RESUMO
Medical procedures, such as radiation therapy, are a vital element in treating many cancers, significantly contributing to improved survival rates. However, a common long-term complication of such exposure is radiation-induced skin fibrosis (RISF), a complex condition that poses substantial physical and psychological challenges. Notably, about 50% of patients undergoing radiation therapy may achieve long-term remission, resulting in a significant number of survivors managing the aftereffects of their treatment. This article delves into the intricate relationship between RISF, reactive oxygen species (ROS), and angiotensin II (Ang II) signaling. It proposes the underlying mechanisms and examines potential treatments for mitigating skin fibrosis. The primary goal is to offer essential insights in order to better care for and improve the quality of life of cancer survivors who face the risk of developing RISF.
Assuntos
Angiotensina II , Fibrose , Espécies Reativas de Oxigênio , Pele , Humanos , Angiotensina II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos da radiação , Pele/patologia , Animais , Lesões por Radiação/etiologia , Radioterapia/efeitos adversos , Transdução de SinaisRESUMO
RATIONALE: Mitochondrial Ca2+ loading augments oxidative metabolism to match functional demands during times of increased work or injury. However, mitochondrial Ca2+ overload also directly causes mitochondrial rupture and cardiomyocyte death during ischemia-reperfusion injury by inducing mitochondrial permeability transition pore opening. The MCU (mitochondrial Ca2+ uniporter) mediates mitochondrial Ca2+ influx, and its activity is modulated by partner proteins in its molecular complex, including the MCUb subunit. OBJECTIVE: Here, we sought to examine the function of the MCUb subunit of the MCU-complex in regulating mitochondria Ca2+ influx dynamics, acute cardiac injury, and long-term adaptation after ischemic injury. METHODS AND RESULTS: Cardiomyocyte-specific MCUb overexpressing transgenic mice and Mcub gene-deleted (Mcub-/-) mice were generated to dissect the molecular function of this protein in the heart. We observed that MCUb protein is undetectable in the adult mouse heart at baseline, but mRNA and protein are induced after ischemia-reperfusion injury. MCUb overexpressing mice demonstrated inhibited mitochondrial Ca2+ uptake in cardiomyocytes and partial protection from ischemia-reperfusion injury by reducing mitochondrial permeability transition pore opening. Antithetically, deletion of the Mcub gene exacerbated pathological cardiac remodeling and infarct expansion after ischemic injury in association with greater mitochondrial Ca2+ uptake. Furthermore, hindlimb remote ischemic preconditioning induced MCUb expression in the heart, which was associated with decreased mitochondrial Ca2+ uptake, collectively suggesting that induction of MCUb protein in the heart is protective. Similarly, mouse embryonic fibroblasts from Mcub-/- mice were more sensitive to Ca2+ overload. CONCLUSIONS: Our studies suggest that Mcub is a protective cardiac inducible gene that reduces mitochondrial Ca2+ influx and permeability transition pore opening after ischemic injury to reduce ongoing pathological remodeling.
Assuntos
Cálcio/metabolismo , Membro Posterior/irrigação sanguínea , Proteínas de Membrana/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Remodelação Ventricular , Animais , Sinalização do Cálcio , Morte Celular , Linhagem Celular , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Precondicionamento Isquêmico , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/patologia , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Proteínas Mitocondriais/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologiaRESUMO
RATIONALE: Congenital heart disease can lead to life-threatening right ventricular (RV) heart failure. Results from clinical trials support expanding cardiac progenitor cell (CPC) based therapies. However, our recent data show that CPCs lose function as they age, starting as early as 1 year. OBJECTIVE: To determine whether the aggregation of child (1-5-year-old) CPCs into scaffold-free spheres can improve differentiation by enhancing Notch signaling, a known regulator of CPC fate. We hypothesized that aggregated (3-dimensional [3D]) CPCs will repair RV heart failure better than monolayer (2-dimensional [2D]) CPCs. METHODS AND RESULTS: Spheres were produced with 1500 CPCs each using a microwell array. CPC aggregation significantly increased gene expression of Notch1 compared with 2D CPCs, accompanied by significant upregulation of cardiogenic transcription factors (GATA4, HAND1, MEF2C, NKX2.5, and TBX5) and endothelial markers (CD31, FLK1, FLT1, VWF). Blocking Notch receptor activation with the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) diminished these effects. To evaluate the therapeutic improvements of CPC aggregation, RV heart failure was induced in athymic rats by pulmonary artery banding, and cells were implanted into the RV free wall. Echocardiographic measurements 28 days postimplantation showed significantly improved RV function with 3D compared with 2D CPCs. Tracking implanted CPCs via DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide)-labeling showed improved retention of 3D CPCs. Transducing 3D CPCs with Notch1-shRNA (short hairpin RNA) did not reduce retention, but significantly reduced RV functional improvements. Histological analyses showed 3D treatment reduced RV fibrosis and increased angiogenesis. Although 3D CPCs formed CD31+ vessel-like cells in vivo, these effects are more likely because of improved 3D CPC exosome function compared with 2D CPC exosomes. CONCLUSIONS: Spherical aggregation improves child CPC function in a Notch-dependent manner. The strong reparative ability of CPC spheres warrants further investigation as a treatment for pediatric heart failure, especially in older children where reparative ability may be reduced.
