of Bias In Nonrandomised Studies of Interventions (ROBINS-1). Overall quality of evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE). RESULTS: Fifteen observational studies were eligible; 7 were prospective observational studies and 8 were case reports/case series. No randomised controlled trials were identified. The 7 prospective observational studies (n = 194) reported on cautious metformin use in patients undergoing maintenance dialysis. Only 3 provided long-term follow-up data. In 2 long-term studies of metformin therapy (≤1000 mg/d) in patients undergoing peritoneal dialysis (PD), 1 reported 6 deaths (6/83; 7%) due to major cardiovascular events (3 MI) and the other reported no deaths (0/35). One long-term study of metformin therapy (250 mg to 500 mg thrice weekly) in patients undergoing haemodialysis reported 4 deaths (4/61; 7%) due to major cardiovascular events (2 MI). These findings provide very low-quality evidence as they come from small observational studies. CONCLUSION: The evidence regarding the safety of metformin in people undergoing dialysis is inconclusive. Appropriately designed randomised controlled trials are needed to resolve this uncertainty.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/efectos adversos , Enfermedades Renales/sangre , Riñón/metabolismo , Metformina/efectos adversos , Diálisis Renal , Acidosis/sangre , Acidosis/inducido químicamente , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Monitoreo de Drogas , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/uso terapéutico , Enfermedades Renales/complicaciones , Enfermedades Renales/terapia , Ácido Láctico/sangre , Metformina/administración & dosificación , Metformina/farmacocinética , Metformina/uso terapéuticoRESUMEN
BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.
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Compuestos de Anilina/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Compuestos de Anilina/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Ingeniería Genética , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Proteínas de la Membrana/metabolismo , Ratones , Morfolinas/farmacología , Morfolinas/uso terapéutico , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Inducción de Remisión , Carcinoma Pulmonar de Células Pequeñas/patología , Sulfonamidas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Orthotopic heart transplantation is the gold-standard long-term treatment for medically refractive end-stage heart failure. However, suitable cardiac donors are scarce. Although donation after circulatory death has been used for kidney, liver, and lung transplantation, it is not used for heart transplantation. We report a case series of heart transplantations from donors after circulatory death. METHODS: The recipients were patients at St Vincent's Hospital, Sydney, Australia. They received Maastricht category III controlled hearts donated after circulatory death from people younger than 40 years and with a maximum warm ischaemic time of 30 min. We retrieved four hearts through initial myocardial protection with supplemented cardioplegia and transferred to an Organ Care System (Transmedics) for preservation, resuscitation, and transportation to the recipient hospital. FINDINGS: Three recipients (two men, one woman; mean age 52 years) with low transpulmonary gradients (<8 mm Hg) and without previous cardiac surgery received the transplants. Donor heart warm ischaemic times were 28 min, 25 min, and 22 min, with ex-vivo Organ Care System perfusion times of 257 min, 260 min, and 245 min. Arteriovenous lactate values at the start of perfusion were 8·3-8·1 mmol/L for patient 1, 6·79-6·48 mmol/L for patient 2, and 7·6-7·4 mmol/L for patient 3. End of perfusion lactate values were 3·6-3·6 mmol/L, 2·8-2·3 mmol/L, and 2·69-2·54 mmol/L, respectively, showing favourable lactate uptake. Two patients needed temporary mechanical support. All three recipients had normal cardiac function within a week of transplantation and are making a good recovery at 176, 91, and 77 days after transplantation. INTERPRETATION: Strict limitations on donor eligibility, optimised myocardial protection, and use of a portable ex-vivo organ perfusion platform can enable successful, distantly procured orthotopic transplantation of hearts donated after circulatory death. FUNDING: NHMRC, John T Reid Charitable Trust, EVOS Trust Fund, Harry Windsor Trust Fund.
