RESUMO
Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis. Sera that produced a phagocytosis magnitude greater than a normal human serum mean + 2 standard deviations were considered phagocytosis-positive. Overall, 42% (8/19) of MHC I alloantibody-negative ITP sera were phagocytosis-positive. The indirect monoclonal antibody immobilization of platelet antigens assay was used to detect immunoglobulin G (IgG) autoantibodies to glycoproteins (GP)IIb/IIIa, GPIb/IX, and GPIa/IIa. Autoantibody-positive sera triggered a higher mean magnitude of phagocytosis than autoantibody-negative sera. Phagocytosis correlated inversely with platelet counts among autoantibody-positive patients but not among autoantibody-negative patients. Select phagocytosis-positive sera were separated into IgG-purified and -depleted fractions via protein G and reassessed for phagocytosis. Phagocytosis was largely retained in the purified IgG fractions. In addition, we assessed serum concentrations of C-reactive protein, serum amyloid P, and pentraxin 3 as potential phagocytosis modulators. Pentraxin 3 concentrations correlated inversely with platelet counts among patients positive for autoantibodies. Taken together, sera from approximately half of the patients with ITP studied triggered macrophage phagocytosis of platelets beyond a normal level. An important role for antiplatelet autoantibodies in phagocytosis is supported; a role for pentraxins such as pentraxin 3 may be suggested.
Assuntos
Púrpura Trombocitopênica Idiopática , Trombocitopenia , Humanos , Púrpura Trombocitopênica Idiopática/diagnóstico , Plaquetas/metabolismo , Trombocitopenia/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Imunoglobulina G , Fagocitose , Macrófagos/metabolismo , AutoanticorposRESUMO
Fc gamma receptors (FcγRs) are critical effector receptors for immunoglobulin G (IgG) antibodies. On macrophages, FcγRs mediate multiple effector functions, including phagocytosis, but the individual contribution of specific FcγRs to phagocytosis has not been fully characterized. Primary human macrophage populations, such as splenic macrophages, can express FcγRI, FcγRIIA, and FcγRIIIA. However, there is currently no widely available monocyte or macrophage cell line expressing all these receptors. Common sources of monocytes for differentiation into macrophages, such as human peripheral blood monocytes and the monocytic leukemia cell line THP-1, generally lack the expression of FcγRIIIA (CD16A). Here, we utilized a lentiviral system to generate THP-1 cells stably expressing human FcγRIIIA (CD16F158). THP-1-CD16A cells treated with phorbol 12-myristate 13-acetate for 24 hours phagocytosed anti-D-opsonized human red blood cells primarily utilizing FcγRI with a lesser but significant contribution of IIIA while phagocytosis of antibody-opsonized human platelets equally utilized FcγRI and Fcγ IIIA. Despite the well-known ability of FcγRIIA to bind IgG in cell free systems, this receptor did not appear to be involved in either RBC or platelet phagocytosis. These transgenic cells may constitute a valuable tool for studying macrophage FcγR utilization and function.
Assuntos
Imunoglobulina G , Receptores de IgG , Humanos , Receptores de IgG/metabolismo , Células THP-1 , Fagocitose , Monócitos/metabolismo , Eritrócitos/metabolismoRESUMO
AIM: Vascular leakage following cardiopulmonary bypass contributes to morbidity. Angiopoietin-1 and -2 are biomarkers of endothelial dysfunction. Our aim was to characterize Ang-1 and -2 association with clinical characteristics and outcomes. METHODS: Observational cohort study measuring Ang-1/-2 with a panel of cytokines in adults undergoing cardiopulmonary bypass. RESULTS: Ang-2 levels increased immediately postop whereas Ang-1 levels decreased over time. No significant correlation was found with other inflammatory mediators. High correlation was found between the hospital length of stay and Ang-2 increase at 24 h (rho = 0.590; p < 0.0001). The predictors of Ang-2 increase were female gender, cross clamp time, transfusion of blood and absence of angiotensin-converting enzyme inhibitor as a pre-op medication. CONCLUSION: Angiopoietins can detect vascular leakage early and could impact patient's management to decrease length of stay after cardiac surgery.
