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1.
J Agric Food Chem ; 70(44): 14235-14246, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36283033

RESUMO

The constituents of germinated brown rice (GBR), brown rice (BRR), and white rice (WHR) and their impact on metabolism, inflammation, and gut microbiota in high fat (HF) diet-fed mice were examined. The contents of total fiber and γ-aminobutyric acid in BRR and GBR were higher than that in WHR (p < 0.05). Male C57 BL/6J mice received HF diet+26 g% of WHR, BRR, or GBR for 12 weeks. BRR and GBR comparably reduced HF diet-induced increases in fasting plasma glucose, lipids, insulin resistance, and inflammatory markers compared to WHR (p < 0.01). The abundance of fecal Bacteroidetes in mice fed HF+GBR or HF+BRR was higher than in HF+WHR-fed mice (p < 0.05). The abundance of fecal Lactobacillus gasseri in GBR-fed mice was greater than that in WHR- or BRR-fed mice (p < 0.05). The results indicated that GBR or BRR attenuated hyperglycemia, insulin resistance, and inflammation in mice. HF+GBR, but not HF+BRR, increased a probiotic bacteria in the gut.


Assuntos
Microbioma Gastrointestinal , Resistência à Insulina , Oryza , Camundongos , Masculino , Animais , Dieta Hiperlipídica/efeitos adversos , Insulina , Inflamação , Camundongos Endogâmicos C57BL
2.
Transplantation ; 85(3): 437-46, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-18301335

RESUMO

BACKGROUND: The authors' previous study has shown that liposome-mediated ex vivo intracoronary interleukin (IL)-4 and IL-10 combined gene therapy suppressed the allo-immune responses and prolonged the cardiac allograft survival by 15 folds. However, the mechanism for promoting long-term allograft survival remains unknown. METHODS: This study tested the hypothesis that this combined cytokine gene targeting may promote alloreactive T-cell apoptosis or prevent apoptosis of cardiac allograft myocytes through Fas/Fas ligand (FasL) pathway. A rabbit functional cervical heterotopic heart transplantation model was used, and plasmid human recombinant IL-4 and IL-10 gene complexed with cationic liposome (GAP/DLRIE) was delivered into cardiac allografts by intracoronary infusion ex vivo. RESULTS: This liposome-mediated IL-4 and IL-10 combined gene therapy significantly increased apoptotic T cells detected by TUNEL staining. The caspase-8 or caspase-3 expressing T cells were also significantly increased. The Fas+ apoptotic T cells dominated in the population of apoptotic CD4+ T cells, but FasL+ CD4+ T-cell population was less effected in the combined gene therapy group. The effect of combined gene therapy on the infiltrative Fas+ CD8+ T-cell population is much less than that on Fas+ CD4+ cells, and there was almost no effect on the FasL+ CD8+ T-cell population. Furthermore, localized IL-4 and IL-10 combined gene therapy protected cardiac allograft myocytes by down-regulating its FasL expression, but not Fas. CONCLUSIONS: These results suggest that this combined gene targeting strategy which induced localized overexpression of exogenous IL-4 and IL-10 may promote alloreactive T-cell apoptosis and prevent myocytes apoptosis through Fas/FasL cell surface interaction, therefore inducing cardiac allograft tolerance.


Assuntos
Apoptose , Proteína Ligante Fas/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Miócitos Cardíacos/metabolismo , Linfócitos T/metabolismo , Receptor fas/metabolismo , Animais , Regulação da Expressão Gênica , Terapia Genética , Sobrevivência de Enxerto/imunologia , Transplante de Coração , Humanos , Interleucina-10/genética , Interleucina-4/genética , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/imunologia , Coelhos , Transdução de Sinais , Linfócitos T/citologia , Fatores de Tempo , Transplante Homólogo/imunologia
3.
J Nucl Med ; 47(4): 706-15, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16595506