Assuntos
Agregação Celular , Cardiopatias Congênitas/patologia , Insuficiência Cardíaca/terapia , Receptores Notch/metabolismo , Esferoides Celulares/metabolismo , Transplante de Células-Tronco/métodos , Disfunção Ventricular Direita/terapia , Animais , Diferenciação Celular , Células Cultivadas , Criança , Pré-Escolar , Cardiopatias Congênitas/complicações , Cardiopatias Congênitas/terapia , Insuficiência Cardíaca/etiologia , Humanos , Lactente , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Transdução de Sinais , Esferoides Celulares/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Disfunção Ventricular Direita/complicaçõesRESUMO
Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.
Assuntos
Adaptação Fisiológica/genética , Microambiente Celular/genética , Fibrose Cística/genética , Pulmão/patologia , Monócitos/patologia , Transcrição Gênica/genética , Adulto , Células Cultivadas , Feminino , Humanos , Masculino , Neutrófilos/patologia , Transdução de Sinais/fisiologiaRESUMO
Nearly 1 in every 120 children born has a congenital heart defect. Although surgical therapy has improved survival, many of these children go on to develop right ventricular heart failure (RVHF). The emergence of cardiovascular regenerative medicine as a potential therapeutic strategy for pediatric HF has provided new avenues for treatment with a focus on repairing or regenerating the diseased myocardium to restore cardiac function. Although primarily tried using adult cells and adult disease models, stem cell therapy is relatively untested in the pediatric population. Here, we investigate the ability of electrical stimulation (ES) to enhance the retention and therapeutic function of pediatric cardiac-derived c-kit+ progenitor cells (CPCs) in an animal model of RVHF. Human CPCs isolated from pediatric patients were exposed to chronic ES and implanted into the RV myocardium of rats. Cardiac function and cellular retention analysis showed electrically stimulated CPCs (ES-CPCs) were retained in the heart at a significantly higher level and longer time than control CPCs and also significantly improved right ventricular functional parameters. ES also induced upregulation of extracellular matrix and adhesion genes and increased in vitro survival and adhesion of cells. Specifically, upregulation of ß1 and ß5 integrins contributed to the increased retention of ES-CPCs. Lastly, we show that ES induces CPCs to release higher levels of pro-reparative factors in vitro. These findings suggest that ES can be used to increase the retention, survival, and therapeutic effect of human c-kit+ progenitor cells and can have implications on a variety of cell-based therapies. Stem Cells 2019;37:1528-1541.
Assuntos
Estimulação Elétrica/métodos , Insuficiência Cardíaca/terapia , Miócitos Cardíacos/citologia , Transplante de Células-Tronco/métodos , Função Ventricular Direita/fisiologia , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Pré-Escolar , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Cardiopatias Congênitas/cirurgia , Humanos , Lactente , Recém-Nascido , Integrina beta1/biossíntese , Masculino , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ratos , Medicina Regenerativa/métodos , Células-Tronco/citologiaRESUMO
Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by recruitment of leucocytes into skin and release of damaging enzymes, resulting in epidermal detachment and blister formation. To better understand the role of leukotriene B4 (LTB4) and other inflammatory factors in BP pathophysiology, we conducted microscopic and immunohistochemical analyses of preserved skin biopsy sections and conducted flow cytometry and ELISA analyses of matched blood and blister fluid from BP patients. Neutrophils predominated in BP blister fluid, which also contained monocytes/macrophages and T cells, but few to no eosinophils and B cells. In contrast, BP skin histology showed a different pattern, with abundant neutrophils but eosinophils being the predominant immune cell type. LTB4 pathway and neutrophil activation markers were prevalent in BP skin lesions and strongly associated with perivascular neutrophils. Blister fluid neutrophils, monocytes/macrophages and eosinophils all exhibited increased surface expression of leukotriene A4 hydrolase and neutrophil elastase (P = .002 for both). Blister fluid was also enriched in interleukins (IL)-1α, IL-1ß, IL-8, IL-10, IL-18, monocyte colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF). Our findings suggest differential leucocyte recruitment from blood into dermis and from dermis into blister, which correlates with disease activity, and presents potential new treatment opportunities for BP.