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Displasia Ventricular Derecha Arritmogénica/terapia , Cardiomiopatía Dilatada/terapia , Trasplante de Corazón/métodos , Miocarditis/terapia , Preservación de Órganos/métodos , Donantes de Tejidos/clasificación , Obtención de Tejidos y Órganos/métodos , Adulto , Displasia Ventricular Derecha Arritmogénica/fisiopatología , Biopsia , Cardiomiopatía Dilatada/fisiopatología , Femenino , Paro Cardíaco Inducido , Humanos , Masculino , Persona de Mediana Edad , Miocardio/patología , Choque/patología , Resultado del Tratamiento , Virosis/terapia , Isquemia TibiaRESUMEN
BACKGROUND: Storage of donor hearts in cardioplegic solutions supplemented with agents that mimic the ischaemic preconditioning response enhanced their post-reperfusion function. The present study examines the minimisation of cell death and activation of pro-survival signalling directed towards maintenance of mitochondrial homeostasis in hearts arrested and stored in two such agents, glyceryl-trinitrate, a nitric oxide donor and cariporide, (a sodium-hydrogen exchange inhibitor). METHODS: After baseline functional measurement, isolated working rat hearts were arrested and stored for 6h at 4°C in either Celsior(®), Celsior(®) containing 0.1mg/ml glyceryl-trinitrate, 10µM cariporide or both agents. After reperfusion, function was remeasured. Hearts were then processed for immunoblotting or histology. RESULTS: Necrotic and apoptotic markers present in the Celsior(®) group post-reperfusion were abolished by glyceryl-trinitrate, cariporide or both. Increased phosphorylation of ERK and Bcl2, after reperfusion in groups stored in glyceryl-trinitrate, cariporide or both along with increased phospho-STAT3 levels in the glyceryl-trinitrate/cariporide group correlated with functional recovery. Inhibition of STAT3 phosphorylation blocked recovery. No phospho-Akt increase was seen in any treatment. CONCLUSIONS: Activation of signalling pathways that favour mitophagy activation (ERK and Bcl2 phosphorylation) and maintenance of mitochondrial transition pore closure after reperfusion (STAT3 and ERK phosphorylation) were crucial for functional recovery of the donor heart.
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Cardiotónicos/farmacocinética , Guanidinas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Nitroglicerina/farmacología , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Sulfonas/farmacología , Animales , Masculino , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas WistarRESUMEN
Hearts from older donors or procured via donation after circulatory death (DCD) can alleviate transplant waitlist; however, these hearts are particularly vulnerable to injury caused by warm ischemic times (WITs) inherent to DCD. This study investigates how the combination of increasing donor age and pharmacologic supplementation affects the ischemic tolerance and functional recovery of DCD hearts and how age impacts cardiac mitochondrial respiratory capacity and oxidative phosphorylation. METHODS: Wistar rats (12-, 18-, and 24-mo-old) were subjected to DCD with 20-min fixed WIT. Hearts were procured, instrumented onto a Langendorff perfusion circuit, flushed with Celsior preservation solution with or without supplementation (glyceryl trinitrate [GTN]/erythropoietin [EPO]/zoniporide [Z]) and perfused (Krebs-Henseleit buffer, 37°C Langendorff 30-min, working 30-min). Cardiac functional recovery of aortic flow (AF), coronary flow (CF), cardiac output (CO), and lactate dehydrogenase release were measured. Native heart tissue (3-, 12-, and 24-mo) were assessed for mitochondrial respiratory capacity. RESULTS: Unsupplemented 18- and 24-month DCD hearts showed a 6-fold decrease in AF recovery relative to unsupplemented 12-month DCD hearts. GTN/EPO/Z supplementation significantly increased AF and CO recovery of 18-month DCD hearts to levels comparable to supplemented 12-month hearts; however, GTN/EPO/Z did not improve 24-month DCD heart recovery. Compared to 12-month heart tissue, 24-month hearts exhibited significantly impaired mitochondrial oxygen flux at complex I, II, and uncoupled maximal respiration stage. CONCLUSIONS: Reduced ischemic tolerance after DCD was associated with increasing age. Pharmacologic supplementation improves functional recovery of rat DCD hearts but only up to age 18 months, possibly attributed to a decline in mitochondrial respiratory capacity with increasing age.
