RESUMO
Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.
Assuntos
Neoplasias Pulmonares/patologia , Fatores de Transcrição NFI/metabolismo , Metástase Neoplásica/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Fatores de Transcrição NFI/genética , Regiões Promotoras Genéticas , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Regulação para CimaRESUMO
Although TP53 is the most commonly mutated gene in human cancers, the p53-dependent transcriptional programs mediating tumor suppression remain incompletely understood. Here, to uncover critical components downstream of p53 in tumor suppression, we perform unbiased RNAi and CRISPR-Cas9-based genetic screens in vivo. These screens converge upon the p53-inducible gene Zmat3, encoding an RNA-binding protein, and we demonstrate that ZMAT3 is an important tumor suppressor downstream of p53 in mouse KrasG12D-driven lung and liver cancers and human carcinomas. Integrative analysis of the ZMAT3 RNA-binding landscape and transcriptomic profiling reveals that ZMAT3 directly modulates exon inclusion in transcripts encoding proteins of diverse functions, including the p53 inhibitors MDM4 and MDM2, splicing regulators, and components of varied cellular processes. Interestingly, these exons are enriched in NMD signals, and, accordingly, ZMAT3 broadly affects target transcript stability. Collectively, these studies reveal ZMAT3 as a novel RNA-splicing and homeostasis regulator and a key component of p53-mediated tumor suppression.
Assuntos
Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/genética , Processamento Alternativo , Animais , Proteínas de Ciclo Celular/metabolismo , Éxons , Perfilação da Expressão Gênica/métodos , Genes Supressores de Tumor , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Interferência de RNA , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin's lymphoma. Although it is well established that PD-1-PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1-PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1-PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.
Assuntos
Neoplasias do Colo/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose , Receptor de Morte Celular Programada 1/metabolismo , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estadiamento de Neoplasias , Fagocitose/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Type Ia supernovae arise from the thermonuclear explosion of white-dwarf stars that have cores of carbon and oxygen. The uniformity of their light curves makes these supernovae powerful cosmological distance indicators, but there have long been debates about exactly how their explosion is triggered and what kind of companion stars are involved. For example, the recent detection of the early ultraviolet pulse of a peculiar, subluminous type Ia supernova has been claimed as evidence for an interaction between a red-giant or a main-sequence companion and ejecta from a white-dwarf explosion. Here we report observations of a prominent but red optical flash that appears about half a day after the explosion of a type Ia supernova. This supernova shows hybrid features of different supernova subclasses, namely a light curve that is typical of normal-brightness supernovae, but with strong titanium absorption, which is commonly seen in the spectra of subluminous ones. We argue that this early flash does not occur through previously suggested mechanisms such as the companion-ejecta interaction. Instead, our simulations show that it could occur through detonation of a thin helium shell either on a near-Chandrasekhar-mass white dwarf, or on a sub-Chandrasekhar-mass white dwarf merging with a less-massive white dwarf. Our finding provides evidence that one branch of previously proposed explosion models-the helium-ignition branch-does exist in nature, and that such a model may account for the explosions of white dwarfs in a mass range wider than previously supposed.
RESUMO
Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or 'efferocytosis'. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-α as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.
Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Aterosclerose/prevenção & controle , Antígeno CD47/imunologia , Fagocitose/efeitos dos fármacos , Animais , Apoptose , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/terapia , Antígeno CD47/biossíntese , Antígeno CD47/metabolismo , Artérias Carótidas/patologia , Vasos Coronários/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , NF-kappa B/metabolismo , Biossíntese de Proteínas , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Gene regulatory networks control tissue homeostasis and disease progression in a cell type-specific manner. Ubiquitously expressed chromatin regulators modulate these networks, yet the mechanisms governing how tissue specificity of their function is achieved are poorly understood. BRD4 (bromodomain-containing protein 4), a member of the BET (bromo- and extraterminal domain) family of ubiquitously expressed acetyl-lysine reader proteins, plays a pivotal role as a coactivator of enhancer signaling across diverse tissue types in both health and disease and has been implicated as a pharmacological target in heart failure. However, the cell-specific role of BRD4 in adult cardiomyocytes remains unknown. METHODS: We combined conditional mouse genetics, unbiased transcriptomic and epigenomic analyses, and classic molecular biology and biochemical approaches to understand the mechanism by which BRD4 in adult cardiomyocyte homeostasis. RESULTS: Here, we show that cardiomyocyte-specific deletion of Brd4 in adult mice leads to acute deterioration of cardiac contractile function with mutant animals demonstrating a transcriptomic signature characterized by decreased expression of genes critical for mitochondrial energy production. Genome-wide occupancy data show that BRD4 enriches at many downregulated genes (including the master coactivators Ppargc1a, Ppargc1b, and their downstream targets) and preferentially colocalizes with GATA4 (GATA binding protein 4), a lineage-determining cardiac transcription factor not previously implicated in regulation of adult cardiac metabolism. BRD4 and GATA4 form an endogenous complex in cardiomyocytes and interact in a bromodomain-independent manner, revealing a new functional interaction partner for BRD4 that can direct its locus and tissue specificity. CONCLUSIONS: These results highlight a novel role for a BRD4-GATA4 module in cooperative regulation of a cardiomyocyte-specific gene program governing bioenergetic homeostasis in the adult heart.