RESUMO

UNLABELLED: 9-(4-(18)F-Fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG) is a sensitive and specific PET reporter probe for imaging the PET reporter genes, herpes simplex 1 thymidine kinase (HSV1-tk) and its mutant HSV1-sr39tk. (18)F-FHBG has suitable pharmacokinetics and dosimetry for clinical applications and imaging of HSV1-TK has been demonstrated in the livers of hepatocellular cancer patients. METHODS: Male and female Sprague-Dawley rats and New Zealand White rabbits were divided into equal groups receiving either 14 microg/kg cold FHBG or carrier solution, for a 14-d acute toxicity assessment. We monitored body weight, food and water consumption, body temperature, cardiovascular electrical and functional indices, respiratory performance and oxygen saturation, comprehensive blood chemistry, complete blood count (CBC), and urinalysis. We conducted daily cage-side examinations for the detection of any clinical abnormalities. Tissues of the animals that were euthanized and necropsied on day 14 were prepared for histopathologic examination. RESULTS: No significant differences in cardiovascular and respiratory parameters, food consumption, body weight, urine components, or clinical signs attributable to test article toxicity were observed between the treatment and control groups. Any differences noted in the blood chemistry and CBC parameters were deemed to be incidental findings unrelated to the administration of the FHBG. CONCLUSION: Acute toxicity evaluation of FHBG at 100 times the expected human dose does not indicate harm to organ function or tissues. The Food and Drug Administration has approved FHBG as an Investigational New Drug.


Assuntos
Guanina/análogos & derivados , Herpesvirus Humano 1/enzimologia , Compostos Radiofarmacêuticos/toxicidade , Timidina Quinase/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Guanina/farmacocinética , Guanina/toxicidade , Masculino , Mutação , Tomografia por Emissão de Pósitrons , Coelhos , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Sprague-Dawley , Timidina Quinase/genética , Testes de Toxicidade Aguda
4.
Circulation ; 109(3): 363-8, 2004 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-14732752

RESUMO

BACKGROUND: Remodeling occurs in both ventricle and atrium in dilated cardiomyopathy and heart failure. However, the alteration of atrial extracellular matrix components during remodeling and its effect on the electrical remodeling and atrial arrhythmia have never been explored. METHODS AND RESULTS: Atrial tissue samples of 53 explanted hearts from patients with dilated cardiomyopathy and end-stage heart failure who underwent heart transplantation were examined. Nineteen patients had permanent atrial fibrillation (PmAF), 18 had persistent AF (PsAF), and 16 had no documented AF (NAF). Sixteen donor left atria (LA) were used as controls (CNs). Western Blot analysis revealed a selective downregulation of tissue inhibitor of metalloproteinase (TIMP)-2 in PmAF and PsAF groups compared with the NAF and CN groups and an upregulation of atrial metalloproteinase (MMP)-2 that was most pronounced in the PmAF group followed by the PsAF and NAF groups. Immunofluorescent staining revealed that in the LA, type I collagen volume fraction (CVF-I) increased significantly in the PmAF group followed by the PsAF and NAF groups compared with that in CN. LA CVF-I significantly correlated with LA dimension and TIMP-2 to MMP-2 ratio. In the PsAF group, CVF-I/CVF-III ratio was significantly correlated with AF duration and the frequency of AF recurrence. CONCLUSIONS: Atrial extracellular matrix remodeling manifested by the selective downregulation of TIMP-2 along with upregulation of MMP-2 and CVF-I in the atrium is associated with the development of sustained atrial fibrillation in patients with cardiomyopathy and heart failure.


Assuntos
Fibrilação Atrial/etiologia , Matriz Extracelular/metabolismo , Átrios do Coração/metabolismo , Fibrilação Atrial/enzimologia , Fibrilação Atrial/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Matriz Extracelular/enzimologia , Feminino , Gelatinases/metabolismo , Átrios do Coração/enzimologia , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Inibidores Teciduais de Metaloproteinases/metabolismo
5.
Transplantation ; 76(1): 137-42, 2003 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-12865799