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BACKGROUND: Storage of donor hearts in cardioplegic solutions supplemented with conditioning agents activating endogenous mitochondrial protective signaling enhanced their postreperfusion recovery. The present study investigates the role of timing and duration of cardiac exposure to cyclosporine A (CsA), another putative mitochondrial protectant, on cardiac functional recovery and potential mechanisms of CsA action in an isolated working rat heart model of donor heart retrieval and storage. METHODS: After measurement of baseline function, hearts were arrested and stored for 6 hours at 4°C in either Celsior alone or Celsior + CsA (0.2 µM), then reperfused for 45 minutes in Krebs solution, when functional recovery was assessed. Two additional groups of Celsior-alone stored hearts were exposed to 0.2 µM CsA for the initial 15 minutes (nonworking period) or the full 45-minute period of reperfusion. Coronary effluent was collected pre- and poststorage for assessment of lactate dehydrogenase release. Tissue samples were collected at the end of each study for immunoblotting and histological studies. RESULTS: CsA supplementation during cold storage or the first 15-minute reperfusion significantly improved functional recovery and significantly increased phospho-AMPKαThr172 and phospho-ULK-1Ser757. Hearts exposed to CsA for 45 minutes at reperfusion recovered poorly with no phospho-AMP-activated protein kinase α activation, decreased phospho-eNOSSer633, and decreased mitochondrial cytochrome c content with increased lactate dehydrogenase release. CONCLUSIONS: Inclusion of CsA during cold storage is cardioprotective. Effects of CsA addition to the perfusate during reperfusion were time dependent, with benefits at 15 minutes but not 45 minutes of reperfusion. The toxic effect with the presence of CsA for the full 45-minute reperfusion is associated with impaired mitochondrial integrity and decreased eNOS phosphorylation.
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Soluciones Cardiopléjicas/farmacología , Ciclosporina/farmacología , Trasplante de Corazón , Corazón/efectos de los fármacos , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Soluciones Cardiopléjicas/toxicidad , Isquemia Fría , Ciclosporina/toxicidad , Disacáridos/farmacología , Disacáridos/toxicidad , Electrólitos/farmacología , Electrólitos/toxicidad , Glutamatos/farmacología , Glutamatos/toxicidad , Glutatión/farmacología , Glutatión/toxicidad , Corazón/fisiopatología , Trasplante de Corazón/efectos adversos , Histidina/farmacología , Histidina/toxicidad , Preparación de Corazón Aislado , Masculino , Manitol/farmacología , Manitol/toxicidad , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Soluciones Preservantes de Órganos/toxicidad , Fosforilación , Ratas Wistar , Recuperación de la Función , Factores de TiempoRESUMEN
BACKGROUND: Transplantation of hearts retrieved from donation after circulatory death (DCD) donors is an evolving clinical practice. OBJECTIVES: The purpose of this study is to provide an update on the authors' Australian clinical program and discuss lessons learned since performing the world's first series of distantly procured DCD heart transplants. METHODS: The authors report their experience of 23 DCD heart transplants from 45 DCD donor referrals since 2014. Donor details were collected using electronic donor records (Donate Life, Australia) and all recipient details were collected from clinical notes and electronic databases at St. Vincent's Hospital. RESULTS: Hearts were retrieved from 33 of 45 DCD donors. A total of 12 donors did not progress to circulatory arrest within the pre-specified timeframe. Eight hearts failed to meet viability criteria during normothermic machine perfusion, and 2 hearts were declined due to machine malfunction. A total of 23 hearts were transplanted between July 2014 and April 2018. All recipients had successful implantation, with mechanical circulatory support utilized in 9 cases. One case requiring extracorporeal membrane oxygenation subsequently died on the sixth post-operative day, representing a mortality of 4.4% over 4 years with a total follow-up period of 15,500 days for the entire cohort. All surviving recipients had normal cardiac function on echocardiogram and no evidence of acute rejection on discharge. All surviving patients remain in New York Heart Association functional class I with normal biventricular function. CONCLUSIONS: DCD heart transplant outcomes are excellent. Despite a higher requirement for mechanical circulatory support for delayed graft function, primarily in recipients with ventricular assist device support, overall survival and rejection episodes are comparable to outcomes from contemporary brain-dead donors.