Assuntos
Metabolismo Energético , Fator de Transcrição GATA4/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Animais , Metabolismo Energético/genética , Fator de Transcrição GATA4/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genótipo , Células HEK293 , Homeostase , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Proteínas Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fenótipo , Ligação Proteica , Ratos Sprague-Dawley , Fatores de Transcrição/genética , Transcriptoma , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular EsquerdaRESUMO
Marfan syndrome (MFS) is a connective tissue disorder that results in aortic root aneurysm formation. Reactive oxygen species (ROS) seem to play a role in aortic wall remodelling in MFS, although the mechanism remains unknown. MFS Fbn1C1039G/+ mouse root/ascending (AS) and descending (DES) aortic samples were examined using DHE staining, lucigenin-enhanced chemiluminescence (LGCL), Verhoeff's elastin-Van Gieson staining (elastin breakdown) and in situ zymography for protease activity. Fbn1C1039G/+ AS- or DES-derived smooth muscle cells (SMC) were treated with anti-TGF-ß antibody, angiotensin II (AngII), anti-TGF-ß antibody + AngII, or isotype control. ROS were detected during early aneurysm formation in the Fbn1C1039G/+ AS aorta, but absent in normal-sized DES aorta. Fbn1C1039G/+ mice treated with the unspecific NADPH oxidase inhibitor, apocynin reduced AS aneurysm formation, with attenuated elastin fragmentation. In situ zymography revealed apocynin treatment decreased protease activity. In vitro SMC studies showed Fbn1C1039G/+ -derived AS SMC had increased NADPH activity compared to DES-derived SMC. AS SMC NADPH activity increased with AngII treatment and appeared TGF-ß dependent. In conclusion, ROS play a role in MFS aneurysm development and correspond anatomically with aneurysmal aortic segments. ROS inhibition via apocynin treatment attenuates MFS aneurysm progression. AngII enhances ROS production in MFS AS SMCs and is likely TGF-ß dependent.
Assuntos
Aneurisma/complicações , Aneurisma/metabolismo , Síndrome de Marfan/complicações , Síndrome de Marfan/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetofenonas/farmacologia , Angiotensina II , Animais , Aorta/metabolismo , Aorta/patologia , Modelos Animais de Doenças , Fibrilina-1/deficiência , Fibrilina-1/metabolismo , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/metabolismoRESUMO
Pathogenic DNM1L mutations cause a mitochondrial disorder with a highly variable clinical phenotype characterized by developmental delay, hypotonia, seizures, microcephaly, poor feeding, ocular abnormalities, and dysarthria. We report the case of an 8-month-old female with autosomal dominant, de novo DNM1L c. 1228G>A (p. E410K) mutation and mitochondrial disorder, septo-optic dysplasia, hypotonia, developmental delay, elevated blood lactate, and severe mitochondrial cardiomyopathy leading to nonischemic congestive heart failure and cardiogenic shock resulting in death. This case suggests that cardiac involvement, previously undescribed, can be a clinically important feature of this syndrome and should be screened for at diagnosis.
Assuntos
Dinaminas/genética , Cardiopatias/diagnóstico , Cardiopatias/genética , Encefalomiopatias Mitocondriais/diagnóstico , Encefalomiopatias Mitocondriais/genética , Mutação , Fenótipo , Adulto , Alelos , Biópsia , Criança , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Imuno-Histoquímica , MasculinoRESUMO
Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 103 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. Stem Cells 2017;35:1994-2000.
