RESUMO
Neuroblastoma (NB) is a cancer of the peripheral nervous system found in children under 15 years of age. It is the most frequently diagnosed cancer during infancy, accounting for ~12% of all cancer-related deaths in children. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a membrane receptor that is associated with the primary tumor formation and metastasis of cancers in the gastrointestinal system. Remarkably, high levels of LGR5 are found in NB tumor cells, and high LGR5 expression is strongly correlated with poor survival. Antibody-drug conjugates (ADCs) are monoclonal antibodies that are covalently linked to cell-killing cytotoxins to deliver the payloads into cancer cells. We generated an ADC with an anti-LGR5 antibody and pyrrolobenzodiazepine (PBD) dimer-based payload SG3199 using a chemoenzymatic conjugation method. The resulting anti-LGR5 ADC was able to inhibit the growth of NB cells expressing LGR5 with high potency and specificity. Importantly, the ADC was able to completely inhibit the growth of NB xenograft tumors in vivo at a clinically relevant dose for the PBD class of ADCs. The findings support the potential of targeting LGR5 using the PBD class of payload for the treatment of high-risk NBs.
RESUMO
BACKGROUND: Burn injury induces multiple signaling pathways leading to a significant inflammatory storm that adversely affects multiple organs, including the heart. Poly (ADP-ribose) polymerase inhibitor 1 (PARP1) inhibition, with specific agents such as N-(5,6-Dihydro-6-oxo-2-phenanthridinyl)-2-acetamide (PJ34), is effective in reducing oxidative stress and cytokine expression in the heart. We hypothesized that PARP1 inhibition would reduce inflammatory signaling and protect against burn injury-induced cardiac dysfunction. STUDY DESIGN: Male Sprague-Dawley rats (8 weeks old, 300 to 350 g) were randomly assigned to sham injury (Sham), 60% total body surface area burn (24 hours post burn), or 60% total body surface area burn with intraperitoneal administration of PJ34 (20 mg/kg, 24 hours post burn + PJ34) and sacrificed 24 hours after injury. Cardiac function was determined using Vevo 2100 echocardiography. Genetic expression of 84 specific toll-like receptor-mediated signal transduction and innate immunity genes were examined using microarray to evaluate cardiac tissue. Qiagen GeneGlobe Data Analysis Center was used to analyze expression, and genetic clustering was performed using TreeView V2.0.8 software. Real-time quantitative polymerase chain reaction was used to validate identified differentially expressed genes. RESULTS: Burn injury significantly altered multiple genes in the toll-like receptor signaling, interleukin-17 signaling, tumor necrosis factor signaling, and nuclear factor-κB signaling pathways and led to significant cardiac dysfunction. PARP1 inhibition with PJ34 normalized these signaling pathways to sham levels as well as improved cardiac function to sham levels. CONCLUSIONS: PARP1 inhibition normalizes multiple inflammatory pathways that are altered after burn injury and improves cardiac dysfunction. PARP1 pathway inhibition may provide a novel methodology to normalize multiple burn injury-induced inflammatory pathways in the heart.
Assuntos
Antineoplásicos , Cardiopatias , Fenantrenos , Ratos , Animais , Masculino , Ratos Sprague-Dawley , Fenantrenos/farmacologia , Fenantrenos/uso terapêutico , Poli(ADP-Ribose) Polimerase-1RESUMO
In 2016, an estimated 486,000 individuals sustained burn injuries requiring medical attention. Severe burn injuries lead to a persistent, hyperinflammatory response that may last up to 2 years. The persistent release of inflammatory mediators contributes to end-organ dysfunction and changes in genome expression. Burn-induced cardiac dysfunction may lead to heart failure and changes in cardiac remodeling. Cytokines promote the inflammatory cascade and promulgate mechanisms resulting in cardiac dysfunction. Here, we review the mechanisms by which TNFα, IL-1 beta, IL-6, and IL-10 cause cardiac dysfunction in post-burn injuries. We additionally review changes in the cytokine transcriptome caused by inflammation and burn injuries.