RESUMO

BACKGROUND: The precise incidence and cause of atrioventricular block (AVB) after heart transplantation remain uncertain. METHODS AND RESULTS: After surgery, immediate and follow-up electrocardiograms from 1047 consecutive patients who underwent heart transplantation were reviewed for AVB and correlated with clinical symptoms, laboratory data, rejection grade, and echocardiogram and coronary angiography findings. A total of 113 patients demonstrated various kinds of AVB; the incidence was 10.8%. First-degree AVB occurred in 87 patients, 37 of whom also demonstrated persistent atrial tachyarrhythmias (ATAs). In 30 patients, first-degree AVB occurred 7 days to 120 months after heart transplantation. Among those, 88% demonstrated cellular rejection, and 20% developed transplant coronary artery disease (TCAD). Fifty patients demonstrated first-degree AVB without ATA, 32 of whom developed AVB from operative day 7 to 156 months. The incidence of cellular rejection was significantly lower (36%, P<0.01), and the rejection was less severe. In 18 patients, AVB occurred early postoperatively (0-7 days), and most were secondary to surgical injury. Second-degree AVB Mobitz I occurred in six patients (four patients with TCAD and two patients undergoing percutaneous transluminal coronary angioplasty). One patient developed Mobitz II during coronary artery stenting. Complete AVB (CAVB) occurred in 19 patients. Nine episodes of CAVB occurred during endomyocardial biopsy or coronary angiography, and four occurred immediately after heart transplantation as the result of surgical insult. CONCLUSION: These results indicate that first-degree AVB is causatively related to cellular rejection and TCAD-induced atrial conduction disturbance. Second-degree AVB and CAVB were mainly the consequences of surgical and catheter intervention injury.


Assuntos
Bloqueio Cardíaco/etiologia , Transplante de Coração/efeitos adversos , Complicações Pós-Operatórias/etiologia , Antiarrítmicos/uso terapêutico , Angiografia Coronária , Seguimentos , Rejeição de Enxerto/epidemiologia , Bloqueio Cardíaco/epidemiologia , Bloqueio Cardíaco/mortalidade , Humanos , Incidência , Procainamida/uso terapêutico , Quinidina/uso terapêutico , Estudos Retrospectivos
6.
Transplantation ; 73(7): 1019-26, 2002 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11965026

RESUMO

We hypothesized that localized IL-10 gene transfer can induce alloreactive T cell apoptosis and tested this hypothesis with liposome-mediated ex vivo intracoronary IL-10 gene transfer using a functional heterotopic allograft heart transplant model in rabbits. Localized IL-10 overexpression prolonged cardiac allograft survival over three folds. In parallel with the time-course of IL-10 overexpression, the percentage of apoptotic CD3+ cells among total CD3+ cells was significantly increased in the gene therapy group (36.5+/-3.9%) compared with that in the control group (6.2+/-2.6%, P<0.01) on postoperative day (POD) 3-6, and it was further increased (45.8+/-5.7%) on POD7-10. Apoptotic CD4+ and CD8+ cells were also significantly increased in the gene group (P<0.01). In contrast, the percentage of apoptotic myocytes significantly decreased from 10.1+/-0.8% in the control group to 3.5+/-0.4% in the gene group on POD7-10 (P<0.01). This reduction was inversely correlated with the increase in the percentages of apoptotic CD4+ and CD8+ cells (P<0.01). The percentage of caspase-3 positive myocytes was significantly reduced, although percentages of caspase-3 positive CD4+ and CD8+ cells were markedly increased in the gene group (P<0.01). Moreover, about 60-80% of apoptotic T lymphocytes expressed Fas in the gene group compared with less than 10% in the control group (P<0.01). These results suggest that localized IL-10 gene transfer induces alloreactive T cell apoptosis via the Fas/FasL pathway that may contribute to the alleviated acute rejection, improved cardiac function, and prolonged survival in the IL-10 gene-treated cardiac allografts.


Assuntos
Apoptose , Terapia Genética , Sobrevivência de Enxerto , Transplante de Coração , Interleucina-10/genética , Glicoproteínas de Membrana/fisiologia , Linfócitos T/fisiologia , Receptor fas/fisiologia , Animais , Caspase 3 , Caspases/fisiologia , Proteína Ligante Fas , Rejeição de Enxerto , Marcação In Situ das Extremidades Cortadas , Coelhos , Transfecção , Transplante Homólogo
7.
J Heart Lung Transplant ; 21(11): 1188-200, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12431492