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Causas de Muerte , Trasplante de Corazón , Choque , Recolección de Tejidos y Órganos , Obtención de Tejidos y Órganos , Adulto , Australia , Selección de Donante/métodos , Femenino , Supervivencia de Injerto , Trasplante de Corazón/métodos , Trasplante de Corazón/estadística & datos numéricos , Humanos , Masculino , Evaluación de Procesos y Resultados en Atención de Salud , Recolección de Tejidos y Órganos/métodos , Recolección de Tejidos y Órganos/estadística & datos numéricos , Obtención de Tejidos y Órganos/métodos , Obtención de Tejidos y Órganos/organización & administraciónRESUMEN
Dysregulation of iron homeostasis is implicated in Alzheimer's disease (AD). In this pilot study, common variants of the apolipoprotein E (APOE) and HFE genes resulting in the iron overload disorder of hereditary hemochromatosis (C282Y, H63D and S65C) were evaluated as factors in sporadic AD in an Ontario sample in which folic acid fortification has been mandatory since 1998. Laboratory studies also were done to search for genetic effects on blood markers of iron status, red cell folates and serum B12. Participants included 58 healthy volunteers (25 males, 33 females) and 54 patients with probable AD (20 males, 34 females). Statistical analyses were interpreted at the 95% confidence level. Contingency table and odds ratio analyses supported the hypothesis that in females of the given age range, E4 significantly predisposed to AD in the presence but not absence of H63D. In males, E4 significantly predisposed to AD in the absence of H63D, and H63D in the absence of E4 appeared protective against AD. Among E4+ AD patients, H63D was associated with significant lowering of red cell folate concentration, possibly as the result of excessive oxidative stress. However, folate levels in the lowest population quartile did not affect the risk of AD. A model is presented to explain the experimental findings.
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Alelos , Enfermedad de Alzheimer/genética , Apolipoproteína E4/genética , Análisis Mutacional de ADN , Ácido Fólico/administración & dosificación , Variación Genética/genética , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Sobrecarga de Hierro/genética , Proteínas de la Membrana/genética , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/tratamiento farmacológico , Biomarcadores/sangre , Eritrocitos/metabolismo , Femenino , Ácido Fólico/sangre , Predisposición Genética a la Enfermedad/genética , Genotipo , Hemocromatosis/sangre , Proteína de la Hemocromatosis , Humanos , Sobrecarga de Hierro/sangre , Masculino , Escala del Estado Mental , Persona de Mediana Edad , Ontario , Factores Sexuales , Vitamina B 12/sangreRESUMEN
The ryanodine receptor antagonist dantrolene inhibits calcium release from the sarcoplasmic reticulum and reduces cardiac ischaemia-reperfusion injury (IRI) in global warm ischaemia models however the cardioprotective potential of dantrolene under hypothermic conditions is unknown. This study addresses whether the addition of dantrolene during cardioplegia and hypothermic storage of the donor heart can improve functional recovery and reduce IRI. Using an ex vivo isolated working heart model, Wistar rat (3 month and 12 month) hearts were perfused to acquire baseline haemodynamic measurements of aortic flow, coronary flow, cardiac output, pulse pressure and heart rate. Hearts were arrested and stored in Celsior preservation solution supplemented with 0.2-40 µM dantrolene for 6 hours at 4°C, then reperfused (15 min Langendorff, 30 min working mode). In 3-month hearts, supplementation with 1 µM dantrolene significantly improved aortic flow and cardiac output compared to unsupplemented controls however lactate dehydrogenase (LDH) release and contraction bands were comparable. In contrast, 40 µM dantrolene-supplementation yielded poor cardiac recovery, increased post-reperfusion LDH but reduced contraction bands. All 3-month hearts stored in dantrolene displayed significantly reduced cleaved-caspase 3 intensities compared to controls. Analysis of cardioprotective signalling pathways showed no changes in AMPKα however dantrolene increased STAT3 and ERK1/2 signaling in a manner unrelated to functional recovery and AKT activity was reduced in 1 µM dantrolene-stored hearts. In contrast to 3-month hearts, no significant improvements were observed in the functional recovery of 12-month hearts following prolonged storage in 1 µM dantrolene. CONCLUSIONS: Dantrolene supplementation at 1 µM during hypothermic heart preservation improved functional recovery of young, but not older (12 month) hearts. Although the molecular mechanisms responsible for dantrolene-mediated cardioprotection are unclear, our studies show no correlation between improved functional recovery and SAFE and RISK pathway activation.
