Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
J Biol Chem ; 291(10): 5185-98, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26702054

RESUMO

Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction.


Assuntos
Aminoquinolinas/farmacologia , Benzamidas/farmacologia , Diabetes Mellitus/metabolismo , Hipoglicemiantes/farmacologia , Obesidade/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Tecido Adiposo/metabolismo , Aminoquinolinas/uso terapêutico , Animais , Apoptose , Benzamidas/uso terapêutico , Glicemia/metabolismo , Linhagem Celular , Diabetes Mellitus/tratamento farmacológico , Estresse do Retículo Endoplasmático , Metabolismo Energético , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Lipogênese , Fígado/metabolismo , Masculino , Camundongos , Camundongos Obesos , Biogênese de Organelas , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos
2.
Lancet ; 387(10024): 1178-86, 2016 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-26803443

RESUMO

BACKGROUND: Sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) activity is deficient in the failing heart. Correction of this abnormality by gene transfer might improve cardiac function. We aimed to investigate the clinical benefits and safety of gene therapy through infusion of adeno-associated virus 1 (AAV1)/SERCA2a in patients with heart failure and reduced ejection fraction. METHODS: We did this randomised, multinational, double-blind, placebo-controlled, phase 2b trial at 67 clinical centres and hospitals in the USA, Europe, and Israel. High-risk ambulatory patients with New York Heart Association class II-IV symptoms of heart failure and a left ventricular ejection fraction of 0·35 or less due to an ischaemic or non-ischaemic cause were randomly assigned (1:1), via an interactive voice and web-response system, to receive a single intracoronary infusion of 1 × 10(13) DNase-resistant particles of AAV1/SERCA2a or placebo. Randomisation was stratified by country and by 6 min walk test distance. All patients, physicians, and outcome assessors were masked to treatment assignment. The primary efficacy endpoint was time to recurrent events, defined as hospital admission because of heart failure or ambulatory treatment for worsening heart failure. Primary efficacy endpoint analyses and safety analyses were done by modified intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01643330. FINDINGS: Between July 9, 2012, and Feb 5, 2014, we randomly assigned 250 patients to receive either AAV1/SERCA2a (n=123) or placebo (n=127); 243 (97%) patients comprised the modified intention-to-treat population. Patients were followed up for at least 12 months; median follow-up was 17·5 months (range 1·8-29·4 months). AAV1/SERCA2a did not improve time to recurrent events compared with placebo (104 vs 128 events; hazard ratio 0·93, 95% CI 0·53-1·65; p=0·81). No safety signals were noted. 20 (16%) patients died in the placebo group and 25 (21%) patients died in the AAV1/SERCA2a group; 18 and 22 deaths, respectively, were adjudicated as being due to cardiovascular causes. INTERPRETATION: CUPID 2 is the largest gene transfer study done in patients with heart failure so far. Despite promising results from previous studies, AAV1/SERCA2a at the dose tested did not improve the clinical course of patients with heart failure and reduced ejection fraction. Although we did not find evidence of improved outcomes at the dose of AAV1/SERCA2a studied, our findings should stimulate further research into the use of gene therapy to treat patients with heart failure and help inform the design of future gene therapy trials. FUNDING: Celladon Corporation.


Assuntos
Cálcio/metabolismo , Terapia Genética/métodos , Insuficiência Cardíaca/terapia , Regulação para Cima , Idoso , Dependovirus/genética , Método Duplo-Cego , Feminino , Vetores Genéticos , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Recidiva , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Resultado do Tratamento
3.
Circ Res ; 114(1): 101-8, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24065463