RESUMO

BACKGROUND: Overexpression of interleukin 10 (IL-10) in the donor heart prolongs allograft survival in animals. Interleukin-10 has many immunosuppressive effects; however, the mechanism(s) of its protective effect on allograft rejection remains unknown. METHODS: Recently, we optimized an ex vivo, intracoronary infusion of the GAP:DLRIE, liposome-mediated, IL-10 gene method using a rabbit, cervical, heterotopic heart transplant model. RESULTS: The efficiency of this new-generation, liposome-mediated, IL-10 gene transfer to the donor hearts was 15% in hypothermic conditions, which represents a 30% increase from the efficiency of other liposomes, such as DOSPA/DOPE, DOGS/DOPE, and DMRIE/DOPE. Cardiac allograft survival was prolonged from 6.0 +/- 0.7 days to 14.3 +/- 1.8 days. Infiltrating lymphocyte sub-populations CD3+, CD4+, and CD8) decreased significantly in the gene therapy group compared with the control group. Local IL-10 overexpression correlated significantly with decreased CD4+ and CD8+ responsiveness and Type-1 helper (Th1) cytokine gene (IL-2, interferon-gamma, and tumor necrosis factor alpha) expression level and correlated inversely with the allograft rejection grade. In the gene therapy group, the cytotoxic activity of infiltrating T cells in the allograft decreased greatly, but the time course of this decrease did not parallel the rejection process. CONCLUSION: We conclude that GAP:DLRIE is the best cationic liposome for ex vivo gene transfection in hypothermic conditions. The effects of IL-10 gene therapy on antigen-specific T-lymphocyte proliferation and Th1-cytokine expression may play an important role in localized immunosuppression and tolerance induction.


Assuntos
Portadores de Fármacos , Éteres , Técnicas de Transferência de Genes , Rejeição de Enxerto/genética , Transplante de Coração/imunologia , Tolerância Imunológica/imunologia , Interleucina-10/genética , Compostos de Amônio Quaternário , Animais , Expressão Gênica , Imuno-Histoquímica , Interleucina-10/metabolismo , Lipossomos , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th1/metabolismo , Transfecção , Tolerância ao Transplante
8.
J Cardiovasc Pharmacol Ther ; 7(1): 29-38, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12000976

RESUMO

Zatebradine, a potent bradycardic agent, is believed to act selectively at the sinoatrial node. The selectivity of such a property relative to various electrophysiologic classes of action is not well defined. To characterize the electrophysiologic properties of zatebradine, the corrected sinus node recovery time, sinoatrial conduction time, conduction intervals, atrial effective refractory period and monophasic action potential duration in the ventricle were measured before and after incremental doses of zatebradine (0.1-1.5 mg/kg) in 15 anesthetized dogs. The electrophysiologic effects of zatebradine developed immediately after a single i.v. bolus dose, reaching steady-steady-state at 15 minutes with the maximum effect evident at 0.75 mg/kg. The IC(50) was 0.23 mg/kg. There was no significant effect on the sinus node recovery time. The PR interval on the electrocardiogram was significantly increased when the dose was higher than 0.25 mg/kg. The duration of the P wave and the PA interval were not changed. Zatebridine greatly increased the AH (from 135 to 178 milliseconds) without changing the HH and HV intervals in His bundle recordings. The EC(50) of this effect was 0.58 mg/kg. The QRS interval was not changed. The QTc was significantly increased from 0.43 to 0.56 s(1/2) (P < 0.05). The action potential duration was significantly increased by high dose zatebradine (> 0.5 mg/kg), the EC(50) for this effect was 0.76 mg/kg. The atrium effective refractory period was significantly increased (31%) with an EC(50) 0.69 mg/kg. These results indicate that zatebradine selectively inhibits sinus node automaticity at low doses. The inhibition of the AV nodal conduction and the lengthening of the refractory period and repolarization in the atria and the ventricles occur at higher dose.