RESUMO
AIMS: Ventilator-induced lung injury (VILI) contributes to mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury (ALI). Absence of activating transcription factor 3 (ATF3) confers susceptibility to ALI/VILI. To identify cell-specific ATF3-dependent mechanisms of susceptibility to ALI/VILI, we generated ATF3 chimera by adoptive bone marrow (BM) transfer and randomized to inhaled saline or lipopolysacharide (LPS) in the presence of mechanical ventilation (MV). Adenovirus vectors to silence or overexpress ATF3 were used in primary human bronchial epithelial cells and murine BM-derived macrophages from wild-type or ATF3-deficient mice. RESULTS: Absence of ATF3 in myeloid-derived cells caused increased pulmonary cellular infiltration. In contrast, absence of ATF3 in parenchymal cells resulted in loss of alveolar-capillary membrane integrity and increased exudative edema. ATF3-deficient macrophages were unable to limit the expression of pro-inflammatory mediators. Knockdown of ATF3 in resident cells resulted in decreased junctional protein expression and increased paracellular leak. ATF3 overexpression abrogated LPS induced membrane permeability. Despite release of ATF3-dependent Nrf2 transcriptional inhibition, mice that lacked ATF3 expression in resident cells had increased Nrf2 protein degradation. INNOVATION: In our model, in the absence of ATF3 in parenchymal cells increased Nrf2 degradation is the result of increased Keap-1 expression and loss of DJ-1 (Parkinson disease [autosomal recessive, early onset] 7), previously not known to play a role in lung injury. CONCLUSION: Results suggest that ATF3 confers protection to lung injury by preventing inflammatory cell recruitment and barrier disruption in a cell-specific manner, opening novel opportunities for cell specific therapy for ALI/VILI.
Assuntos
Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/metabolismo , Pulmão/citologia , Fator 2 Relacionado a NF-E2/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Quimera , Células Epiteliais , Feminino , Humanos , Inflamação/metabolismo , Pulmão/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Estresse Oxidativo , Transdução de SinaisRESUMO
OBJECTIVE: Recent evidence suggests that mesenchymal stem cells may attenuate lung inflammation and fibrosis in acute lung injury. However, so far, no study has investigated the effects of mesenchymal stem cell therapy on the time course of the structural, mechanical, and remodeling properties in pulmonary or extrapulmonary acute lung injury. DESIGN: Prospective randomized controlled experimental study. SETTING: University research laboratory. SUBJECTS: One hundred forty-three females and 24 male C57BL/6 mice. INTERVENTIONS: Control mice received saline solution intratracheally (0.05 mL, pulmonary control) or intraperitoneally (0.5 mL, extrapulmonary control). Acute lung injury mice received Escherichia coli lipopolysaccharide intratracheally (2 mg/kg in 0.05 mL of saline/mouse, pulmonary acute lung injury) or intraperitoneally (20 mg/kg in 0.5 mL of saline/mouse, extrapulmonary acute lung injury). Mesenchymal stem cells were intravenously injected (IV, 1 × 10 cells in 0.05 mL of saline/mouse) 1 day after lipopolysaccharide administration. MEASUREMENTS AND MAIN RESULTS: At days 1, 2, and 7, static lung elastance and the amount of alveolar collapse were similar in pulmonary and extrapulmonary acute lung injury groups. Inflammation was markedly increased at day 2 in both acute lung injury groups as evidenced by neutrophil infiltration and levels of cytokines in bronchoalveolar lavage fluid and lung tissue. Conversely, collagen deposition was only documented in pulmonary acute lung injury. Mesenchymal stem cell mitigated changes in elastance, alveolar collapse, and inflammation at days 2 and 7. Compared with extrapulmonary acute lung injury, mesenchymal stem cell decreased collagen deposition only in pulmonary acute lung injury. Furthermore, mesenchymal stem cell increased metalloproteinase-8 expression and decreased expression of tissue inhibitor of metalloproteinase-1 in pulmonary acute lung injury, suggesting that mesenchymal stem cells may have an effect on the remodeling process. This change may be related to a shift in macrophage phenotype from M1 (inflammatory and antimicrobial) to M2 (wound repair and inflammation resolution) phenotype. CONCLUSIONS: Mesenchymal stem cell therapy improves lung function through modulation of the inflammatory and remodeling processes. In pulmonary acute lung injury, a reduction in collagen fiber content was observed associated with a balance between metalloproteinase-8 and tissue inhibitor of metalloproteinase-1 expressions.