RESUMO

RATIONALE: The Calcium Up-Regulation by Percutaneous Administration of Gene Therapy In Cardiac Disease (CUPID 1) study was a phase 1/phase 2 first-in-human clinical gene therapy trial using an adeno-associated virus serotype 1 (AAV1) vector carrying the sarcoplasmic reticulum calcium ATPase gene (AAV1/SERCA2a) in patients with advanced heart failure. The study explored potential benefits of the therapy at 12 months, and results were previously reported. OBJECTIVE: To report long-term (3-year) clinical effects and transgene expression in the patients in CUPID 1. METHODS AND RESULTS: A total of 39 patients with advanced heart failure who were on stable, optimal heart failure therapy were randomized to receive intracoronary infusion of AAV1/SERCA2a in 1 of 3 doses (low-dose, 6×10(11) DNase-resistant particles; mid-dose, 3×10(12) DNase-resistant particles; and high-dose, 1×10(13) DNase-resistant particles) versus placebo. The following recurrent cardiovascular and terminal events were tracked for 3 years in all groups: myocardial infarction, worsening heart failure, heart failure-related hospitalization, ventricular assist device placement, cardiac transplantation, and death. The number of cardiovascular events, including death, was highest in the placebo group, high but delayed in the low- and mid-dose groups, and lowest in the high-dose group. Evidence of long-term transgene presence was also observed in high-dose patients. The risk of prespecified recurrent cardiovascular events was reduced by 82% in the high-dose versus placebo group (P=0.048). No safety concerns were noted during the 3-year follow-up. CONCLUSIONS: After a single intracoronary infusion of AAV1/SERCA2a in patients with advanced heart failure, positive signals of cardiovascular events persist for years.


Assuntos
Terapia Genética , Insuficiência Cardíaca/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Adulto , Idoso , Dependovirus/genética , Método Duplo-Cego , Feminino , Vetores Genéticos/genética , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/fisiopatologia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Tempo , Transgenes/genética , Resultado do Tratamento
4.
Circulation ; 128(5): 512-23, 2013 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-23804254

RESUMO

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by dysregulated proliferation of pulmonary artery smooth muscle cells leading to (mal)adaptive vascular remodeling. In the systemic circulation, vascular injury is associated with downregulation of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) and alterations in Ca(2+) homeostasis in vascular smooth muscle cells that stimulate proliferation. We, therefore, hypothesized that downregulation of SERCA2a is permissive for pulmonary vascular remodeling and the development of PAH. METHODS AND RESULTS: SERCA2a expression was decreased significantly in remodeled pulmonary arteries from patients with PAH and the rat monocrotaline model of PAH in comparison with controls. In human pulmonary artery smooth muscle cells in vitro, SERCA2a overexpression by gene transfer decreased proliferation and migration significantly by inhibiting NFAT/STAT3. Overexpresion of SERCA2a in human pulmonary artery endothelial cells in vitro increased endothelial nitric oxide synthase expression and activation. In monocrotaline rats with established PAH, gene transfer of SERCA2a via intratracheal delivery of aerosolized adeno-associated virus serotype 1 (AAV1) carrying the human SERCA2a gene (AAV1.SERCA2a) decreased pulmonary artery pressure, vascular remodeling, right ventricular hypertrophy, and fibrosis in comparison with monocrotaline-PAH rats treated with a control AAV1 carrying ß-galactosidase or saline. In a prevention protocol, aerosolized AAV1.SERCA2a delivered at the time of monocrotaline administration limited adverse hemodynamic profiles and indices of pulmonary and cardiac remodeling in comparison with rats administered AAV1 carrying ß-galactosidase or saline. CONCLUSIONS: Downregulation of SERCA2a plays a critical role in modulating the vascular and right ventricular pathophenotype associated with PAH. Selective pulmonary SERCA2a gene transfer may offer benefit as a therapeutic intervention in PAH.


Assuntos
Hipertensão Pulmonar/terapia , Monocrotalina/toxicidade , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/uso terapêutico , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Hipertensão Pulmonar Primária Familiar , Técnicas de Transferência de Genes , Células HEK293 , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/enzimologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/biossíntese , Resultado do Tratamento
5.
Circ Res ; 111(11): 1434-45, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-22931954