Assuntos
Benzazepinas/farmacologia , Cardiotônicos/farmacologia , Sistema de Condução Cardíaco/efeitos dos fármacos , Potenciais de Ação , Animais , Cães , Relação Dose-Resposta a Droga , Eletrocardiografia , Eletrofisiologia , Feminino , Sistema de Condução Cardíaco/fisiologia , Injeções Intravenosas
9.
Am J Physiol Heart Circ Physiol ; 292(1): H277-84, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16951047

RESUMO

Perforin is a cytolytic mediator produced by cytotoxic T cells (CD8(+) cells) and natural killer cells. We previously reported that ex vivo IL-10 gene therapy induced apoptosis of allogenic infiltrative CD8(+) cells and significantly prolonged cardiac allograft survival. To further test the hypothesis that localized IL-10 overexpression in cardiac allografts may also effect the alloreactive CD8(+) T cell function by downregulating its perforin production, we used a rabbit functional heterotopic allograft heart transplant model. Human recombinant IL-10 gene complexed with liposome was intracoronary delivered into the cardiac allografts ex vivo. The percentage of apoptotic infiltrative CD8(+) cells in cardiac allografts was increased 6-fold in the gene therapy group vs. the control group, whereas the percentage of perforin-positive CD8(+) cells was decreased 2.9-fold (P < 0.01). Perforin expression level in the allograft myocardium of the gene therapy group was deceased 3.2-fold (P < 0.01). The amount of infiltrative perforin-positive CD8(+) cells and perforin expression level were inversely correlated with IL-10 transgene and protein expression level in the myocardium of cardiac allografts (P < 0.01), the percentage of apoptotic cardiac myocytes (P < 0.01), and the peak left ventricular systolic pressure of cardiac allografts (P < 0.01) but significantly correlated with the infiltrative T cell cytotoxicity (P < 0.01) and allograft rejection score (P < 0.01). These results suggest that localized IL-10 gene therapy prolongs cardiac allograft survival, at least in part, through downregulation of perforin production by activated allogenic CD8(+) T cells. Reduction of cytolytic function of cytotoxic effector cells prevents the apoptosis of cardiac myocytes.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Sobrevivência de Enxerto/fisiologia , Transplante de Coração/métodos , Interleucina-10/metabolismo , Glicoproteínas de Membrana/metabolismo , Miocárdio/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animais , Linfócitos T CD8-Positivos/patologia , Transplante de Coração/patologia , Interleucina-10/genética , Ativação Linfocitária/fisiologia , Miocárdio/patologia , Perforina , Coelhos , Transplante Homólogo , Regulação para Cima
10.
J Immunol ; 174(11): 6983-92, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15905541

RESUMO

Localized gene transfer has the potential to introduce immunosuppressive molecules only into the transplanted allograft, which would limit systemic side effects, and prolong allograft survival. However, an applicable gene transfer strategy is not available, and the feasible therapeutic gene(s) has not yet been determined. We developed an ex vivo liposome-mediated gene therapy strategy that is able to intracoronary deliver the combination of IL-4 and IL-10 cDNA expression vectors to the allograft simultaneously. We examined the efficiency, efficacy, and cardiac adverse effects of this combinatorial gene therapy protocol using a rabbit functional cervical heterotopic heart transplant model. Although the efficiency was moderate, the expression of both transgenes was long lasting and localized only in the target organ. The mean survival of cardiac allograft was prolonged from 7 to >100 days. Synergism of overexpressed IL-4 and IL-10 in the inhibition of T lymphocyte infiltration and cytoxicity, and modulation of Th1/Th2 cytokine production promote long-term survival of cardiac allografts.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Citocinas/administração & dosagem , Citocinas/genética , Facilitação Imunológica de Enxerto/métodos , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Imunossupressores/administração & dosagem , Adjuvantes Imunológicos/biossíntese , Animais , Inibição de Migração Celular , Citocinas/biossíntese , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Combinação de Medicamentos , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Facilitação Imunológica de Enxerto/efeitos adversos , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/genética , Transplante de Coração/efeitos adversos , Transplante de Coração/patologia , Humanos , Imunossupressores/efeitos adversos , Interleucina-10/administração & dosagem , Interleucina-10/biossíntese , Interleucina-10/genética , Interleucina-4/administração & dosagem , Interleucina-4/biossíntese , Interleucina-4/genética , Lipossomos , Coelhos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Transplante Heterotópico
11.
Mol Ther ; 12(1): 49-57, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15963920