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Dantroleno/farmacología , Corazón/efectos de los fármacos , Corazón/fisiología , Animales , Frío , Criopreservación , Suplementos Dietéticos , Hemodinámica , Técnicas In Vitro , Masculino , Preservación de Órganos , Soluciones Preservantes de Órganos , Ratas , Ratas Wistar , Daño por Reperfusión/prevención & control , Transducción de SeñalRESUMEN
Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it.Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance.Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance "free" cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes.Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197-208. ©2017 AACR.
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Proteína 11 Similar a Bcl2/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Terapia Molecular Dirigida , Compuestos de Anilina/farmacología , Animales , Apoptosis/genética , Ciclo Celular/genética , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Receptores ErbB/genética , Humanos , Ratones , Mutación , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: Inhibition of poly(ADP-ribose) polymerase 1 (PARP) has been shown to be effective in minimizing cardiac ischemia reperfusion injury. We investigated the cardioprotective effect of the PARP inhibitor, INO-1153, in isolated working rat hearts after 6 hr of hypothermic storage in Celsior. METHODS: Hearts were treated with 1 muM INO-1153 before hypothermic storage, at cardioplegia and storage or after hypothermic storage. Hearts not exposed to INO-1153 served as controls. Another group was pretreated with the phosphatidylinositol 3-kinase inhibitor Wortmannin (0.1 muM) before storage in INO-1153-supplemented Celsior. After baseline measurement of aortic flow, heart rate, coronary flow, and cardiac output were obtained, hearts were arrested and stored in Celsior at 2-3 degrees C for 6 hr. After storage, hearts were reperfused for 15 min before performing work for a further 30 min, at which time poststorage indices of cardiac function were remeasured then heart tissue was stored at -80 degrees C for Western blot analysis. RESULTS: The presence of INO-1153 during prestorage perfusion or during cardioplegia and storage significantly improved poststorage cardiac function. Functional improvements produced by INO-1153 were completely abolished by Wortmnanin pretreatment. Western blots showed a significant increase in phospho-Akt in presence of INO-1153, which was inhibited by Wortmannin. CONCLUSION: Activation of the prosurvival phosphatidylinositol 3-kinase-Akt pathway was involved in the protective action of PARP inhibition in this model of donor heart procurement and hypothermic storage. Importantly for the logistics of clinical organ procurement, maximum protection is observed when the PARP inhibitor is included in the cardioplegic storage solution.
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Trasplante de Corazón/fisiología , Preservación de Órganos/métodos , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Androstadienos/farmacología , Animales , Disacáridos/farmacología , Electrólitos/farmacología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Glutamatos/farmacología , Glutatión/farmacología , Corazón/efectos de los fármacos , Corazón/fisiología , Histidina/farmacología , Masculino , Manitol/farmacología , Modelos Animales , Miocardio/enzimología , Soluciones Preservantes de Órganos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Daño por Reperfusión/fisiopatología , Daño por Reperfusión/prevención & control , Transducción de Señal/efectos de los fármacos , WortmaninaRESUMEN
Allopurinol is the drug most widely used to lower the blood concentrations of urate and, therefore, to decrease the number of repeated attacks of gout. Allopurinol is rapidly and extensively metabolised to oxypurinol (oxipurinol), and the hypouricaemic efficacy of allopurinol is due very largely to this metabolite. The pharmacokinetic parameters of allopurinol after oral dosage include oral bioavailability of 79 +/- 20% (mean +/- SD), an elimination half-life (t((1/2))) of 1.2 +/- 0.3 hours, apparent oral clearance (CL/F) of 15.8 +/- 5.2 mL/min/kg and an apparent volume of distribution after oral administration (V(d)/F) of 1.31 +/- 0.41 L/kg. Assuming that 90 mg of oxypurinol is formed from every 100mg of allopurinol, the pharmacokinetic parameters of oxypurinol in subjects with normal renal function are a t((1/2)) of 23.3 +/- 6.0 hours, CL/F of 0.31 +/- 0.07 mL/min/kg, V(d)/F of 0.59 +/- 0.16 L/kg, and renal clearance (CL(R)) relative to creatinine clearance of 0.19 +/- 0.06. Oxypurinol is cleared almost entirely by urinary excretion and, for many years, it has been recommended that the dosage of allopurinol should be reduced in renal impairment. A reduced initial target dosage in renal impairment is still reasonable, but recent data on the toxicity of allopurinol indicate that the dosage may be increased above the present guidelines if the reduction in plasma urate concentrations is inadequate. Measurement of plasma concentrations of oxypurinol in selected patients, particularly those with renal impairment, may help to decrease the risk of toxicity and improve the hypouricaemic response. Monitoring of plasma concentrations of oxypurinol should also help to identify patients with poor adherence. Uricosuric drugs, such as probenecid, have potentially opposing effects on the hypouricaemic efficacy of allopurinol. Their uricosuric effect lowers the plasma concentrations of urate; however, they increase the CL(R) of oxypurinol, thus potentially decreasing the influence of allopurinol. The net effect is an increased degree of hypouricaemia, but the interaction is probably limited to patients with normal renal function or only moderate impairment.