Assuntos
Lesão Pulmonar Aguda/terapia , Remodelação das Vias Aéreas/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Lesão Pulmonar Aguda/fisiopatologia , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Feminino , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/patologia , Masculino , Metaloproteinases da Matriz/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mecânica RespiratóriaRESUMO
Although bone marrow-derived mesenchymal stem cell (MSC) systemic administration reduces sepsis-associated inflammation, organ injury, and mortality in clinically relevant models of polymicrobial sepsis, the cellular and molecular mechanisms mediating beneficial effects are controversial. This study identifies the molecular mechanisms of MSC-conferred protection in sepsis by interrogating transcriptional responses of target organs to MSC therapy. Sepsis was induced in C57Bl/6J mice by cecal ligation and puncture, followed 6 hours later by an i.v. injection of either MSCs or saline. Total RNA from lungs, hearts, kidneys, livers, and spleens harvested 28 hours after cecal ligation and puncture was hybridized to mouse expression bead arrays. Common transcriptional responses were analyzed using a network knowledge-based approach. A total of 4751 genes were significantly changed between placebo- and MSC-treated mice (adjusted P ≤ 0.05). Transcriptional responses identified three common effects of MSC administration in all five organs examined: i) attenuation of sepsis-induced mitochondrial-related functional derangement, ii down-regulation of endotoxin/Toll-like receptor innate immune proinflammatory transcriptional responses, and iii) coordinated expression of transcriptional programs implicated in the preservation of endothelial/vascular integrity. Transcriptomic analysis indicates that the protective effect of MSC therapy in sepsis is not limited to a single mediator or pathway but involves a range of complementary activities affecting biological networks playing critical roles in the control of host cell metabolism and inflammatory response.
Assuntos
Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Sepse/genética , Sepse/terapia , Transcrição Gênica , Animais , Ceco/lesões , Comunicação Celular/genética , Metabolismo Energético/genética , Feminino , Regulação da Expressão Gênica , Imunidade/genética , Inflamação/genética , Inflamação/patologia , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Especificidade de Órgãos/genética , Sepse/induzido quimicamente , Sepse/prevenção & controle , Transdução de Sinais/genéticaRESUMO
OBJECTIVES: We hypothesized that resveratrol administration would reverse sepsis-dependent downregulation of peroxisome proliferator activated receptor-γ coactivator 1α, preserve mitochondrial integrity, and rescue animals from sepsis-induced myocardial failure. SETTING: Teaching hospital research laboratory. INTERVENTIONS: Cecal ligation and puncture in mice was performed to induce sepsis. Mice that underwent cecal ligation and puncture were randomly assigned to receive resveratrol (30 mg/kg or 60 mg/kg) or vehicle 1 mL sodium chloride 0.9% subcutaneously in the scruff of the neck directly after surgery and at 16, 24, and 40 hrs, respectively. MEASUREMENTS AND RESULTS: Forty-eight hrs after cecal ligation and puncture, cardiac performance was established using echocardiography. Mitochondrial integrity was evaluated with electron microscopy, and changes in gene expression were evaluated with microarray analysis. Survival at 48 hrs was just under 50% and comparable between groups. Myocardial contractile function significantly improved after resveratrol treatment. Resveratrol-treated mice developed focal areas of edema, whereas vehicle-treated mice developed significant, diffuse myocardial edema. Electron microscopy revealed widespread swollen mitochondria with ruptured outer membranes, autophagosomes, and vacuolation of the internal compartment, which were significantly attenuated in resveratrol-treated animals. Resveratrol treatment significantly increased cardiac expression of peroxisome proliferator-activated receptor-γ coactivator 1a. Microarray analysis revealed that resveratrol treatment resulted in upregulation of the peroxisome proliferator-activated receptor-γ coactivator gene set containing genes known to be regulated by this transcriptional coactivator. Our data strongly suggest that administration of resveratrol modulates bioenergy metabolism, substrate utilization, oxidative stress, and detoxification pathways associated with both mitochondrial and cardiac pathological conditions, but does not alter mortality from sepsis. CONCLUSIONS: The salutary effects of resveratrol on cecal ligation and puncture-induced myocardial dysfunction are associated with increased peroxisome proliferator-activated receptor-γ coactivator 1a abundance and function. Preservation of myocardial energy production capacity, prevention of secondary injury, mitigation of inflammation, and reversal of sepsis-induced myocardial remodeling are likely to underlie its beneficial effects. This however, does not result in improved survival.