RESUMO
Immune cascade is one of major factors leading to cardiac dysfunction after burn injury. TLRs are a class of pattern-recognition receptors (PRRs) that initiate the innate immune response by sensing conserved molecular patterns for early immune recognition of a pathogen. The Rat Toll-Like Receptor (TLR) Signaling Pathway RT² Profiler PCR Array profiles the expression of 84 genes central to TLR-mediated signal transduction and innate immunity, and is a validated tool for identifying differentially expressed genes (DEGs). We employed the PCR array to identify burn-induced cardiac TLR-signaling-related DEGs. A total of 38 up-regulated DEGs and 19 down-regulated DEGs were identified. Network analysis determined that all DEGS had 10 clusters, while up-regulated DEGs had 6 clusters and down-regulated DEGs had 5 clusters. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were involved in TLR signaling, the RIG-I-Like receptor signaling pathway, the IL-17 signaling pathway, and the NFkB signaling pathway. Function analysis indicated that DEGs were associated with Toll-like receptor 2 binding, Lipopeptide binding, Toll-like receptor binding, and NAD(P)+ nucleosidase activity. The validation of 18 up-regulated DEGs (≥10-fold change) and 6 down-regulated DEGs (≤5-fold change) demonstrated that the PCR array is a trusted method for identifying DEGs. The analysis of validated DEG-derived protein-protein interaction networks will guide our future investigations. In summary, this study not only identified the TLR-signaling-pathway-related DEGs after burn injury, but also confirmed that the burn-induced cardiac cytokine cascade plays an important role in burn-induced heart dysfunction. The results will provide the novel therapeutic targets to protect the heart after burn injury.
RESUMO
BACKGROUND: Our previous studies have found that burn injury induces cardiac dysfunction through interruption of the antioxidant-response element (ARE) pathway in cardiac mitochondria. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator that activates many antioxidant enzymes. Oltipraz (Olti) is a Nrf2 activator and a well-known inducer of NQO1 along with other enzymes that comprise the Nrf2-associated antioxidants. We propose that Nrf2 activation will induce the ARE pathway, leading to abrogation of burn-induced cardiac dysfunction. STUDY DESIGN: In this study, we investigated the effect of Nrf2-deficiency in mice on burn-induced cardiac dysfunction. Wild-type (WT) and Nrf2-deficient mice received 30% total body surface area burn injury and were treated with or without Olti and then harvested at 3 hours and 24 hours post burn (3 hpb and 24 hpb). RESULTS: As expected, Nrf2-deficient mice exhibited exacerbated cardiac dysfunction after burn injury, as measured by Vevo 2100 echocardiography. Electron microscopy showed that Nrf2 depletion worsened burn injury-induced cardiac mitochondrial damage. In addition, Nrf2 depletion increased cardiac mitochondrial dysfunction and myocardial fibrosis after burn injury. Treatment with Olti ameliorated the heart dysfunction in burned Nrf2-/+ mice, improved cardiac mitochondrial structure and oxidative phosphorylation, as well as decreased cardiac fibrosis. These results suggest that Nrf2 and its downstream targets modulate cardiac function after burn injury. CONCLUSIONS: In summary, Nrf2 depletion worsens cardiac dysfunction after burn injury. Nrf2 activation, with a drug such as Olti, offers a promising therapeutic strategy for abrogating burn-induced cardiac dysfunction.
Assuntos
Cardiopatias , Fator 2 Relacionado a NF-E2 , Animais , Elementos de Resposta Antioxidante , Antioxidantes , Queimaduras/metabolismo , Cardiopatias/etiologia , Camundongos , Transdução de SinaisRESUMO
BACKGROUND: Imbalance of oxidants/antioxidants results in heart failure, contributing to mortality after burn injury. Cardiac mitochondria are a prime source of reactive oxygen species (ROS), and a mitochondrial-specific antioxidant may improve burn-induced cardiomyopathy. We hypothesize that the mitochondrial-specific antioxidant, Triphenylphosphonium chloride (Mito-TEMPO), could protect cardiac function after burn. STUDY DESIGN: Male rats had a 60% total body surface area (TBSA) scald burn injury and were treated with/without Mito-TEMPO (7 mg/kg-1, intraperitoneal) and harvested at 24 hours post-burn. Echocardiography (ECHO) was used for measurement of heart function. Masson Trichrome and hematoxylin and eosin (H & E) staining were used for cardiac fibrosis and immune response. Qualitative polymerase chain reaction (qPCR) was used for mitochondrial DNA replication and gene expression. RESULTS: Burn-induced cardiac dysfunction, fibrosis, and mitochondrial damage were assessed by measurement of mitochondrial function, DNA replication, and DNA-encoded electron transport chain-related gene expression. Mito-TEMPO partially improved the abnormal parameters. Burn-induced cardiac dysfunction was associated with crosstalk between the NFE2L2-ARE pathway, PDE5A-PKG pathway, PARP1-POLG-mtDNA replication pathway, and mitochondrial SIRT signaling. CONCLUSIONS: Mito-TEMPO reversed burn-induced cardiac dysfunction by rescuing cardiac mitochondrial dysfunction. Mitochondria-targeted antioxidants may be an effective therapy for burn-induced cardiac dysfunction.