RESUMO

We developed a clinically applicable approach for noninvasive monitoring of reporter-therapeutic linked gene expression in the whole heart of large animals using PET imaging and further validated the efficacy and cardiac adverse effects of reporter-therapeutic linked gene transfer in a rabbit cervical heterotopic functional heart transplant model. Cationic liposome complexed with a vector containing a herpes simplex virus type 1 mutant thymidine kinase (HSV1-sr39tk) as the reporter gene and a recombinant human immunosuppressive cytokine, interleukin-10 (hIL-10), as the therapeutic gene was ex vivo intracoronarily delivered into cardiac allografts before implantation. Long-term HSV1-sr39tk and hIL-10 transgene and protein overexpression associated with myocardial PET reporter probe 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) accumulation was observed in the allografts. The expression of the HSV1-sr39tk gene was significantly correlated with the hIL-10 gene expression and the total myocardial [18F]FHBG accumulation quantified as a percentage of intravenously injected [18F]FHBG dose. A homogeneous distribution of [18F]FHBG accumulation was seen in the whole heart similar to the distribution of [18F]fluorodeoxyglucose, a PET glucose metabolism probe. The immunosuppressive therapeutic efficacy remained the same in allografts treated with reporter-therapeutic linked gene and therapeutic gene only. No cardiac adverse effect was found. Our results demonstrate for the first time that PET reporter-therapeutic linked gene imaging is applicable for noninvasively monitoring ex vivo intracoronarily delivered therapeutic transgene expression in the whole heart.


Assuntos
Técnicas de Transferência de Genes , Coração/diagnóstico por imagem , Animais , Genes Reporter , Vetores Genéticos , Humanos , Hibridização In Situ , Lipossomos , Tomografia por Emissão de Pósitrons , Coelhos , Radiografia , Transgenes
12.
Am J Physiol Heart Circ Physiol ; 283(5): H2119-29, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12384490

RESUMO

To evaluate the effects of liothyronine (3,5,3'-triiodo-L-thyronine, T(3)) on Na(+) channel current (I(Na)) properties, I(Na) was recorded in adult guinea pig ventricular myocytes. T(3) (1 nM) acutely increased whole cell I(Na) and shifted the steady-state I(Na) inactivation curve dose dependently. When the pipette solution contained 100 microM GTP or GTPgammaS, the effect of T(3) on the whole cell I(Na) was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T(3) increased the open probability of single I(Na) by reducing the null probability. In the inside-out patch, T(3) effect was 10 times faster than that in whole cell and cell-attached patches while GTPgammaS was present and could be completely washed out. T(3) alone slightly increased the channel open probability by increasing the closed state to open state rate constant (k(CO)) and reducing the null probability. GTPgammaS exposure only increased the number of functional channels. T(3) and GTPgammaS synergistically enhanced the channel open probability 5.8 +/- 0.5-fold by increasing k(CO), decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T(3) acts on the cytosolic side of the membrane and acutely activates I(Na). Pertussis toxin-sensitive G protein modulation greatly magnifies the T(3) effects on the channel kinetics and null probability, thereby increasing the channel open probability.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Sódio/metabolismo , Tri-Iodotironina/farmacologia , Animais , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cobaias , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Toxina Pertussis/farmacologia
13.
Am J Physiol Heart Circ Physiol ; 285(6): H2832-41, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12907417