Assuntos
Insuficiência Cardíaca/prevenção & controle , Contração Miocárdica/efeitos dos fármacos , Sepse/complicações , Estilbenos/farmacologia , Transativadores/metabolismo , Vasodilatadores/farmacologia , Animais , Cardiomiopatias/etiologia , Ceco , Regulação para Baixo/efeitos dos fármacos , Edema/etiologia , Expressão Gênica/efeitos dos fármacos , Insuficiência Cardíaca/etiologia , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Distribuição Aleatória , Resveratrol , Transativadores/genética , Fatores de TranscriçãoRESUMO
RATIONALE: Ventilator-induced lung injury (VILI) contributes to the mortality in patients with acute lung injury by increasing inflammation. Recent evidence suggests that stimulation of the cholinergic antiinflammatory pathway may be an attractive way to attenuate inflammatory injury. OBJECTIVES: To determine the role of vagus nerve signaling in VILI and establish whether stimulation of the vagus reflex can mitigate VILI. METHODS: We performed bilateral vagotomy in a mouse model of high-tidal volume-induced lung injury. We performed pharmacological and electrical vagus nerve stimulation in a rat model of VILI following ischemia/reperfusion injury. To determine the contribution of the alpha 7 acetylcholine nicotinic receptor to pulmonary cell injury, we exposed human bronchial epithelial cells to cyclic stretch in the presence of specific agonist or antagonist of the alpha 7 receptor. MEASUREMENTS AND MAIN RESULTS: Vagotomy exacerbates lung injury from VILI in mice as demonstrated by increased wet-to-dry ratio, infiltration of neutrophils, and increased IL-6. Vagal stimulation attenuates lung injury in rats after ischemia/reperfusion injury ventilated with high-volume strategies. Treatment of both mice and rats with the vagus mimetic drug semapimod resulted in decreased lung injury. Vagotomy also increased pulmonary apoptosis, whereas vagus stimulation (electrical and pharmacological) attenuated VILI-induced apoptosis. In vitro studies suggest that vagus-dependent effects on inflammation and apoptosis are mediated via the α7 nicotinc acetylcholine receptor-dependent effects on cyclic stretch-dependent signaling pathways c-jun N-terminal kinase and tumor necrosis factor receptor superfamily, member 6. CONCLUSIONS: Stimulation of the cholinergic antiinflammatory reflex may represent a promising alternative for the treatment of VILI.
Assuntos
Neuroimunomodulação/imunologia , Lesão Pulmonar Induzida por Ventilação Mecânica/imunologia , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/imunologia , Animais , Apoptose , Células Cultivadas , Modelos Animais de Doenças , Estimulação Elétrica , Humanos , Hidrazonas/administração & dosagem , Imunossupressores/administração & dosagem , Mediadores da Inflamação/imunologia , Interleucina-6/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/imunologia , Traumatismo por Reperfusão , Nervo Vago/efeitos dos fármacos , Nervo Vago/imunologia , Lesão Pulmonar Induzida por Ventilação Mecânica/complicaçõesRESUMO
BACKGROUND AND PURPOSE: Downregulation of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is thought to be a novel neuroprotective strategy in ischemic stroke, but the underlying mechanisms remain unclear. In this study, we aimed to validate the use of PTEN regulation of gamma-aminobutyric acid subtype A receptors (GABA(A)Rs) as a molecular target for the treatment of ischemic stroke. Because suppression of GABA(A)Rs contributes to ischemic neuron death, describing the intracellular signaling that interacts with GABA(A)Rs in ischemic neurons would provide a molecular basis for novel stroke therapies. METHODS: We measured surface GABA(A)R expression by immunocytochemical labeling and surface protein biotinylation assay. Knockdown and overexpression approaches were used to test the effects of PTEN on the expression and function of GABA(A)Rs. Neuronal death was detected in both in vitro and in vivo stroke models. RESULTS: The knockdown and overexpression approaches provided the first evidence that PTEN negatively regulated membrane expression and function of GABA(A)Rs in rat hippocampal neurons. Importantly, we demonstrated that a PTEN inhibitor prevented the reduction of surface GABA(A)Rs in injured hippocampal neurons subjected to oxygen-glucose deprivation, an in vitro insult that mimics ischemic injury, whereas a GABA(A)R antagonist significantly reduced this PTEN inhibitor-induced neuroprotection in both the in vitro and in vivo ischemic stroke models. CONCLUSIONS: Our study provides direct evidence that downregulation of PTEN protects against ischemic neuron death by preserving GABA(A)R function. Targeting this pathway may be an effective strategy for development of selective, potent stroke treatments.