RESUMO

RATIONALE: There is growing evidence that the myocardium responds to injury by recruiting c-kit(+) cardiac progenitor cells to the damage tissue. Even though the ability of exogenously introducing c-kit(+) cells to injured myocardium has been established, the capability of recruiting these cells through modulation of local signaling pathways by gene transfer has not been tested. OBJECTIVE: To determine whether stem cell factor gene transfer mediates cardiac regeneration in a rat myocardial infarction model, through survival and recruitment of c-kit(+) progenitors and cell-cycle activation in cardiomyocytes, and explore the mechanisms involved. METHODS AND RESULTS: Infarct size, cardiac function, cardiac progenitor cells recruitment, fibrosis, and cardiomyocyte cell-cycle activation were measured at different time points in controls (n=10) and upon stem cell factor gene transfer (n=13) after myocardial infarction. We found a regenerative response because of stem cell factor overexpression characterized by an enhancement in cardiac hemodynamic function: an improvement in survival; a reduction in fibrosis, infarct size and apoptosis; an increase in cardiac c-kit(+) progenitor cells recruitment to the injured area; an increase in cardiomyocyte cell-cycle activation; and Wnt/ß-catenin pathway induction. CONCLUSIONS: Stem cell factor gene transfer induces c-kit(+) stem/progenitor cell expansion in situ and cardiomyocyte proliferation, which may represent a new therapeutic strategy to reverse adverse remodeling after myocardial infarction.


Assuntos
Proliferação de Células , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismo , Adenoviridae/genética , Animais , Western Blotting , Contagem de Células , Expressão Gênica , Terapia Genética/métodos , Humanos , Estimativa de Kaplan-Meier , Masculino , Microscopia Confocal , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Células-Tronco/genética , Células-Tronco/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo
6.
Mol Ther ; 20(3): 565-71, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22215018

RESUMO

SERCA2a gene therapy improves contractile and energetic function of failing hearts and has been shown to be associated with benefits in clinical outcomes, symptoms, functional status, biomarkers, and cardiac structure in a phase 2 clinical trial. In an effort to enhance the efficiency and homogeneity of gene uptake in cardiac tissue, we examined the effects of nitroglycerin (NTG) in a porcine model following AAV1.SERCA2a gene delivery. Three groups of Göttingen minipigs were assessed: (i) group A: control intracoronary (IC) AAV1.SERCA2a (n = 6); (ii) group B: a single bolus IC injection of NTG (50 µg) immediately before administration of intravenous (IV) AAV1.SERCA2a (n = 6); and (iii) group C: continuous IV NTG (1 µg/kg/minute) during the 10 minutes of AAV1.SERCA2a infusion (n = 6). We found that simultaneous IV infusion of NTG and AAV1.SERCA2a resulted in increased viral transduction efficiency, both in terms of messenger RNA (mRNA) as well as SERCA2a protein levels in the whole left ventricle (LV) compared to control animals. On the other hand, IC NTG pretreatment did not result in enhanced gene transfer efficiency, mRNA or protein levels when compared to control animals. Importantly, the transgene expression was restricted to the heart tissue. In conclusion, we have demonstrated that IV infusion of NTG significantly improves cardiac gene transfer efficiency in porcine hearts.


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Miocárdio/metabolismo , Nitroglicerina/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Células Cultivadas , Circulação Coronária/efeitos dos fármacos , Expressão Gênica , Hemodinâmica/efeitos dos fármacos , Infusões Intra-Arteriais , Infusões Intravenosas , Masculino , Miócitos Cardíacos/metabolismo , Nitroglicerina/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suínos , Transdução Genética
7.
Circulation ; 124(3): 304-13, 2011 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-21709064