Assuntos
Exsudatos e Transudatos/citologia , Leucotrieno B4/metabolismo , Penfigoide Bolhoso/sangue , Penfigoide Bolhoso/patologia , Pele/patologia , Idoso , Idoso de 80 Anos ou mais , Eosinófilos , Epóxido Hidrolases/metabolismo , Exsudatos e Transudatos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Interleucinas/metabolismo , Elastase de Leucócito/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/enzimologia , Masculino , Pessoa de Meia-Idade , Monócitos/enzimologia , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Penfigoide Bolhoso/imunologia , Fatores Raciais , Fatores Sexuais , Pele/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
RATIONALE: Neutrophils are recruited to the airways of individuals with cystic fibrosis (CF). In adolescents and adults with CF, airway neutrophils actively exocytose the primary granule protease elastase (NE), whose extracellular activity correlates with lung damage. During childhood, free extracellular NE activity is measurable only in a subset of patients, and the exocytic function of airway neutrophils is unknown. OBJECTIVES: To measure NE exocytosis by airway neutrophils in relation to free extracellular NE activity and lung damage in children with CF. METHODS: We measured lung damage using chest computed tomography coupled with the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis scoring system. Concomitantly, we phenotyped blood and BAL fluid leukocytes by flow and image cytometry, and measured free extracellular NE activity using spectrophotometric and Förster resonance energy transfer assays. Children with airway inflammation linked to aerodigestive disorder were enrolled as control subjects. MEASUREMENTS AND MAIN RESULTS: Children with CF but not disease control children harbored BAL fluid neutrophils with high exocytosis of primary granules, before the detection of bronchiectasis. This measure of NE exocytosis correlated with lung damage (R = 0.55; P = 0.0008), whereas the molecular measure of free extracellular NE activity did not. This discrepancy may be caused by the inhibition of extracellular NE by BAL fluid antiproteases and its binding to leukocytes. CONCLUSIONS: NE exocytosis by airway neutrophils occurs in all children with CF, and its cellular measure correlates with early lung damage. These findings implicate live airway neutrophils in early CF pathogenesis, which should instruct biomarker development and antiinflammatory therapy in children with CF.
Assuntos
Fibrose Cística/fisiopatologia , Exocitose/fisiologia , Lesão Pulmonar/fisiopatologia , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Pré-Escolar , Feminino , Humanos , Lactente , MasculinoRESUMO
RATIONALE: Studies have demonstrated that exosomes can repair cardiac tissue post-myocardial infarction and recapitulate the benefits of cellular therapy. OBJECTIVE: We evaluated the role of donor age and hypoxia of human pediatric cardiac progenitor cell (CPC)-derived exosomes in a rat model of ischemia-reperfusion injury. METHODS AND RESULTS: Human CPCs from the right atrial appendages from children of different ages undergoing cardiac surgery for congenital heart defects were isolated and cultured under hypoxic or normoxic conditions. Exosomes were isolated from the culture-conditioned media and delivered to athymic rats after ischemia-reperfusion injury. Echocardiography at day 3 post-myocardial infarction suggested statistically improved function in neonatal hypoxic and neonatal normoxic groups compared with saline-treated controls. At 28 days post-myocardial infarction, exosomes derived from neonatal normoxia, neonatal hypoxia, infant hypoxia, and child hypoxia significantly improved cardiac function compared with those from saline-treated controls. Staining showed decreased fibrosis and improved angiogenesis in hypoxic groups compared with controls. Finally, using sequencing data, a computational model was generated to link microRNA levels to specific outcomes. CONCLUSIONS: CPC exosomes derived from neonates improved cardiac function independent of culture oxygen levels, whereas CPC exosomes from older children were not reparative unless subjected to hypoxic conditions. Cardiac functional improvements were associated with increased angiogenesis, reduced fibrosis, and improved hypertrophy, resulting in improved cardiac function; however, mechanisms for normoxic neonatal CPC exosomes improved function independent of those mechanisms. This is the first study of its kind demonstrating that donor age and oxygen content in the microenvironment significantly alter the efficacy of human CPC-derived exosomes.