Assuntos
Células-Tronco Pluripotentes/patologia , Radiação Ionizante , Teratoma/radioterapia , Apoptose/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Humanos , Células-Tronco Pluripotentes/efeitos da radiação , Teratoma/patologiaRESUMO
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) describes a group of developmental disorders affecting the lungs with its pulmonary vasculature. Mutations in the FOXF1 gene have been reported in most cases, and extrapulmonary findings were described. We present two patients with ACDMPV and FOXF1 mutations that illustrate the variability in presentation and outcome of their disease. Patient 1 was a full-term infant with imperforate anus and pulmonary hypertension. He required Extracorporeal Membrane Oxygenation on day of life (DOL) 3, and passed away on DOL 13 after no clinical improvement. Postmortem findings were consistent with ACDMPV. FOXF1 testing revealed a heterozygous pathogenic frameshift de novo mutation, c.1057_1078dup, p.(Gly360Valfs*58). Patient 2 is a 6-month-old female, with a small omphalocele. She had intermittent retractions at 1 week of age. She was admitted with pulmonary hypertension at 7 weeks of age. Lung biopsy confirmed ACDMPV. FOXF1 testing revealed a de novo, heterozygous likely pathogenic missense mutation c.253T>C, p.(Phe85Leu]). Our two patients had different presentations, ages of onset, and progression of their disease. Our second patient had patchy lung involvement on biopsy, which may explain the relatively delayed onset and longer survival. ACDMPV is an important consideration for full-term infants with worsening pulmonary hypertension early in life.
Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Fatores de Transcrição Forkhead/genética , Mutação , Síndrome da Persistência do Padrão de Circulação Fetal/diagnóstico , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Alvéolos Pulmonares/anormalidades , Veias Pulmonares/anormalidades , Autopsia , Biomarcadores , Biópsia , Hibridização Genômica Comparativa , Análise Citogenética , Ecocardiografia , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
RATIONALE: Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease, but its mechanism remains unknown. OBJECTIVE: To determine whether this association is secondary to an increase in atherosclerosis, or it is the result of a separate angiogenesis-related mechanism. METHODS AND RESULTS: Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under nonatherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hindlimb ischemia and digital autoamputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell to support the developing neovessel. Microarray studies identified impaired transforming growth factor ß (TGFß) signaling in cultured cyclin-dependent kinase inhibitor 2B (CDKN2B)-deficient cells, as well as TGFß1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFß activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFß1-induced-1, which is a TGFß-rheostat known to have antagonistic effects on the endothelial cell and smooth muscle cell. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. CONCLUSIONS: These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis but may also impair TGFß signaling and hypoxic neovessel maturation.
Assuntos
Aterosclerose/enzimologia , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Neovascularização Fisiológica , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/mortalidade , Aterosclerose/patologia , Artérias Carótidas/enzimologia , Artérias Carótidas/patologia , Hipóxia Celular , Células Cultivadas , Cromossomos Humanos Par 9 , Vasos Coronários/enzimologia , Vasos Coronários/patologia , Inibidor de Quinase Dependente de Ciclina p15/deficiência , Inibidor de Quinase Dependente de Ciclina p15/genética , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Membro Posterior , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/fisiopatologia , Neovascularização Patológica , Fenótipo , Interferência de RNA , Proteína Smad7/metabolismo , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta1/genéticaRESUMO
BACKGROUND: Our aim was to develop a model of acute right heart failure (ARHF) in the setting of pulmonary hypertension and to characterize acute right ventricular lesions that develop early after hemodynamic restoration. METHODS AND RESULTS: We used a described piglet model of chronic pulmonary hypertension (cPH) induced by pulmonary artery occlusions. We induced ARHF in animals with cPH (ARHF-cPH group, n = 9) by volume loading and iterative acute pulmonary embolism until hemodynamic compromise followed by dobutamine infusion for hemodynamic restoration before sacrifice for right ventricular tissue evaluation. The median duration of ARHF before sacrifice was 162 (135-189) minutes. Although ventriculoarterial coupling (measured with multibeat pressure-volume loops) and stroke volume decreased after iterative pulmonary embolism and improved with dobutamine, relative pulmonary to systemic pressure increased by 2-fold and remained similarly increased with dobutamine. Circulating high-sensitivity troponin I increased after hemodynamic restoration. We found an increase in right ventricular subendocardial and subepicardial focal ischemic lesions and in expression of autophagy-related protein LC3-II (Western blot) in the ARHF-cPH group compared with the cPH (n = 5) and control (n = 5) groups. CONCLUSIONS: We developed and phenotyped a novel large animal model of ARHF on cPH in which right ventricular ischemic lesions were observed early after hemodynamic restoration.