Assuntos
Isquemia Encefálica/prevenção & controle , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Receptores de GABA-A/fisiologia , Acidente Vascular Cerebral/prevenção & controle , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Células Cultivadas , Técnicas de Silenciamento de Genes/métodos , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Compostos Organometálicos/farmacologia , Compostos Organometálicos/uso terapêutico , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologiaRESUMO
Oleylethanolamide (OEA) is a natural fatty acid ethanolamide produced in the heart, but its biological actions in myocardium have not yet been defined. This study was carried out to determine whether OEA could be used to prevent the development of heart failure or improve evolving heart failure. We studied in vivo and in vitro actions of OEA in cardiac muscle. In an animal model of doxorubicin cardiomyopathy, OEA showed robust effects and attenuated the progression of systolic/diastolic dysfunction and ventricular remodeling. During evolving doxorubicin cardiomyopathy, a therapeutic course of OEA treatment partially restored myocardial function. The preventive and therapeutic effects of OEA were associated with significant improvement of survival. To investigate the mechanism of OEA action in cardiac muscle, we have carried out in vitro experiments in cultured cardiomyocytes. The results showed that OEA, through activation of Ras-Raf-1-Mek-Erk signaling, inhibited doxorubicin-induced apoptosis. Additional experiments showed that OEA activation of the Erk pathway involved activation of Neu/ErbB2 receptor, which suggests OEA actions in cardiac muscle might require activation of Neu/ErbB2. In summary, OEA improved ventricular remodeling and augmented cardiac function in doxorubicin cardiomyopathy, possibly involving activation of Neu/ErbB2 and Ras-Erk signaling. These findings suggest OEA is a novel cardioprotective compound that may be used to develop new strategies for the management of cardiomyopathy.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cardiopatias/prevenção & controle , Miócitos Cardíacos/metabolismo , Ácidos Oleicos/metabolismo , Proteínas ras/metabolismo , Análise de Variância , Animais , Cardiotônicos/metabolismo , Modelos Animais de Doenças , Doxorrubicina , Cardiopatias/induzido quimicamente , Cardiopatias/metabolismo , Masculino , Ácidos Oleicos/farmacologia , Ratos , Receptor ErbB-2/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Remodelação Ventricular/fisiologiaRESUMO
High throughput gene expression profiling with DNA microarray provides an opportunity to analyze transcriptional regulation of hundreds or thousands of similarly regulated genes. Transcriptional regulation of gene expression plays an important role in myocardial remodeling. We have studied cardiac muscle gene expression with DNA microarray and used a computational strategy to identify common promoter motifs that respond to insulin-like growth factor 1 (IGF-1) stimulation in cardiac muscle cells. The analysis showed that the Sp1 binding site is a likely target of IGF-1 action. Further experiments with gel shift assay indicated that IGF-1 regulated the Sp1 site in cardiomyocytes, by increasing the abundance of Sp1 and Sp3 proteins. Using firefly luciferase as reporter gene, additional experiments showed that IGF-1 activated the promoter of cyclin D3 and Glut1. Both promoters contain one Sp1 site. The effect of IGF-1 on these two promoters was abolished with siRNA for Sp1. Thus, the transcriptional activation of these two promoters by IGF-1 requires the induction of Sp1 protein. These experiments suggest that the global transcriptional regulatory actions of IGF-1 involve activation of the Sp1 site in cardiac muscle. The computational model we have developed is a prototypical method that may be further developed to identify unique cis- and trans-acting elements in response to hormonal stimulation during cardiac muscle growth, repair, and remodeling in normal and abnormal cardiac muscle.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fator de Transcrição Sp1/metabolismo , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Ciclina D3 , Ciclinas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Regulação da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 1 , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Transfecção , Regulação para Cima/efeitos dos fármacosRESUMO