Assuntos
Exossomos/fisiologia , MicroRNAs/fisiologia , Miócitos Cardíacos/fisiologia , Traumatismo por Reperfusão/terapia , Células-Tronco/fisiologia , Fatores Etários , Animais , Hipóxia Celular/fisiologia , Células Cultivadas , Criança , Pré-Escolar , Compreensão , Método Duplo-Cego , Exossomos/transplante , Humanos , Lactente , Recém-Nascido , MicroRNAs/administração & dosagem , Miócitos Cardíacos/transplante , Distribuição Aleatória , Ratos , Ratos Nus , Traumatismo por Reperfusão/fisiopatologia , Transplante de Células-Tronco/métodosRESUMO
BACKGROUND: Allergic and autoimmune diseases comprise a group of inflammatory disorders caused by aberrant immune responses in which CD25+ Forkhead box P3-positive (FOXP3+) T regulatory (Treg) cells that normally suppress inflammatory events are often poorly functioning. This has stimulated an intensive investigative effort to find ways of increasing Tregs as a method of therapy for these conditions. One such line of investigation includes the study of how ligation of Toll-like receptors (TLRs) by CpG oligonucleotides (ODN) results in an immunostimulatory cascade that leads to induction of T-helper (Th) type 1 and Treg-type immune responses. OBJECTIVE: The present study investigated the mechanisms by which calf thymus mammalian double-stranded DNA (CT-DNA) and a synthetic methylated DNA CpG ODN sequence suppress in vitro lymphoproliferative responses to antigens, mitogens, and alloantigens when measured by [3H]-thymidine incorporation and promote FoxP3 expression in human CD4+ T cells in the presence of transforming growth factor (TGF) beta and interleukin-2 (IL-2). METHODS: Lymphoproliferative responses of peripheral blood mononuclear cells from four healthy subjects or nine subjects with systemic lupus erythematosus to CT-DNA or phytohemagglutinin (PHA) was measured by tritiated thymidine ([3H]-TdR) incorporation expressed as a stimulation index. Mechanisms of immunosuppressive effects of CT-DNA were evaluated by measurement of the degree of inhibition to lymphoproliferative responses to streptokinase-streptodornase, phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), or alloantigens by a Con A suppressor assay. The effects of CpG methylation on induction of FoxP3 expression in human T cells were measured by comparing inhibitory responses of synthetic methylated and nonmethylated 8-mer CpG ODN sequences by using cell sorting, in vitro stimulation, and suppressor assay. RESULTS: Here, we showed that CT-DNA and a synthetic methylated DNA 8-mer sequence could suppress antigen-, mitogen-, and alloantigen-induced lymphoproliferation in vitro when measured by [3H]-thymidine. The synthetic methylated DNA CpG ODN but not an unmethylated CpG ODN sequence was shown to promote FoxP3 expression in human CD4+ T cells in the presence of TGF beta and IL-2. The induction of FoxP3+ suppressor cells is dose dependent and offers a potential clinical therapeutic application in allergic and autoimmune and inflammatory diseases. CONCLUSION: The use of this methylated CpG ODN offers a broad clinical application as a novel therapeutic method for Treg induction and, because of its low cost and small size, should facilitate delivery via nasal, respiratory, gastrointestinal routes, and/or by injection, routes of administration important for vaccine delivery to target sites responsible for respiratory, gastrointestinal, and systemic forms of allergic and autoimmune disease.
Assuntos
Linfócitos T CD4-Positivos/imunologia , DNA/imunologia , Imunoterapia/métodos , Lúpus Eritematoso Sistêmico/imunologia , Linfócitos T Reguladores/imunologia , Animais , Bovinos , Proliferação de Células , Células Cultivadas , Ilhas de CpG/genética , DNA/genética , Metilação de DNA/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Terapia de Imunossupressão , Isoantígenos/imunologia , Lúpus Eritematoso Sistêmico/terapia , Ativação Linfocitária , Fator de Crescimento Transformador beta/metabolismoRESUMO
Myocardial infarction (MI) is the most common cause of heart failure. Excessive production of ROS plays a key role in the pathogenesis of cardiac remodeling after MI. NADPH with NADPH oxidase (Nox)2 as the catalytic subunit is a major source of superoxide production, and expression is significantly increased in the infarcted myocardium, especially by infiltrating macrophages. While microRNAs (miRNAs) are potent regulators of gene expression and play an important role in heart disease, there still lacks efficient ways to identify miRNAs that target important pathological genes for treating MI. Thus, the overall objective was to establish a miRNA screening and delivery system for improving heart function after MI using Nox2 as a critical target. With the use of the miRNA-target screening system composed of a self-assembled cell microarray (SAMcell), three miRNAs, miR-106b, miR-148b, and miR-204, were identified that could regulate Nox2 expression and its downstream products in both human and mouse macrophages. Each of these miRNAs were encapsulated into polyketal (PK3) nanoparticles that could effectively deliver miRNAs into macrophages. Both in vitro and in vivo studies in mice confirmed that PK3-miRNAs particles could inhibit Nox2 expression and activity and significantly improve infarct size and acute cardiac function after MI. In conclusion, our results show that miR-106b, miR-148b, and miR-204 were able to improve heart function after myocardial infarction in mice by targeting Nox2 and possibly altering inflammatory cytokine production. This screening system and delivery method could have broader implications for miRNA-mediated therapeutics for cardiovascular and other diseases.NEW & NOTEWORTHY NADPH oxidase (Nox)2 is a promising target for treating cardiovascular disease, but there are no specific inhibitors. Finding endogenous signals that can target Nox2 and other inflammatory molecules is of great interest. In this study, we used high-throughput screening to identify microRNAs that target Nox2 and improve cardiac function after infarction.