Assuntos
Modelos Animais de Doenças , Insuficiência Cardíaca/diagnóstico por imagem , Hipertensão Pulmonar/diagnóstico por imagem , Embolia Pulmonar/diagnóstico por imagem , Disfunção Ventricular Direita/diagnóstico por imagem , Animais , Animais Recém-Nascidos , Biomarcadores/sangue , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/fisiopatologia , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/fisiopatologia , Embolia Pulmonar/sangue , Embolia Pulmonar/fisiopatologia , Suínos , Disfunção Ventricular Direita/sangue , Disfunção Ventricular Direita/fisiopatologiaRESUMO
RATIONALE: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. OBJECTIVE: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. METHODS AND RESULTS: We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. CONCLUSIONS: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.
Assuntos
Células-Tronco Embrionárias/transplante , Sobrevivência de Enxerto , Transplante de Coração/métodos , Infarto do Miocárdio/cirurgia , Miócitos Cardíacos/transplante , Músculos Papilares/transplante , Engenharia Tecidual/métodos , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Conexina 43/metabolismo , Modelos Animais de Doenças , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/metabolismo , Transplante de Coração/efeitos adversos , Xenoenxertos , Humanos , Imunossupressores/farmacologia , Masculino , Contração Miocárdica , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Músculos Papilares/imunologia , Músculos Papilares/metabolismo , Músculos Papilares/patologia , Músculos Papilares/fisiopatologia , Ratos Nus , Ratos Sprague-Dawley , Volume Sistólico , Fatores de Tempo , TransfecçãoRESUMO
The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.
Assuntos
Nervo Femoral/fisiologia , Membro Posterior/fisiologia , Regeneração , Nervo Isquiático/fisiologia , Animais , Sequência de Bases , Primers do DNA , Camundongos , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: Rupture and dissection of aortic root aneurysms remain the leading causes of death in patients with the Marfan syndrome, a hereditary connective tissue disorder that affects 1 in 5000 individuals worldwide. In the present study, we use a Marfan mouse model (Fbn1(C1039G/+)) to investigate the biological importance of apoptosis during aneurysm development in Marfan syndrome. APPROACH AND RESULTS: Using in vivo single-photon emission computed tomographic-imaging and ex vivo autoradiography for Tc99m-annexin, we discovered increased apoptosis in the Fbn1(C1039G/+) ascending aorta during early aneurysm development peaking at 4 weeks. Immunofluorescence colocalization studies identified smooth muscle cells (SMCs) as the apoptotic cell population. As biological proof of concept that early aortic wall apoptosis plays a role in aneurysm development in Marfan syndrome, Fbn1(C1039G/+) mice were treated daily from 2 to 6 weeks with either (1) a pan-caspase inhibitor, Q-VD-OPh (20 mg/kg), or (2) vehicle control intraperitoneally. Q-VD-OPh treatment led to a significant reduction in aneurysm size and decreased extracellular matrix degradation in the aortic wall compared with control mice. In vitro studies using Fbn1(C1039G/+) ascending SMCs showed that apoptotic SMCs have increased elastolytic potential compared with viable cells, mostly because of caspase activity. Moreover, in vitro (1) cell membrane isolation, (2) immunofluorescence staining, and (3) scanning electron microscopy studies illustrate that caspases are expressed on the exterior cell surface of apoptotic SMCs. CONCLUSIONS: Caspase inhibition attenuates aneurysm development in an Fbn1(C1039G/+) Marfan mouse model. Mechanistically, during apoptosis, caspases are expressed on the cell surface of SMCs and likely contribute to elastin degradation and aneurysm development in Marfan syndrome.