Assuntos
Antioxidantes/administração & dosagem , Queimaduras/terapia , Insuficiência Cardíaca/tratamento farmacológico , Compostos Organofosforados/administração & dosagem , Piperidinas/administração & dosagem , Animais , Queimaduras/complicações , Modelos Animais de Doenças , Ecocardiografia , Coração/efeitos dos fármacos , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Humanos , Injeções Intraperitoneais , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Miocárdio/citologia , Miocárdio/patologia , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Burn-induced cardiac dysfunction is thought to involve mitochondrial dysfunction, although the mechanisms responsible are unclear. In this study, we used our established model of in vivo burn injury to understand the genetic evidence of burn-induced mitochondrial confusion dysfunction by describing cardiac mitochondrial metabolism-related gene expression after burn. Cardiac tissue was collected at 24 hours after burn injury. An O2K respirometer system was utilized to measure the cardiac mitochondrial function. Oxidative phosphorylation complex activities were determined using enzyme activity assays. RT Profiler PCR array was used to identify the differential regulation of genes involved in mitochondrial biogenesis and metabolism. The quantitative qPCR and Western blotting were applied to validate the differentially expressed genes. Burn-induced cardiac mitochondrial dysfunction was supported by the finding of decreased state 3 respiration, decreased mitochondrial electron transport chain activity in complex I, III, IV, and V, and decreased mitochondrial DNA-encoded gene expression as well as decreased levels of the corresponding proteins after burn injury. Eighty-four mitochondrial metabolism-related gene profiles were measured. The mitochondrial gene profile showed that 29 genes related to mitochondrial energy and metabolism was differentially expressed. Of these 29 genes, 16 were more than 2-fold upregulated and 13 were more than 2-fold downregulated. All genes were validated using qPCR and partial genes were correlated with their protein levels. This study provides preliminary evidence that a large percentage of mitochondrial metabolism-related genes in cardiomyocytes were significantly affected by burn injury.
RESUMO
Background: Severe burn injury initiates a feedback cycle of inflammation, fibrosis, oxidative stress and cardiac mitochondrial damage via the PDE5A-cGMP-PKG pathway. Aim: To test if the PDE5A-cGMP-PKG pathway may contribute to burn-induced heart dysfunction. Methods: Sprague-Dawley rats were divided four groups: sham; sham/sildenafil; 24 h post burn (60% total body surface area scald burn, harvested at 24 h post burn); and 24 h post burn/sildenafil. We monitored heart function and oxidative adducts, as well as cardiac inflammatory, cardiac fibrosis and cardiac remodeling responses in vivo. Results: Sildenafil inhibited the burn-induced PDE5A mRNA level and increased the cGMP level and PKG activity, leading to the normalization of PKG down-regulated genes (IRAG, PLB, RGS2, RhoA and MYTP), a decreased ROS level (H2O2), decreased oxidatively modified adducts (malonyldialdehyde [MDA], carbonyls), attenuated fibrogenesis as well as fibrosis gene expression (ANP, BNP, COL1A2, COL3A2, αSMA and αsk-Actin), and reduced inflammation and related gene expression (RELA, IL-18 and TGF-ß) after the burn. Additionally, sildenafil treatment preserved left ventricular heart function (CO, EF, SV, LVvol at systolic, LVPW at diastolic and FS) and recovered the oxidant/antioxidant balance (total antioxidant, total SOD activity and Cu,ZnSOD activity). Conclusions: The PDE5A-cGMP-PKG pathway mediates burn-induced heart dysfunction. Sildenafil treatment recovers burn-induced cardiac dysfunction.
Assuntos
Queimaduras/complicações , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/etiologia , Citrato de Sildenafila/uso terapêutico , Animais , Antioxidantes/metabolismo , Cardiomiopatias/genética , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxidantes/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Citrato de Sildenafila/farmacologiaRESUMO
Burn-induced heart dysfunction is a key factor for patient mortality. However, the molecular mechanisms are not yet fully elucidated. This study sought to understand whether burn-induced heart dysfunction is associated with cardiac mitochondrial dysfunction and interruption of the PDE5A-cGMP-PKG pathway. Sixty percent total body surface area (TBSA) scald burned rats (±sildenafil) were used in this study. A transmission electron microscope (TEM), real-time qPCR, O2K-respirometer, and electron transport chain assays were used to characterized molecular function. Cardiac mitochondrial morphological shapes were disfigured with a decline in mitochondrial number, area, and size, resulting in deficiency of cardiac mitochondrial replication. Burn induced a decrease in all mitDNA encoded genes. State 3 oxygen consumption was significantly decreased. Mitochondrial complex I substrate-energized or complex II substrate-energized and both of respiratory control ratio (RCRs) were decreased after burn. All mitochondrial complex activity except complex II were decreased in the burn group, correlating with decreases in mitochondrial ATP and MnSOD activity. Sildenafil, a inhibitor of the PDE5A-cGMP-PKG pathway, preserved the mitochondrial structure, respiratory chain efficiency and energy status in cardiac tissue. Furthermore, sildenafil treatment significantly restored ADP-conjugated respiration in burned groups. In conclusion, cardiac mitochondrial damage contributes to burn-induced heart dysfunction via the PDE5A-cGMP-PKG pathway.