RESUMO

Abnormal regulation of apoptosis is observed in ischemic injury and may contribute to the pathogenesis of atherosclerosis. However, its role in cardiac allograft vasculopathy (CAV), the fundamental lesion of chronic rejection (CR) in heart transplantation, remains uncertain. To clarify this issue, apoptosis was quantitated in myocardium and coronary arteries from 5 cardiac allograft donors (NL) and explanted hearts of 24 patients with ischemic cardiomyopathy (IsCM) and 15 patients with CR. Tissue samples were analyzed via end-labeling fragmented DNA [via deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)] and immunoblotting for activated caspase-3 and -9. Myocyte apoptosis assessed by TUNEL was similarly increased over NL (0.21%) in both the CR (0.88%; P < 0.01) and IsCM (0.88%; P < 0.01) groups. Activated caspase-9 levels were significantly higher in CR (14.7%) compared with IsCM (6.9%; P < 0.01) and NL (0%) groups, whereas activated caspase-3 levels were similarly elevated in both CR and IsCM (7.8 and 6.5% vs. 0% in NL; P < 0.01 and P < 0.05) groups. Expression of myocardial Bcl-2 and Bax was increased in CR compared with both NL (Bax, 4.3-fold; P < 0.01; Bcl-2, 5.9-fold; P < 0.01) and IsCM (IsCM: Bax, 2.2-fold; P < 0.05; Bcl-2, 3.2-fold; P < 0.01) groups. The rate of apoptosis and the Bcl-2/Bax ratio independently correlated to graft survival in CR (activation of caspase-9: r = 0.87; P < 0.01; Bcl-2/Bax: r = 0.57; P = 0.05). Compared with native atherosclerosis, coronary arteries with CAV showed more medial apoptosis (7.8-fold; P < 0.01) and higher Bcl-2 levels (5.1-fold; P < 0.01) with lower Bax levels (threefold; P < 0.05) in the intima. These results indicate that abnormal Bcl-2 and Bax expression in myocardium and coronary arteries of cardiac allografts with CR is distinct from that in IsCM and suggest that balancing Bcl-2 to Bax in transplanted hearts promotes long-term graft survival.


Assuntos
Cardiomiopatias/metabolismo , Fragmentação do DNA/fisiologia , Sobrevivência de Enxerto/fisiologia , Transplante de Coração , Mitocôndrias/metabolismo , Adolescente , Adulto , Idoso , Cardiomiopatias/patologia , Cardiomiopatias/cirurgia , Caspase 3 , Caspases/metabolismo , Criança , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/cirurgia , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transplante Homólogo , Proteína X Associada a bcl-2 , Proteína bcl-X
14.
Am J Physiol Heart Circ Physiol ; 285(3): H964-73, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12915385

RESUMO

Activation-induced cell death and cytokine deprivation are demonstrated by peripheral T cell populations at the conclusion of natural immune responses, and each of these processes is modulated by the immunosuppressive cytokine interleukin (IL)-10 in vitro. This study employs a clinically relevant in vivo model of IL-10 gene transfer with heterotopically transplanted cardiac allografts to determine the mechanisms of the effects of IL-10 on T cell survival. IL-10 protein overexpression within allografts 4-5 days after gene transfer augments apoptosis of CD4+ and CD8+ graft-infiltrating lymphocytes by 7.1-fold (P < 0.001) and 6.0-fold (P < 0.001), respectively. Graft-infiltrating T cells express 10-fold more proapoptotic Fas (P < 0.01) and 30-fold more Bax (P < 0.01) than controls. The fractions of activated caspase-8 (FADD-like IL-1beta-converting enzyme) and activated caspase-9 were increased 7- and 2.3-fold, respectively, in IL-10 gene-treated allografts at postoperative day 4-5. These changes in the Fas-Fas ligand pathway and Bcl-2 mitochondrial apoptosis regulation are enhanced by complete suppression of antiapoptotic FADD-like IL-1beta-converting enzyme inhibitory protein (FLIP) (from 30.5 to 0.0%, P < 0.01) and Bcl-xL (from 22.5 to 0.1%, P = 0.03) expression among these cells from the earliest days after gene transfer. Although changes in proteins of Fas- and Bcl-2-mediated apoptosis signaling occur, only the levels of Fas and FLIP correlate to the rate of apoptosis of graft-infiltrating CD3 lymphocytes and histological rejection scores. These results indicate that dichotomous apoptosis-regulatory pathways are affected by IL-10 gene therapy, but Fas-mediated mechanisms of activation-induced cell death more substantially contribute to the greater cell death of graft-infiltrating T cells after ex vivo IL-10 gene transfer.


Assuntos
Apoptose/imunologia , Transplante de Coração/imunologia , Interleucina-10/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/citologia , Receptor fas/metabolismo , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Complexo CD3/análise , Proteínas de Transporte/metabolismo , Caspase 8 , Caspase 9 , Caspases/metabolismo , Terapia Genética/métodos , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Rejeição de Enxerto/terapia , Sobrevivência de Enxerto/imunologia , Humanos , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Coelhos , Linfócitos T/química , Linfócitos T/metabolismo , Transplante Homólogo , Regulação para Cima/imunologia , Proteína X Associada a bcl-2 , Proteína bcl-X
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