Assuntos
Aneurisma Aórtico/etiologia , Apoptose , Caspases/metabolismo , Membrana Celular/enzimologia , Síndrome de Marfan/complicações , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Remodelação Vascular , Animais , Aorta/enzimologia , Aneurisma Aórtico/diagnóstico , Aneurisma Aórtico/enzimologia , Aneurisma Aórtico/genética , Aneurisma Aórtico/prevenção & controle , Apoptose/efeitos dos fármacos , Autorradiografia , Inibidores de Caspase/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Elastina/metabolismo , Feminino , Fibrilina-1 , Fibrilinas , Imunofluorescência , Masculino , Síndrome de Marfan/genética , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica de Varredura , Músculo Liso Vascular/diagnóstico por imagem , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/ultraestrutura , Mutação , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/ultraestrutura , Fatores de Tempo , Tomografia Computadorizada de Emissão de Fóton Único , Remodelação Vascular/efeitos dos fármacosRESUMO
The presence of eosinophils within the neutrophilic infiltrates of acute febrile neutrophilic dermatosis (Sweet syndrome) is documented in the literature. Here, the authors describe a case of eosinophil-rich acute febrile neutrophilic dermatosis in the setting of new onset enteropathy-associated T-cell lymphoma (EATL), type 1. Histopathologic evaluation of the skin biopsies demonstrated a mixed superficial perivascular and inflammatory infiltrate composed of neutrophils, lymphocytes, and abundant eosinophils. EATL, type 1 is an aggressive although rare primary intestinal lymphoma that may be associated with celiac disease. This lymphoma is associated with a poor prognosis due to treatment resistance or bowel perforation. To the authors' knowledge, Sweet syndrome has not been reported in a patient with EATL.
Assuntos
Linfoma de Células T Associado a Enteropatia/complicações , Síndrome de Sweet/etiologia , Idoso de 80 Anos ou mais , Linfoma de Células T Associado a Enteropatia/patologia , Linfoma de Células T Associado a Enteropatia/fisiopatologia , Humanos , Masculino , Síndrome de Sweet/patologia , Síndrome de Sweet/fisiopatologiaRESUMO
Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.
Assuntos
Regeneração Óssea , Células-Tronco Pluripotentes/transplante , Animais , Regeneração Óssea/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Masculino , Camundongos , Camundongos Nus , Osteogênese/fisiologia , Células-Tronco Pluripotentes/citologia , Crânio/diagnóstico por imagem , Crânio/lesões , Crânio/patologia , Nicho de Células-Tronco/fisiologia , Teratoma/patologia , Alicerces Teciduais , Transplante Heterólogo , Microtomografia por Raio-XRESUMO
RATIONALE: Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models. OBJECTIVE: To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia. METHODS AND RESULTS: Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery. CONCLUSIONS: In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.
Assuntos
Transplante de Células , Endotélio Vascular/citologia , Endotélio Vascular/transplante , Coração/fisiopatologia , Técnicas Analíticas Microfluídicas , Infarto do Miocárdio/terapia , Comunicação Parácrina/fisiologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Ecocardiografia , Endotélio Vascular/fisiologia , Feminino , Imageamento por Ressonância Magnética , Camundongos , Camundongos SCID , Modelos Animais , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/patologia , Neovascularização Fisiológica , Células-Tronco Pluripotentes/fisiologia , Suínos , Porco MiniaturaRESUMO
Allogeneic transplantation of pancreatic islets for patients with difficult-to-control diabetes mellitus is severely hampered by the requirement for continuous immunosuppression and its associated morbidity. We report that allogeneic transplantation of genetically engineered (B2M-/-, CIITA-/-, CD47+), primary, hypoimmune, pseudo-islets (p-islets) results in their engraftment into a fully immunocompetent, diabetic non-human primate wherein they provide stable endocrine function and enable insulin independence without inducing any detectable immune response in the absence of immunosuppression. Hypoimmune primary p-islets may provide a curative cell therapy for type 1 diabetes mellitus.
Assuntos
Diabetes Mellitus Tipo 1 , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Animais , Humanos , Insulina/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/metabolismo , Primatas , Diabetes Mellitus Tipo 1/terapia , Transplante HomólogoRESUMO
Genetic engineering of allogeneic cell therapeutics that fully prevents rejection by a recipient's immune system would abolish the requirement for immunosuppressive drugs or encapsulation and support large-scale manufacturing of off-the-shelf cell products. Previously, we generated mouse and human hypoimmune pluripotent (HIP) stem cells by depleting HLA class I and II molecules and overexpressing CD47 (B2M-/-CIITA-/-CD47+). To determine whether this strategy is successful in non-human primates, we engineered rhesus macaque HIP cells and transplanted them intramuscularly into four allogeneic rhesus macaques. The HIP cells survived unrestricted for 16 weeks in fully immunocompetent allogeneic recipients and differentiated into several lineages, whereas allogeneic wild-type cells were vigorously rejected. We also differentiated human HIP cells into endocrinologically active pancreatic islet cells and showed that they survived in immunocompetent, allogeneic diabetic humanized mice for 4 weeks and ameliorated diabetes. HIP-edited primary rhesus macaque islets survived for 40 weeks in an allogeneic rhesus macaque recipient without immunosuppression, whereas unedited islets were quickly rejected.