Assuntos
Terapia Genética/métodos , Glicoproteínas de Membrana/genética , MicroRNAs/genética , MicroRNAs/uso terapêutico , Infarto do Miocárdio/genética , NADPH Oxidases/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Ensaios de Triagem em Larga Escala , Humanos , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/administração & dosagem , NADPH Oxidase 2 , NADPH Oxidases/antagonistas & inibidores , Nanopartículas , Superóxidos/metabolismoRESUMO
PURPOSE: We previously identified small molecules that fit into a BRCA1-binding pocket within estrogen receptor-alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity ("BRCA1-mimetics"), and overcome antiestrogen resistance. One such compound, the hydrochloride salt of NSC35446 ("NSC35446.HCl"), also inhibited the growth of antiestrogen-resistant LCC9 tumor xenografts. The purpose of this study was to investigate the down-stream effects of NSC35446.HCl and its mechanism of action. METHODS: Here, we studied antiestrogen-resistant (LCC9, T47DCO, MCF-7/RR, LY2), ERα-negative (MDA-MB-231, HCC1806, MDA-MB-468), and antiestrogen-sensitive (MCF-7) cell lines. Techniques utilized include RNA-seq, qRT-PCR, cell growth analysis, cell-cycle analysis, Western blotting, luciferase reporter assays, TUNEL assays, in silico analysis of the IKKB gene, and ChIP assays. RESULTS: SC35446.HCl inhibited proliferation and induced apoptosis in antiestrogen-resistant LCC9, T47DCO, MCF-7/RR, and LY2 cells but not in ERα-negative breast cancer cell lines. IKKB (IKKß, IKBKB), an upstream activator of NF-κB, was identified as a BRCA1-mimetic-regulated gene based on an RNA-seq analysis. NSC35446.HCl inhibited IKKB, IKKA, and IKKG/NEMO mRNA and protein expression in LCC9 cells. NSC35446.HCl also inhibited NF-κB activity and expression of NF-κB target genes. In silico analysis of the IKKB promoter identified nine estrogen response element (ERE) half-sites and one ERE-like full-site. ChIP assays revealed that ERα was recruited to the ERE-like full-site and five of the nine half-sites and that ERα recruitment was inhibited by NSC35446.HCl in LCC9 and T47DCO cells. CONCLUSIONS: These studies identify functional EREs in the IKKB promoter and identify IKKB as an ERα and NSC35446.HCl-regulated gene, and they suggest that NF-κB and IKKB, which were previously linked to antiestrogen resistance, are targets for NSC35446.HCl in reversing antiestrogen resistance.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Antagonistas de Estrogênios/administração & dosagem , Receptor alfa de Estrogênio/genética , Quinase I-kappa B/genética , Apoptose/genética , Proteína BRCA1/antagonistas & inibidores , Proteína BRCA1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Estrogênios/genética , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , NF-kappa B/genética , Regiões Promotoras GenéticasRESUMO
RATIONALE: Myocardial infarction is a leading cause of death in developed nations, and there remains a need for cardiac therapeutic systems that mitigate tissue damage. Cardiac progenitor cells (CPCs) and other stem cell types are attractive candidates for treatment of myocardial infarction; however, the benefit of these cells may be as a result of paracrine effects. OBJECTIVE: We tested the hypothesis that CPCs secrete proregenerative exosomes in response to hypoxic conditions. METHODS AND RESULTS: The angiogenic and antifibrotic potential of secreted exosomes on cardiac endothelial cells and cardiac fibroblasts were assessed. We found that CPC exosomes secreted in response to hypoxia enhanced tube formation of endothelial cells and decreased profibrotic gene expression in TGF-ß-stimulated fibroblasts, indicating that these exosomes possess therapeutic potential. Microarray analysis of exosomes secreted by hypoxic CPCs identified 11 miRNAs that were upregulated compared with exosomes secreted by CPCs grown under normoxic conditions. Principle component analysis was performed to identify miRNAs that were coregulated in response to distinct exosome-generating conditions. To investigate the cue-signal-response relationships of these miRNA clusters with a physiological outcome of tube formation or fibrotic gene expression, partial least squares regression analysis was applied. The importance of each up- or downregulated miRNA on physiological outcomes was determined. Finally, to validate the model, we delivered exosomes after ischemia-reperfusion injury. Exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis. CONCLUSIONS: These data provide a foundation for subsequent research of the use of exosomal miRNA and systems biology as therapeutic strategies for the damaged heart.