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Alopurinol/farmacocinética , Oxipurinol/farmacocinética , Alopurinol/metabolismo , Humanos , Redes y Vías Metabólicas , Modelos Teóricos , Oxipurinol/metabolismo , FarmacocinéticaRESUMEN
Congenital heart disease (CHD), comprising structural or functional abnormalities present at birth, is the most common birth defect in humans. Reduced expression of connexin40 (Cx40) has been found in association with atrial fibrillation, and deletion of Cx40 in a mouse model causes various structural heart abnormalities in 18% of heterozygotes. We screened 505 unrelated CHD cases for deletions or duplications of the Cx40 gene (GJA5) by real-time quantitative PCR, in order to determine whether altered copy number of this gene may be associated with a cardiac phenotype in humans. Dosage of Cx40 flanking genes (ACPL1 and Cx50 gene, GJA8) was determined by real-time PCR for all apparent positive cases. In total, 3 cases were found to carry deletions on chromosome 1q21.1 spanning ACPL1, Cx40, and Cx50 genes. Absence of heterozygosity was observed in all 3 index cases over a 1.5- to 3-Mb region. Samples from the parents of two cases were obtained, and microsatellites across 1q21.1 were genotyped. One of the apparently unaffected parents was found to carry this deletion. All 3 index cases presented with obstruction of the aortic arch as the common structural cardiac malformation, and had no consistent dysmorphic features. Genotyping of 520 unrelated normal controls for this deletion was negative. We hypothesize that this 1q21.1 multigene deletion is associated with a range of cardiac defects, with anomalies of the aortic arch being a particular feature.
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Aorta Torácica/anomalías , Cromosomas Humanos Par 1/genética , Conexinas/genética , Eliminación de Gen , Cardiopatías Congénitas/genética , Fosfatasa Ácida/genética , Adolescente , Adulto , Animales , Aorta Torácica/embriología , Niño , Preescolar , Cromosomas Humanos Par 1/ultraestructura , Sistemas de Computación , Conexinas/deficiencia , Proteínas del Ojo/genética , Femenino , Cardiopatías Congénitas/embriología , Humanos , Lactante , Recién Nacido , Pérdida de Heterocigocidad , Masculino , Ratones , Repeticiones de Microsatélite , Modelos Animales , Penetrancia , Reacción en Cadena de la Polimerasa , Proteína alfa-5 de Unión ComunicanteRESUMEN
The success of organ transplantation is critically dependent on the quality of the donor organ. Donor organ quality, in turn, is determined by a variety of factors including donor age and preexisting disease, the mechanism of brain death, donor management prior to organ procurement, the duration of hypothermic storage, and the circumstances of reperfusion. It has been recognized for some time that both the short- and long-term outcomes after cadaveric organ transplantation are significantly inferior to those obtained when the transplanted organ is obtained from a living donor, regardless of whether the donor is related or unrelated to the recipient. Brain death results in a series of hemodynamic, neurohormonal, and pro-inflammatory perturbations, all of which are thought to contribute to donor organ dysfunction. The process of transplantation exposes the donor organ to an obligatory period of ischemia and reperfusion. Traditionally, hypothermic storage of the donor organ has been used to protect it from ischemic injury, but donor organs differ markedly in their capacity to withstand hypothermic ischemia. Data from the Registry of the International Society for Heart and Lung Transplantation indicate that the risk of primary graft failure and death rises dramatically for both the heart and lung as ischemic time increases. Based on these data, maximum recommended ischemic times for the donor heart and lung are 6 and 8 h, respectively. In this chapter, strategies aimed at minimizing the adverse consequences of brain death and ischemia/reperfusion injury to the donor heart and lung are discussed. These strategies are likely to become increasingly important as the reliance on marginal donors increases to meet the growing demand for organ transplantation.