RESUMO

BACKGROUND: Adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase was assessed in a randomized, double-blind, placebo-controlled, phase 2 study in patients with advanced heart failure. METHODS AND RESULTS: Thirty-nine patients received intracoronary adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase or placebo. Seven efficacy parameters were assessed in 4 domains: symptoms (New York Heart Association class, Minnesota Living With Heart Failure Questionnaire), functional status (6-minute walk test, peak maximum oxygen consumption), biomarker (N-terminal prohormone brain natriuretic peptide), and left ventricular function/remodeling (left ventricular ejection fraction, left ventricular end-systolic volume), plus clinical outcomes. The primary end point success criteria were prospectively defined as achieving efficacy at 6 months in the group-level (concordant improvement in 7 efficacy parameters and no clinically significant worsening in any parameter), individual-level (total score for predefined clinically meaningful changes in 7 efficacy parameters), or outcome end points (cardiovascular hospitalizations and time to terminal events). Efficacy in 1 analysis had to be associated with at least a positive trend in the other 2 analyses. This combination of requirements resulted in a probability of success by chance alone of 2.7%. The high-dose group versus placebo met the prespecified criteria for success at the group-level, individual-level, and outcome analyses (cardiovascular hospitalizations) at 6 months (confirmed at 12 months) and demonstrated improvement or stabilization in New York Heart Association class, Minnesota Living With Heart Failure Questionnaire, 6-minute walk test, peak maximum oxygen consumption, N-terminal prohormone brain natriuretic peptide levels, and left ventricular end-systolic volume. Significant increases in time to clinical events and decreased frequency of cardiovascular events were observed at 12 months (hazard ratio=0.12; P=0.003), and mean duration of cardiovascular hospitalizations over 12 months was substantially decreased (0.4 versus 4.5 days; P=0.05) on high-dose treatment versus placebo. There were no untoward safety findings. CONCLUSIONS: The Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) study demonstrated safety and suggested benefit of adeno-associated virus type 1/sarcoplasmic reticulum Ca(2+)-ATPase in advanced heart failure, supporting larger confirmatory trials. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT00454818.


Assuntos
Cálcio/metabolismo , Terapia Genética/métodos , Cardiopatias/terapia , Insuficiência Cardíaca/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Regulação para Cima/fisiologia , Adenoviridae/genética , Adulto , Idoso , Progressão da Doença , Método Duplo-Cego , Teste de Esforço , Feminino , Terapia Genética/efeitos adversos , Cardiopatias/fisiopatologia , Humanos , Injeções Intra-Arteriais , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio/fisiologia , Índice de Gravidade de Doença , Resultado do Tratamento
8.
J Vis Exp ; (189)2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36440832

RESUMO

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.


Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Humanos , Camundongos , Animais , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/etiologia , Artéria Pulmonar , Hemodinâmica , Hipóxia , Modelos Animais de Doenças
9.
J Card Fail ; 15(3): 171-81, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19327618

RESUMO

BACKGROUND: SERCA2a deficiency is commonly seen in advanced heart failure (HF). This study is designed to investigate safety and biological effects of enzyme replacement using gene transfer in patients with advanced HF. METHODS AND RESULTS: A total of 9 patients with advanced HF (New York Heart Association [NYHA] Class III/IV, ejection fraction [EF] < or = 30%, maximal oxygen uptake [VO2 max] <16 mL.kg.min, with maximal pharmacological and device therapy) received a single intracoronary infusion of AAV1/SERCA2a in the open-label portion of this ongoing study. Doses administered ranged from 1.4 x 10(11) to 3 x 10(12) DNase resistant particles per patient. We present 6- to 12-month follow-up data for these patients. AAV1/SERCA2a demonstrated an acceptable safety profile in this advanced HF population. Of the 9 patients treated, several demonstrated improvements from baseline to month 6 across a number of parameters important in HF, including symptomatic (NYHA and Minnesota Living with Heart Failure Questionnaire, 5 patients), functional (6-minute walk test and VO2 max, 4 patients), biomarker (NT-ProBNP, 2 patients), and LV function/remodeling (EF and end-systolic volume, 5 patients). Of note, 2 patients who failed to improve had preexisting anti-AAV1 neutralizing antibodies. CONCLUSIONS: Quantitative evidence of biological activity across a number of parameters important for assessing HF status could be detected in several patients without preexisting neutralizing antibodies in this open-label study, although the number of patients in each cohort is too small to conduct statistical analyses. These findings support the initiation of the Phase 2 double-blind, placebo-controlled portion of this study.