Assuntos
Exossomos/fisiologia , MicroRNAs/fisiologia , Miócitos Cardíacos/fisiologia , Células-Tronco/fisiologia , Biologia de Sistemas/métodos , Animais , Animais Recém-Nascidos , Hipóxia Celular/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
The treatment of triple negative breast cancer (TNBC) is a significant challenge to cancer research. The lack of hormone receptors limits the treatment options available to patients with this diagnosis, forcing them to endure prolonged radiation and chemotherapy. Anti-angiogenesis is a chemotherapeutic strategy that targets the vasculature of tumors. Combretastatin A-4 (CA-4) is a well-known vasculature-disrupting agent, which has been shown to effectively kill a variety of cancers through inhibition of tubulin polymerization. Due to its toxicity, small molecule analogues of CA-4 have been sought out. We have designed a novel dual action CA-4 prodrug, YK-5-252, which releases the drug through a disulfide bond cleavage mechanism and contains a near-infrared (NIR) fluorophore, which allows fluorescence monitoring of cleavage. This disulfide linkage causes CA-4 to become effective only when released by glutathione (GSH) reducing the toxicity of the drug while simultaneously releasing the NIR fluorophore. Therefore the prodrug, YK-5-252, represents a novel CA-4 analogue which has reduced toxicity and can be used for theranostics imaging.
Assuntos
Benzopiranos/uso terapêutico , Estilbenos/uso terapêutico , Nanomedicina Teranóstica , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Benzopiranos/química , Benzopiranos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Espectrometria de Massas , Espectroscopia de Prótons por Ressonância Magnética , Estilbenos/química , Estilbenos/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Moduladores de TubulinaRESUMO
Bacteria colonize cystic fibrosis (CF) airways, and although T cells with appropriate Ag specificity are present in draining lymph nodes, they are conspicuously absent from the lumen. To account for this absence, we hypothesized that polymorphonuclear neutrophils (PMNs), recruited massively into the CF airway lumen and actively exocytosing primary granules, also suppress T cell function therein. Programmed death-ligand 1 (PD-L1), which exerts T cell suppression at a late step, was expressed bimodally on CF airway PMNs, delineating PD-L1(hi) and PD-L1(lo) subsets, whereas healthy control (HC) airway PMNs were uniformly PD-L1(hi). Blood PMNs incubated in CF airway fluid lost PD-L1 over time; in coculture, Ab blockade of PD-L1 failed to inhibit the suppression of T cell proliferation by CF airway PMNs. In contrast with PD-L1, arginase 1 (Arg1), which exerts T cell suppression at an early step, was uniformly high on CF and HC airway PMNs. However, arginase activity was high in CF airway fluid and minimal in HC airway fluid, consistent with the fact that Arg1 activation requires primary granule exocytosis, which occurs in CF, but not HC, airway PMNs. In addition, Arg1 expression on CF airway PMNs correlated negatively with lung function and positively with arginase activity in CF airway fluid. Finally, combined treatment with arginase inhibitor and arginine rescued the suppression of T cell proliferation by CF airway fluid. Thus, Arg1 and PD-L1 are dynamically modulated upon PMN migration into human airways, and, Arg1, but not PD-L1, contributes to early PMN-driven T cell suppression in CF, likely hampering resolution of infection and inflammation.
Assuntos
Arginase/imunologia , Antígeno B7-H1/imunologia , Fibrose Cística/imunologia , Neutrófilos/imunologia , Linfócitos T/imunologia , Adulto , Apoptose/imunologia , Arginase/biossíntese , Antígeno B7-H1/antagonistas & inibidores , Proliferação de Células , Exocitose/imunologia , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Pulmão/imunologia , Pulmão/patologia , Ativação Linfocitária/imunologia , Masculino , Testes de Função Respiratória , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Adulto JovemRESUMO
Cytotoxic chemotherapies are used to treat breast cancer, but are limited by systemic toxicity. The key to addressing this important issue is the development of a nontoxic, tissue selective, and molecular specific delivery system. In order to potentially increase the therapeutic index of clinical reagents, we designed an Aminopeptidase P (APaseP) targeting tissue-specific construct conjugated to a homing peptide for selective binding to human breast-derived cancer cells. Homing peptides are short amino acid sequences derived from phage display libraries that have the unique property of localizing to specific organs. Our molecular construct allows for tissue-specific drug delivery, by binding to APaseP in the vascular endothelium. The breast homing peptide evaluated in our studies is a cyclic nine-amino-acid peptide with the sequence CPGPEGAGC, referred to as PEGA. We show by confocal microscopy that the PEGA peptide and similar peptide conjugates distribute to human breast tissue xenograft specifically and evaluate the interaction with the membrane-bound proline-specific APaseP (KD = 723 ± 3 nM) by binding studies. To achieve intracellular breast cancer cell delivery, the incorporation of the Tat sequence, a cell-penetrating motif derived from HIV, was conjugated with the fluorescently labeled PEGA peptide sequence. Ultimately, tissue specific peptides and their conjugates can enhance drug delivery and treatment by their ability to discriminate between tissue types. Tissue specific conjugates as we have designed may be valuable tools for drug delivery and visualization, including the potential to treat breast cancer, while simultaneously minimizing systemic toxicity.