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Muerte Encefálica/fisiopatología , Preservación de Órganos , Predicción , Supervivencia de Injerto , Trasplante de Corazón/mortalidad , Humanos , Isquemia/fisiopatología , Donadores Vivos , Trasplante de Pulmón/mortalidad , Daño por Reperfusión/prevención & control , Factores de TiempoRESUMEN
Small cell lung cancer (SCLC) is an aggressive tumor type with high mortality. One promising approach for SCLC treatment would be to utilize agents targeting molecular abnormalities regulating resistance to apoptosis. BH3 mimetic antagonists, such as ABT-737 and its orally available derivative ABT-263 (navitoclax) have been developed to block the function of pro-survival BCL-2 family members. The sensitivity of SCLC to these drugs varies over a broad range in vitro and in clinical trials. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is a critical determinant of sensitivity to ABT-737. Thus, pharmacological up-regulation of Noxa could enhance cell death induced by the BH3 mimetics. We find that the combination of ABT-263 and a HDAC inhibitor, vorinostat, efficiently induces apoptosis in a variety of SCLC cell lines, including ABT-263 resistant cell lines. Cell death induced by combined treatment is Noxa- and/or BIM-dependent in some cell lines but in others appears to be mediated by down-regulation of BCL-XL and release of BAK from BCL-XL and MCL-1. These results suggest that combination of HDAC inhibitors and BCL-2 inhibitors could be an alternative and effective regimen for SCLC treatment.
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Compuestos de Anilina/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Sulfonamidas/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Vorinostat , Proteína Destructora del Antagonista Homólogo bcl-2/biosíntesis , Proteína bcl-X/biosíntesisRESUMEN
Noxa, a BH3-only pro-apoptotic BCL-2 family protein, causes apoptosis by specifically interacting with the anti-apoptotic protein MCL-1 to induce its proteasome-mediated degradation. We show here that the DNA damaging agents cisplatin and etoposide upregulate Noxa expression, which is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, proteasome-dependent degradation, and apoptosis. Noxa-induced MCL-1 phosphorylation at these sites occurs at the mitochondria and is primarily regulated by CDK2. MCL-1 and CDK2 form a stable complex and Noxa binds to this complex to facilitate the phosphorylation of MCL-1. When Ser64 and Thr70 of MCL-1 are substituted with alanine, the mutated MCL-1 is neither phosphorylated nor ubiquitinated, and becomes more stable than the wild-type protein. As a consequence, this mutant can inhibit apoptosis induced by Noxa overexpression or cisplatin treatment. These results indicate that Noxa-mediated MCL-1 phosphorylation followed by MCL-1 degradation is critical for apoptosis induced by DNA damaging agents through regulation of the Noxa/MCL-1/CDK2 complex.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Cisplatino/farmacología , Quinasa 2 Dependiente de la Ciclina/genética , Daño del ADN , Etopósido/farmacología , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Fosforilación/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARNRESUMEN
BACKGROUND: Hearts from older donors are increasingly being referred for transplantation. However, these hearts are more susceptible to ischemia-reperfusion injury (IRI), reflected in higher rates of primary graft dysfunction. We assessed a strategy of pharmacologic conditioning, supplementing Celsior (Genzyme, Naarden, The Netherlands) preservation solution with glyceryl trinitrate (GTN; Hospira Australia Pty, Ltd, Mulgrave, VIC, Australia), erythropoietin (EPO; Eprex; Janssen-Cilag, North Ryde, NSW, Australia), and zoniporide (ZON; Pfizer, Inc., Groton, CT), to protect older hearts against IRI and improve graft function. METHODS: Wistar rats, aged 3, 12, and 18 months old, were used to represent adolescent, 30-year-old, and 45-year-old human donors, respectively. Animals were subjected to brain death (BD) and hearts stored for 6 hours at 2° to 3°C in Celsior or Celsior supplemented with GTN+EPO+ZON. Cardiac function and