Assuntos
Terapia Genética , Insuficiência Cardíaca/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Teste de Esforço , Feminino , Insuficiência Cardíaca/genética , Humanos , Infusões Intra-Arteriais , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/sangue , Consumo de Oxigênio , Fragmentos de Peptídeos/sangue , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/administração & dosagem , Volume Sistólico , Sístole , Regulação para Cima , Remodelação Ventricular
10.
J Card Fail ; 14(5): 355-67, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18514926

RESUMO

BACKGROUND: Heart failure (HF) remains a major cause of morbidity and mortality in North America. With an aging population and an unmet clinical need by current pharmacologic and device-related therapeutic strategies, novel treatment options for HF are being explored. One such promising strategy is gene therapy to target underlying molecular anomalies in the dysfunctional cardiomyocyte. Prior animal and human studies have documented decreased expression of SERCA2a, a major cardiac calcium cycling protein, as a major defect found in HF. METHODS AND RESULTS: We hypothesize that increasing the activity of SERCA2a in patients with moderate to severe HF will improve their cardiac function, disease status, and quality of life. Gene transfer of SERCA2a will be performed via an adeno-associated viral (AAV) vector, derived from a nonpathogenic virus with long-term transgene expression as well as a clinically established favorable safety profile. CONCLUSIONS: We describe the design of a phase 1 clinical trial of antegrade epicardial coronary artery infusion (AECAI) administration of AAVI/SERCA2a (MYDICAR) to subjects with HF divided into 2 stages: in Stage 1, subjects will be assigned open-label MYDICAR in one of up to 4 sequential dose escalation cohorts; in Stage 2, subjects will be randomized in parallel to 2 or 3 doses of MYDICAR or placebo in a double-blinded manner.


Assuntos
Dependovirus , Terapia Genética , Vetores Genéticos , Insuficiência Cardíaca/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Transdução Genética/métodos , Adolescente , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Idoso , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Terapia Combinada , Vasos Coronários , Diuréticos/uso terapêutico , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Proteínas de Fluorescência Verde , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Angiotensina/agonistas , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transgenes
11.
J Am Coll Cardiol ; 67(17): 2032-46, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-27126531

RESUMO

BACKGROUND: Pulmonary hypertension (PH) is characterized by pulmonary arterial remodeling that results in increased pulmonary vascular resistance, right ventricular (RV) failure, and premature death. Down-regulation of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) in the pulmonary vasculature leads to perturbations in calcium ion (Ca(2+)) homeostasis and transition of pulmonary artery smooth muscle cells to a proliferative phenotype. OBJECTIVES: We assessed the feasibility of sustained pulmonary vascular SERCA2a gene expression using aerosolized delivery of adeno-associated virus type 1 (AAV1) in a large animal model of chronic PH and evaluated the efficacy of gene transfer regarding progression of pulmonary vascular and RV remodeling. METHODS: A model of chronic post-capillary PH was created in Yorkshire swine by partial pulmonary vein banding. Development of chronic PH was confirmed hemodynamically, and animals were randomized to intratracheal administration of aerosolized AAV1 carrying the human SERCA2a gene (n = 10, AAV1.SERCA2a group) or saline (n = 10). Therapeutic efficacy was evaluated 2 months after gene delivery. RESULTS: Transduction efficacy after intratracheal delivery of AAV1 was confirmed by ß-galactosidase detection in the distal pulmonary vasculature. Treatment with aerosolized AAV1.SERCA2a prevented disease progression as evaluated by mean pulmonary artery pressure, vascular resistance, and limited vascular remodeling quantified by histology. Therapeutic efficacy was supported further by the preservation of RV ejection fraction (p = 0.014) and improvement of the RV end-diastolic pressure-volume relationship in PH pigs treated with aerosolized AAV1.SERCA2a. CONCLUSIONS: Airway-based delivery of AAV vectors to the pulmonary arteries was feasible, efficient, and safe in a clinically relevant chronic PH model. Vascular SERCA2a overexpression resulted in beneficial effects on pulmonary arterial remodeling, with attendant improvements in pulmonary hemodynamics and RV performance, and might offer therapeutic benefit by modifying fundamental pathophysiology in pulmonary vascular diseases.