The development of doxorubicin cardiomyopathy involves apoptosis of cardiac muscle cells. This study was carried out to define the roles of two heat-shock proteins, Hsp10 and Hsp60, on doxorubicin-induced apoptosis in primary cardiomyocytes. Doxorubicin induces apoptosis of cardiomyocytes by activating mitochondria apoptosis signaling. Transducing cardiomyocytes with Hsp10 or Hsp60 with adenoviral vector suppressed the occurrence of apoptosis in the doxorubicin-treated cardiomyocytes. Overexpression of Hsp10 and Hsp60 increased the abundance of the anti-apoptotic Bcl-xl and Bcl-2, and reduced the protein content of the pro-apoptotic Bax. Hsp60 overexpression also significantly reduced doxorubicin induction of Bad, whereas overexpression of Hsp10 did not alter the expression of Bad in the doxorubicin-treated cells. Overexpression of Hsp10 and Hsp60, respectively, stabilized mitochondrial cross-membrane potential, inhibited Caspase 3, and suppressed PARP. These findings indicate that overexpression of Hsp10 and Hsp60 differentially modulated Bcl-2 family and in turn attenuate doxorubicin-induced cardiac muscle death. The effects of Hsp10 and Hsp60 on Bcl-2 family could not be explained by the abundance of Bcl-2 family mRNA levels. Hsp60 interacted with Bcl-xl and Bax in the cardiomyocytes in vivo. The effect of Hsp10 and Hsp60 on the abundance of Bcl-xl could not be blocked by cycloheximide. Moreover, Hsp10 and Hsp60 inhibited ubiquitination of Bcl-xl. These findings suggest that Hsp10 and Hsp60 modulated post-translational modification of Bcl-xl. Antisense Hsp60 reduced the abundance of endogenous Hsp60 in cardiomyocytes and amplified the cytotoxicity of doxorubicin. These data provide a novel link between Hsp10/Hsp60 and cardiac protection in doxorubicin cardiomyopathy.
Assuntos
Apoptose , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Células Cultivadas , Chaperonina 60/efeitos dos fármacos , Doxorrubicina/farmacologia , Doxorrubicina/toxicidade , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/análise , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteína X Associada a bcl-2 , Proteína de Morte Celular Associada a bcl , Proteína bcl-XRESUMO
We have investigated the effects of two heat shock proteins, Hsp10 and Hsp60, on insulin-like growth factor-1 receptor (IGF-1R) signaling in cardiac muscle cells. Neonatal cardiomyocytes were transduced with Hsp10 or Hsp60 via adenoviral vector. Compared with the cells transduced with a control vector, overexpression of Hsp10 or Hsp60 increased the abundance of IGF-1R and IGF-1-stimulated receptor autophosphorylation. Thus, Hsp10 and Hsp60 overexpression increased the number of functioning receptors and amplified activation of IGF-1R signaling. IGF-1 stimulation of MEK, Erk, p90Rsk, and Akt were accordingly augmented. Transducing cardiomyocytes with antisense Hsp60 oligonucleotides reduced Hsp60 expression, decreased the abundance of IGF-1R, attenuated IGF-1R autophosphorylation, and suppressed the pro-survival action of IGF-1 in cardiomyocytes. Using cycloheximide to inhibit protein synthesis did not alter the effect of Hsp60 on IGF-1R signaling, and IGF-1R mRNA levels were not up-regulated by Hsp10 or Hsp60. Additional experiments showed that Hsp10 and Hsp60 suppressed polyubiquitination of IGF-1 receptor. These data indicate that Hsp10 and Hsp60 can modulate IGF-1R signaling through post-translational modification. In animal models of diabetes, diabetic myocardium is associated with decreased abundance of Hsp60, increased ubiquitination of IGF-1R, and lower level of IGF-1R protein. Declined myocardial protection is a major feature of diabetic cardiomyopathy. These data suggest that decreased Hsp60 expression and subsequent decline of IGF-1R signaling may be a fundamental mechanism underlying the development of diabetic cardiomyopathy.