Assuntos
Aminopeptidases/metabolismo , Mama/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Animais , Mama/patologia , Transformação Celular Neoplásica , Corantes Fluorescentes/química , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Especificidade de ÓrgãosRESUMO
DIM (3,3'-diindolylmethane), a small molecule compound, is a proposed cancer preventive agent that can be safely administered to humans in repeated doses. We report that administration of DIM in a multidose schedule protected rodents against lethal doses of total body irradiation up to 13 Gy, whether DIM dosing was initiated before or up to 24 h after radiation. Physiologic submicromolar concentrations of DIM protected cultured cells against radiation by a unique mechanism: DIM caused rapid activation of ataxia-telangiectasia mutated (ATM), a nuclear kinase that regulates responses to DNA damage (DDR) and oxidative stress. Subsequently, multiple ATM substrates were phosphorylated, suggesting that DIM induces an ATM-dependent DDR-like response, and DIM enhanced radiation-induced ATM signaling and NF-κB activation. DIM also caused activation of ATM in rodent tissues. Activation of ATM by DIM may be due, in part, to inhibition of protein phosphatase 2A, an upstream regulator of ATM. In contrast, DIM did not protect human breast cancer xenograft tumors against radiation under the conditions tested. In tumors, ATM was constitutively phosphorylated and was not further stimulated by radiation and/or DIM. Our findings suggest that DIM is a potent radioprotector and mitigator that functions by stimulating an ATM-driven DDR-like response and NF-κB survival signaling.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ativação Enzimática/efeitos dos fármacos , Indóis/farmacologia , Lesões Experimentais por Radiação/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Ensaio Cometa , Feminino , Proteínas de Fluorescência Verde , Imunoprecipitação , Indóis/uso terapêutico , Estimativa de Kaplan-Meier , Luciferases , Camundongos , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , RNA Interferente Pequeno/genética , Lesões Experimentais por Radiação/tratamento farmacológico , Radiação Ionizante , Ratos , Ratos Sprague-DawleyRESUMO
Activation of STAT3 in cancers leads to gene expression promoting cell proliferation and resistance to apoptosis, as well as tumor angiogenesis, invasion, and migration. In the characterization of effects of ST3-H2A2, a selective inhibitor of the STAT3 N-terminal domain (ND), we observed that the compound induced apoptotic death in cancer cells associated with robust activation of proapoptotic genes. Using ChIP and tiling human promoter arrays, we found that activation of gene expression in response to ST3-H2A2 is accompanied by altered STAT3 chromatin binding. Using inhibitors of STAT3 phosphorylation and a dominant-negative STAT3 mutant, we found that the unphosphorylated form of STAT3 binds to regulatory regions of proapoptotic genes and prevents their expression in tumor cells but not normal cells. siRNA knockdown confirmed the effects of ST3-HA2A on gene expression and chromatin binding to be STAT3 dependent. The STAT3-binding region of the C/EBP-homologous protein (CHOP) promoter was found to be localized in DNaseI hypersensitive site of chromatin in cancer cells but not in nontransformed cells, suggesting that STAT3 binding and suppressive action can be chromatin structure dependent. These data demonstrate a suppressive role for the STAT3 ND in the regulation of proapoptotic gene expression in cancer cells, providing further support for targeting STAT3 ND for cancer therapy.
Assuntos
Apoptose/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/metabolismo , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Fosforilação , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genéticaRESUMO
BACKGROUND: The mechanisms underlying glucocorticoid responsiveness are largely unknown. Although redox regulation of the glucocorticoid receptor (GR) has been reported, it has not been studied in asthmatic patients. OBJECTIVE: We characterized systemic cysteine oxidation and its association with inflammatory and clinical features in healthy children and children with difficult-to-treat asthma. We hypothesized that cysteine oxidation would be associated with increased markers of oxidative stress and inflammation, increased features of asthma severity, decreased clinically defined glucocorticoid responsiveness, and impaired GR function. METHODS: PBMCs were collected from healthy children (n = 16) and children with asthma (n = 118) aged 6 to 17 years. Children with difficult-to-treat asthma underwent glucocorticoid responsiveness testing with intramuscular triamcinolone. Cysteine, cystine, and inflammatory chemokines and reactive oxygen species generation were quantified, and expression and activity of the GR were assessed. RESULTS: Cysteine oxidation was present in children with difficult-to-treat asthma and accompanied by increased reactive oxygen species generation and increased CCL3 and CXCL1 mRNA expression. Children with the greatest extent of cysteine oxidation had more features of asthma severity, including poorer symptom control, greater medication use, and less glucocorticoid responsiveness despite inhaled glucocorticoid therapy. Cysteine oxidation also modified the GR protein by decreasing available sulfhydryl groups and decreasing nuclear GR expression and activity. CONCLUSIONS: A highly oxidized cysteine redox state promotes a posttranslational modification of the GR that might inhibit its function. Given that cysteine oxidation is prevalent in children with difficult-to-treat asthma, the cysteine redox state might represent a potential therapeutic target for restoration of glucocorticoid responsiveness in this population.