Assuntos
Técnicas de Transferência de Genes , Hipertensão Pulmonar/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Aerossóis , Animais , Dependovirus , Modelos Animais de Doenças , Estudos de Viabilidade , Vetores Genéticos , Pulmão/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/farmacologia , Volume Sistólico , Suínos , Remodelação Vascular , Remodelação Ventricular , beta-Galactosidase/metabolismo
12.
Circ Heart Fail ; 8(1): 167-74, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25342737

RESUMO

BACKGROUND: Stem cell factor (SCF), a ligand of the c-kit receptor, is a critical cytokine, which contributes to cell migration, proliferation, and survival. It has been shown that SCF expression increases after myocardial infarction (MI) and may be involved in cardiac repair. The aim of this study was to determine whether gene transfer of membrane-bound human SCF improves cardiac function in a large animal model of MI. METHODS AND RESULTS: A transmural MI was created by implanting an embolic coil in the left anterior descending artery in Yorkshire pigs. One week after the MI, the pigs received direct intramyocardial injections of either a recombinant adenovirus encoding for SCF (Ad.SCF, n=9) or ß-gal (Ad.ß-gal, n=6) into the infarct border area. At 3 months post-MI, ejection fraction increased by 12% relative to baseline after Ad.SCF therapy, whereas it decreased by 4.2% (P=0.004) in pigs treated with Ad.ß-gal. Preload-recruitable stroke work was significantly higher in pigs after SCF treatment (Ad.SCF, 55.5±11.6 mm Hg versus Ad.ß-gal, 31.6±12.6 mm Hg, P=0.005), indicating enhanced cardiac function. Histological analyses confirmed the recruitment of c-kit(+) cells as well as a reduced degree of apoptosis 1 week after Ad.SCF injection. In addition, increased capillary density compared with pigs treated with Ad.ß-gal was found at 3 months and suggests an angiogenic role of SCF. CONCLUSIONS: Local overexpression of SCF post-MI induces the recruitment of c-kit(+) cells at the infarct border area acutely. In the chronic stages, SCF gene transfer was associated with improved cardiac function in a preclinical model of ischemic cardiomyopathy.


Assuntos
Terapia Genética/métodos , Infarto do Miocárdio/terapia , Miocárdio/metabolismo , Fator de Células-Tronco/metabolismo , Volume Sistólico , Função Ventricular Esquerda/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Suínos
13.
Int Arch Allergy Immunol ; 99(2-4): 323-325, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-34167212

RESUMO

We studied the activity of recombinant human stem cell factor (rhSCF) on the release of mediators from human skin mast cells. High concentrations of rhSCF (1 ng/ml-1 µg/ml) induced a rapid and sustained rise in intracellular Ca2+ levels that was accompanied by release of histamine and prostaglandin D2 (PGD2). A brief incubation (10 min) with lower concentrations of rhSCF (0.1 pg/ml-1 ng/ml) enhanced anti-IgE-induced histamine release, but not the release of PGD2. Since some of the effects of rhSCF on mast cell mediator release in vitro occur at concentrations of cytokine similar to those found in the serum of normal subjects it is possible that SCF contributes to the modulation of mast cell function under physiological conditions.

14.
JACC Heart Fail ; 2(1): 84-92, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24622121

RESUMO

OBJECTIVES: Impaired cardiac isoform of sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA2a) activity is a key abnormality in heart failure patients with reduced ejection fraction. The CUPID 2 (Calcium Up-Regulation by Percutaneous Administration of Gene Therapy in Cardiac Disease Phase 2b) trial is designed to evaluate whether increasing SERCA2a activity via gene therapy improves clinical outcome in these patients. BACKGROUND: Intracoronary delivery of recombinant adeno-associated virus serotype 1 (AAV1)/SERCA2a improves intracellular Ca(2+) handling by increasing SERCA2a protein levels and, as a consequence, restores systolic and diastolic function. In a previous phase 2a trial, this therapy improved symptoms, functional status, biomarkers, and left ventricular function, and reduced cardiovascular events in advanced heart failure patients. METHODS: CUPID 2 is a phase 2b, double-blind, placebo-controlled, multinational, multicenter, randomized event-driven study in up to 250 patients with moderate-to-severe heart failure with reduced ejection fraction and New York Heart Association functional class II to IV symptoms despite optimal therapy. Enrolled patients will be at high risk for recurrent heart-failure hospitalizations by virtue of having elevated N-terminal pro-B-type natriuretic peptide/BNP (>1,200 pg/ml, or >1,600 pg/ml if atrial fibrillation is present) and/or recent heart failure hospitalization. The primary endpoint of time-to-recurrent event (heart failure-related hospitalizations in the presence of terminal events [all-cause death, heart transplant, left ventricular assist device implantation or ambulatory worsening heart failure]) will be assessed using the joint frailty model. This ongoing trial is expected to complete recruitment in 2014, with the required number of 186 recurrent events estimated to occur by mid 2015. RESULTS: Available data indicate that calcium up-regulation by AAV1/SERCA2a gene therapy is safe and of potential benefit in advanced heart failure patients. CONCLUSIONS: The CUPID 2 trial is designed to study the effects of this therapy on clinical outcome in these patients. (Calcium Up-Regulation by Percutaneous Administration of Gene Therapy in Cardiac Disease Phase 2b [CUPID-2b]; NCT01643330).