lactate dehydrogenase before and after storage were assessed during ex vivo perfusion. Western blots and histopathology were also analyzed. RESULTS: After BD, 18-month hearts demonstrated impaired aortic flow, coronary flow, and cardiac output compared with 3-month hearts (p < 0.001 to p < 0.0001). After storage in Celsior, the recovery of aortic flow, coronary flow, and cardiac output in 18-month BD hearts was further impaired (p < 0.01 vs 3-month hearts). Percentage functional recovery of 18-month BD hearts stored in Celsior supplemented with GTN+EPO+ZON was equivalent to that of 3-month hearts and significantly improved compared with 18-month hearts stored in Celsior alone (p < 0.01 to p < 0.001), with reduced lactate dehydrogenase release (p < 0.01) and myocardial edema (p < 0.05) and elevated phosphorylated extracellular signal-related kinase 1/2 (p < 0.05) and phosphorylated Akt (p < 0.01). CONCLUSIONS: Older hearts are more susceptible to IRI induced by BD and prolonged hypothermic storage. Supplemented Celsior activates cell survival signaling in older hearts, reduces IRI, and enhances donor heart preservation.
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Corazón , Animales , Trasplante de Corazón , Preservación de Órganos , Soluciones Preservantes de Órganos , Ratas , Ratas WistarRESUMEN
BACKGROUND: Donation after circulatory death (DCD) provides an alternative pathway to deceased organ transplantation. Although clinical DCD lung, liver, and kidney transplantation are well established, transplantation of hearts retrieved from DCD donors has reached clinical translation only recently. Progress has been limited by concern regarding the viability of DCD hearts. The aim of this study was to document the pathophysiological changes that occur in the heart and circulation during withdrawal of life (WLS) support. METHODS: In a porcine asphyxia model, we characterized the hemodynamic, volumetric, metabolic, biochemical, and endocrine changes after WLS for up to 40 minutes. Times to circulatory arrest and electrical asystole were recorded. RESULTS: After WLS, there was rapid onset of profound hypoxemia resulting in acute pulmonary hypertension and right ventricular distension. Concurrently, progressive systemic hypotension occurred with a fall in left atrial pressure and little change in left ventricular volume. Mean times to circulatory arrest and electrical asystole were 8 ± 1 and 16 ± 2 minutes, respectively. Hemodynamic changes were accompanied by a rapid fall in pH, and rise in blood lactate, troponin-T, and potassium. Plasma noradrenaline and adrenaline levels rose rapidly with dramatic increases in coronary sinus levels indicative of myocardial release. CONCLUSIONS: These findings provide insight into the nature and tempo of the damaging events that occur in the heart and in particular the right ventricle during WLS, and give an indication of the limited timeframe for the implementation of potential postmortem interventions that could be applied to improve organ viability.
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Muerte , Trasplante de Corazón/métodos , Donantes de Tejidos , Obtención de Tejidos y Órganos/métodos , Animales , Asfixia/patología , Análisis de los Gases de la Sangre , Presión Sanguínea , Epinefrina/sangre , Corazón , Paro Cardíaco/fisiopatología , Hemodinámica , Concentración de Iones de Hidrógeno , Lactatos/sangre , Miocardio/patología , Norepinefrina/sangre , Potasio/sangre , Porcinos , Factores de Tiempo , Supervivencia Tisular , Troponina T/sangre , Isquemia TibiaRESUMEN
Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199. This sensitivity occurs in part through low anti-apoptotic BCL-xL expression, high pro-apoptotic NOXA expression, and paradoxical, MYCN-driven upregulation of NOXA. Screening for enhancers of ABT-199 sensitivity in MYCN-amplified neuroblastomas, we demonstrate that the Aurora Kinase A inhibitor MLN8237 combines with ABT-199 to induce widespread apoptosis. In diverse models of MYCN-amplified neuroblastoma, including a patient-derived xenograft model, this combination uniformly induced tumor shrinkage, and in multiple instances led to complete tumor regression.