Assuntos
Queimaduras/patologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Mitocôndrias Cardíacas/patologia , Transdução de Sinais , Animais , Queimaduras/complicações , Queimaduras/metabolismo , Masculino , Mitocôndrias Cardíacas/metabolismo , Consumo de Oxigênio , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Mitochondrial oxidative stress plays a prominent role in the development of burn-induced cardiac dysfunction. AMP-activated kinase (AMPK), an energy sensor, has a central role in the pathogenesis of heart failure. However, its role in cardiac dysfunction after burn injury is unclear. Our hypothesis is that burn injury acts through the AMPK-sirtuin 1-PGC1α-nuclear factor erythroid 2-related factor 2 (NFE2L2)-ARE signaling pathway, leading to cardiac mitochondrial impairment, resulting in cardiac dysfunction. STUDY DESIGN: Male Sprague-Dawley rats underwent sham procedure or 60% total body surface area full-thickness burn. Echocardiograms were performed 24 hours post burn. Heart tissue was harvested at 24 hours post burn for biochemistry/molecular biologic analysis. AC16 cardiomyocytes were treated with either sham or burned rat serum (±AMPK inhibitor/AMPK activator/PGC1α activator) for evaluation of cardiomyocyte mitochondrial function by using seahorse in vitro. RESULTS: Burn injury-induced cardiac dysfunction was measured by echocardiogram. Burn injury suppressed cardiac AMPK, sirtuin 1, and PGC1 expression, leading to acetylation of cardiomyocyte proteins. In addition, burn injury caused NFE2L2 and NFE2L2 regulated antioxidants (heme oxygenase 1, NADH quinone oxidoreductase 1, glutamatecysteine ligase catalytic subunit, manganese superoxide dismutase, and glutathione peroxidase) to decrease, resulting in cardiac oxidative stress. In vitro, AMPK1 activator and PGC1α agonist treatment improved Ac16 cell mitochondrial dysfunction, and AMPK1 inhibitor treatment worsened Ac16 cellular damage. CONCLUSIONS: Burn-induced cardiac dysfunction and cardiac mitochondrial damage occur via the AMPK-sirtuin 1-PGC1α-NFE2L2-ARE signaling pathway. AMPK and PGC1α agonists might be promising therapeutic agents to reverse cardiac dysfunction after burn injury.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Elementos de Resposta Antioxidante/fisiologia , Queimaduras/complicações , Cardiopatias/etiologia , Fator 2 Relacionado a NF-E2/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/fisiologia , Transdução de Sinais/fisiologia , Sirtuína 1/fisiologia , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
In this study, we have investigated the effects of manganese superoxide dismutase (SOD2 or MnSOD) deficiency on mitochondrial function and oxidative stress during Chagas disease. For this, C57BL/6 wild type (WT) and MnSOD+/- mice were infected with Trypanosoma cruzi (Tc), and evaluated at 150 days' post-infection that corresponded to chronic disease phase. Genetic deletion of SOD2 decreased the expression and activity of MnSOD, but it had no effect on the expression of other members of the SOD family. The myocardial expression and activity of MnSOD were significantly decreased in chronically infected WT mice, and it was further worsened in MnSOD+/- mice. Chronic T. cruzi infection led to a decline in mitochondrial complex I and complex II driven, ADP-coupled respiration and ATP synthesis in the myocardium of WT mice. The baseline oxidative phosphorylation (OXPHOS) capacity in MnSOD+/- mice was decreased, and it had an additive effect on mitochondrial dysregulation of ATP synthesis capacity in chagasic myocardium. Further, MnSOD deficiency exacerbated the mitochondrial rate of reactive oxygen species (ROS) production and myocardial oxidative stress (H2O2, protein carbonyls, malondialdehyde, and 4-hydroxynonenal) in Chagas disease. Peripheral and myocardial parasite burden and inflammatory response (myeloperoxidase, IL-6, lactate dehydrogenase, inflammatory infiltrate) were increased in all chagasic WT and MnSOD+/- mice. We conclude that MnSOD deficiency exacerbates the loss in mitochondrial function and OXPHOS capacity and enhances the myocardial oxidative damage in chagasic cardiomyopathy. Mitochondria targeted, small molecule mitigators of MnSOD deficiency will offer potential benefits in averting the mitochondrial dysfunction and chronic oxidative stress in Chagas disease.