Assuntos
Terapia Genética/métodos , Insuficiência Cardíaca/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/administração & dosagem , Adenoviridae , Administração Cutânea , Cálcio/metabolismo , Método Duplo-Cego , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Infusões Intralesionais , Proteínas Recombinantes/administração & dosagem , Recidiva , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Resultado do Tratamento , Regulação para Cima
15.
J Biomol Screen ; 19(2): 215-22, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24436077

RESUMO

We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/isolamento & purificação , Animais , Proteínas de Fluorescência Verde/química , Células HEK293 , Hepatócitos/metabolismo , Humanos , Proteínas Luminescentes/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Bibliotecas de Moléculas Pequenas , Proteína Vermelha Fluorescente
16.
J Biomol Screen ; 18(1): 97-107, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22923787

RESUMO

Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarcoplasmic reticulum Ca-ATPase (SERCA) by its cardiac regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca(2+) regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20 000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 hits (0.2%), 31 (72%) were found to be false-positives upon more thorough FRET testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and preclinical testing. We were concerned about the high rate of false-positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HTS.


Assuntos
Ativadores de Enzimas/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Regulação Alostérica , Animais , Proteínas de Ligação ao Cálcio/fisiologia , Células Cultivadas , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/fisiologia , Coelhos , Ratos , Ratos Sprague-Dawley , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/enzimologia , Estimulação Química
17.
J Am Coll Cardiol ; 51(11): 1112-9, 2008 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-18342232

RESUMO

OBJECTIVES: The aim of this study was to examine the effects of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) gene transfer in a swine heart failure (HF) model. BACKGROUND: Reduced expression and activity of SERCA2a have been documented in HF. Prior studies have reported the beneficial effects of short-term SERCA2a overexpression in rodent models. However, the effects of long-term expression of SERCA2a in pre-clinical large animal models are not known. METHODS: Yorkshire-Landrace pigs were used (n = 16) to create volume overload by percutaneously severing chordae tendinae of the mitral apparatus with a bioptome to induce mitral regurgitation. At 2 months, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 1 (rAAV1) carrying SERCA2a under a cytomegalovirus promoter (rAAV1.SERCA2a) (n = 10; group 1) or saline (n = 6; group 2). RESULTS: At 2 months, study animals were found to be in a compensated state of volume-overload HF (increased left ventricular internal diastolic and systolic diameters [LVIDd and LVIDs]). At 4 months, gene transfer resulted in: 1) positive left ventricular (LV) inotropic effects (adjusted peak left ventricular pressure rate of rise (dP/dt)max/P, 21.2 +/- 3.2 s(-1) group 1 vs. 15.5 +/- 3.0 s(-1) group 2; p < 0.01); 2) improvement in LV remodeling (% change in LVIDs -3.0 +/- 10% vs. +15 +/- 11%, respectively; p < 0.01). At follow-up, brain natriuretic peptide levels remained stable in group 1 after gene transfer, in contrast to rising levels in group 2. Further, cardiac SERCA2a expression was significantly decreased in group 2 whereas in group 1 it was restored to normal levels. There was no histopathological evidence of acute myocardial inflammation or necrosis. CONCLUSIONS: Using a large-animal, volume-overload model of HF, we report that long-term overexpression of SERCA2a by in vivo rAAV1-mediated intracoronary gene transfer preserved systolic function, potentially prevented diastolic dysfunction, and improved ventricular remodeling.