Assuntos
Asma/tratamento farmacológico , Glucocorticoides/uso terapêutico , Leucócitos Mononucleares/imunologia , Processamento de Proteína Pós-Traducional , Receptores de Glucocorticoides/imunologia , Triancinolona/uso terapêutico , Administração por Inalação , Adolescente , Asma/genética , Asma/imunologia , Asma/patologia , Quimiocina CCL3/genética , Quimiocina CCL3/imunologia , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Criança , Cisteína/química , Cisteína/imunologia , Cistina/química , Cistina/imunologia , Monitoramento de Medicamentos , Feminino , Expressão Gênica , Humanos , Injeções Intramusculares , Leucócitos Mononucleares/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Masculino , Oxirredução , Estresse Oxidativo , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genéticaRESUMO
Gonadal hypofunction is described in male and female patients with sickle cell anemia (SCA) after bone marrow transplant (BMT) and in males treated with hydroxyurea (HU). Anti-Müllerian hormone (AMH) is a serum marker of ovarian reserve. This study describes AMH and follicle-stimulating hormone (FSH) levels in female SCA subjects treated with supportive care (SCA-SC), HU (SCA-HU) and BMT (SCA-BMT). SCA (SS/Sß(0)) subjects not on HU, on HU and status-post BMT, ages 10-21 years were recruited. SCA-HU subjects were treated with HU ≥ 20 mg/kg for ≥ 12 consecutive months. SCA-BMT subjects had received busulfan and cyclophosphamide. Serum AMH and random FSH levels were obtained. Diminished ovarian reserve (DOR) was defined as AMH level <5th percentile for age-matched controls. Subjects also with FSH >40 IU/L were classified as having premature ovarian insufficiency (POI). 14 SCA-SC (14.5 ± 2.7 years), 33 SCA-HU (14.4 ± 2.4 years) and 9 SCA-BMT (14.3 ± 2.7 years) females were included. AMH was undetectable in all SCA-BMT subjects and <5th percentile in 24% of SCA-HU subjects. FSH was menopausal (>40 IU/L) in 88.9% of SCA-BMT subjects. All SCA-BMT subjects and 24% of subjects on HU had DOR; 89% of SCA-BMT subjects had POI. AMH and FSH may be useful tools in assessing ovarian reserve and function.
Assuntos
Anemia Falciforme/terapia , Hormônio Antimülleriano/sangue , Antidrepanocíticos/uso terapêutico , Transplante de Medula Óssea , Hidroxiureia/uso terapêutico , Insuficiência Ovariana Primária/terapia , Adolescente , Anemia Falciforme/sangue , Anemia Falciforme/complicações , Anemia Falciforme/diagnóstico , Biomarcadores/sangue , Bussulfano/uso terapêutico , Estudos de Casos e Controles , Criança , Ciclofosfamida/uso terapêutico , Feminino , Hormônio Foliculoestimulante/sangue , Hemoglobina Falciforme/metabolismo , Heterozigoto , Homozigoto , Humanos , Menarca/fisiologia , Agonistas Mieloablativos/uso terapêutico , Reserva Ovariana/efeitos dos fármacos , Insuficiência Ovariana Primária/sangue , Insuficiência Ovariana Primária/complicações , Insuficiência Ovariana Primária/diagnóstico , Adulto JovemRESUMO
The discovery and development of small molecules that antagonize neuronal nicotinic acetylcholine receptors may provide new ligands for evaluation in models of depression or addiction. We discovered a small molecule, VMY-2-95, a nAChR ligand with picomolar affinity and high selectivity for α4ß2 receptors. In this study, we investigated its preclinical profile in regards to solubility, lipophilicity, metabolic stability, intestinal permeability, bioavailability, and drug delivery to the rat brain. Metabolic stability of VMY-2-95·2HCl was monitored on human liver microsomes, and specific activity of VMY-2-95·2HCl on substrate metabolism by CYP1A2, 2C9, 2C19, 2D6, and 3A4 was tested in a high-throughput manner. The intestinal transport of VMY-2-95·2HCl was studied through Caco-2 cell monolayer permeability. VMY-2-95·2HCl was soluble in water and chemically stable, and the apparent partition coefficient was 0.682. VMY-2-95·2HCl showed significant inhibition of CYP2C9 and 2C19, but weak or no effect on 1A2, 2D6, and 3A4. The Caco-2 cell model studies revealed that VMY-2-95·2HCl was highly permeable with efflux ratio of 1.11. VMY-2-95·2HCl achieved a maximum serum concentration of 0.56 mg/mL at 0.9 h and was orally available with a half-life of â¼9 h. Furthermore, VMY-2-95·2HCl was detected in the rat brain after 3 mg/kg oral administration and achieved a maximal brain tissue concentration of 2.3 µg/g within 60 min. Overall, the results demonstrate that VMY-2-95·2HCl has good drug like properties and can penetrate the blood-brain barrier with oral administration.