Assuntos
Técnicas de Transferência de Genes , Insuficiência Cardíaca/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Disfunção Ventricular Esquerda/terapia , Animais , Citomegalovirus/genética , Modelos Animais de Doenças , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca Diastólica/terapia , Insuficiência Cardíaca Sistólica/terapia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suínos , Remodelação Ventricular
18.
J Mol Cell Cardiol ; 42(4): 852-61, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17300800

RESUMO

The aim of this study was to examine whether short- and long-term gene transfer of Ca(2+) handling proteins restore left ventricular (LV) mechanoenergetics in aortic banding-induced failing hearts. Aortic-banded rats received recombinant adenoviruses carrying sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) (Banding+SERCA), parvalbumin (Banding+Parv) or beta-galactosidase (Banding+betagal), or an adeno-associated virus carrying SERCA2a (Banding+AAV.SERCA) by a catheter-based technique. LV mechanoenergetic function was measured in cross-circulated hearts. "Banding", "Banding+betagal" and "Banding+saline" groups showed lower end-systolic pressure at 0.1 ml intraballoon water (ESP(0.1)), higher end-diastolic pressure at 0.1 ml intraballoon water (EDP(0.1)) and slower LV relaxation rate, compared with "Normal" and "Sham". However, "Banding+SERCA" and "Banding+Parv" showed high ESP(0.1), low EDP(0.1) and fast LV relaxation rate. In "Banding", "Banding+betagal" and "Banding+saline", slope of relation between cardiac oxygen consumption and systolic pressure-volume area, O(2) cost of total mechanical energy, was twice higher than normal value, whereas slope in "Baning+SERCA" and "Banding+Parv" was similar to normal value. Furthermore, O(2) cost of LV contractility in the 3 control banding groups was approximately 3 times higher than normal value, whereas O(2) cost of contractility in "Banding+SERCA", "Banding+AAV.SERCA" and "Banding+Parv" was as low as normal value. Thus, high O(2) costs of total mechanical energy and of LV contractility in failing hearts indicate energy wasting both in chemomechanical energy transduction and in calcium handling. Improved calcium handling by both short- and long-term overexpression of SERCA2a and parvalbumin transforms the inefficient energy utilization into a more efficient state. Therefore enhancement of calcium handling either by resequestration into the SR or by intracellular buffering improves not only mechanical but energetic function in failing hearts.


Assuntos
Aorta/enzimologia , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Adenoviridae/genética , Animais , Cálcio , Terapia Genética , Masculino , Consumo de Oxigênio , Ratos , Ratos Wistar , Retículo Sarcoplasmático/metabolismo , Função Ventricular Esquerda , beta-Galactosidase/metabolismo
19.
Curr Protoc Immunol ; Chapter 6: 6.17.1-6.17.11, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18432808

RESUMO

Stem cell factor (SCF) is an early-acting, hematopoietic growth factor that binds to the receptor encoded by the proto-oncogene c-kit. It is a potent growth factor for primitive bone marrow cells as well as thymocytes. This unit describes three protocols for detecting human and murine SCF. In the first, human or rodent SCF is measured by its ability to stimulate proliferation of the human megakaryoblastic leukemia cell line, UT-7. Because rat and mouse SCF bind well to human c-kit, human and rodent SCF can both be measured using the first basic protocol. In an Alternate Protocol, rodent SCF is assayed by its ability to stimulate proliferation of the clonal murine mast cell line, MC/9. Human SCF is not very active on rodent cells and thus cannot be measured using this protocol. Both of the cell proliferation assays lack specificity because they are capable of detecting other cytokines in addition to SCF. The third protocol is a radioreceptor assay using the human erythroleukemia cell line, OCIM1; it specifically measures murine or human SCF and not other cytokines. Support protocols describe maintenance of UT-7 and MC/9 cells and preparation of plasma membranes from OCIM1 cells.


Assuntos
Bioensaio/métodos , Mastócitos/metabolismo , Ensaio Radioligante/métodos , Fator de Células-Tronco/análise , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Humanos , Camundongos , Proto-Oncogene Mas , Fator de Células-Tronco/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA