RESUMO
A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X.
Assuntos
Fibrose Cística , Células-Tronco Pluripotentes Induzidas , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Mutação , Fibrose Cística/terapia , Fibrose Cística/tratamento farmacológico , Códon sem Sentido , Células EpiteliaisRESUMO
Hidradenitis suppurativa (HS) is a complex inflammatory skin disease with undefined mechanistic underpinnings. Here, we investigated HS epithelial cells and demonstrated that HS basal progenitors modulate their lineage restriction and give rise to pathogenic keratinocyte clones, resulting in epidermal hyperproliferation and dysregulated inflammation in HS. When comparing to healthy epithelial stem/progenitor cells, in HS, we identified changes in gene signatures that revolve around the mitotic cell cycle, DNA damage response and repair, as well as cell-cell adhesion and chromatin remodeling. By reconstructing cell differentiation trajectory and CellChat modeling, we identified a keratinocyte population specific to HS. This population is marked by S100A7/8/9 and KRT6 family members, triggering IL1, IL10, and complement inflammatory cascades. These signals, along with HS-specific proinflammatory cytokines and chemokines, contribute to the recruitment of certain immune cells during the disease progression. Furthermore, we revealed a previously uncharacterized role of S100A8 in regulating the local chromatin environment of target loci in HS keratinocytes. Through the integration of genomic and epigenomic datasets, we identified genome-wide chromatin rewiring alongside the switch of transcription factors (TFs), which mediated HS transcriptional profiles. Importantly, we identified numerous clinically relevant inflammatory enhancers and their coordinated TFs in HS basal CD49fhigh cells. The disruption of the S100A enhancer using the CRISPR/Cas9-mediated approach or the pharmacological inhibition of the interferon regulatory transcription factor 3 (IRF3) efficiently reduced the production of HS-associated inflammatory regulators. Our study not only uncovers the plasticity of epidermal progenitor cells in HS but also elucidates the epigenetic mechanisms underlying HS pathogenesis.
Assuntos
Hidradenite Supurativa , Humanos , Hidradenite Supurativa/genética , Pele/metabolismo , Epigenômica , Epigênese Genética , Células-Tronco/metabolismo , Cromatina/metabolismoAssuntos
Anemia Falciforme , Braço , Anemia Falciforme/genética , Anemia Falciforme/terapia , Animais , Terapia Genética , CamundongosRESUMO
Expanding the utility of immune-based cancer treatments is a clinical challenge due to tumor-intrinsic factors that suppress the immune response. Here we report the identification of tumoral ring finger protein 2 (RNF2), the core subunit of polycomb repressor complex 1, as a negative regulator of antitumor immunity in various human cancers, including breast cancer. In syngeneic murine models of triple-negative breast cancer, we found that deleting genes encoding the polycomb repressor complex 1 subunits Rnf2, BMI1 proto-oncogene, polycomb ring finger (Bmi1), or the downstream effector of Rnf2, remodeling and spacing factor 1 (Rsf1), was sufficient by itself to induce durable tumor rejection and establish immune memory by enhancing infiltration and activation of natural killer and CD4+ T cells, but not CD8+ T cells, into the tumor and enabled their cooperativity. These findings uncover an epigenetic reprogramming of the tumor-immune microenvironment, which fosters durable antitumor immunity and memory.
Assuntos
Neoplasias , Complexo Repressor Polycomb 1/metabolismo , Microambiente Tumoral , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Humanos , Camundongos , Neoplasias/genética , Proteínas Nucleares , Complexo Repressor Polycomb 1/genética , Proteínas do Grupo Polycomb , Transativadores , Microambiente Tumoral/genéticaRESUMO
Hypoxic tumor niches are chief causes of treatment resistance and tumor recurrence. Sickle erythrocytes' (SSRBCs') intrinsic oxygen-sensing functionality empowers them to access such hypoxic niches wherein they form microaggregates that induce focal vessel closure. In search of measures to augment the scale of SSRBC-mediated tumor vaso-occlusion, we turned to the vascular disrupting agent, combretastatin A-4 (CA-4). CA-4 induces selective tumor endothelial injury, blood stasis, and hypoxia but fails to eliminate peripheral tumor foci. In this article, we show that introducing deoxygenated SSRBCs into tumor microvessels treated with CA-4 and sublethal radiation (SR) produces a massive surge of tumor vaso-occlusion and broadly propagated tumor infarctions that engulfs treatment-resistant hypoxic niches and eradicates established lung tumors. Tumor regression was histologically corroborated by significant treatment effect. Treated tumors displayed disseminated microvessels occluded by tightly packed SSRBCs along with widely distributed pimidazole-positive hypoxic tumor cells. Humanized HbS-knockin mice (SSKI) but not HbA-knockin mice (AAKI) showed a similar treatment response underscoring SSRBCs as the paramount tumoricidal effectors. Thus, CA-4-SR-remodeled tumor vessels license SSRBCs to produce an unprecedented surge of tumor vaso-occlusion and infarction that envelops treatment-resistant tumor niches resulting in complete tumor regression. Strategically deployed, these innovative tools constitute a major conceptual advance with compelling translational potential.
Assuntos
Anemia Falciforme/sangue , Antineoplásicos Fitogênicos/administração & dosagem , Eritrócitos Anormais/transplante , Neoplasias Pulmonares/terapia , Recidiva Local de Neoplasia/terapia , Animais , Adesão Celular , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Terapia Combinada/métodos , Feminino , Técnicas de Introdução de Genes , Hemoglobina Falciforme/genética , Humanos , Pulmão/irrigação sanguínea , Pulmão/diagnóstico por imagem , Pulmão/efeitos dos fármacos , Pulmão/patologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Transgênicos , Microvasos/citologia , Microvasos/efeitos dos fármacos , Microvasos/patologia , Recidiva Local de Neoplasia/irrigação sanguínea , Recidiva Local de Neoplasia/diagnóstico por imagem , Recidiva Local de Neoplasia/patologia , Estilbenos/administração & dosagem , Transplante Heterólogo/métodos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin ß chain (ß93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the ß93 cysteine had been replaced with alanine (ß93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (ß93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with ß93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in ß93C and ß93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between ß93C and ß93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in ß93C and ß93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the ß93 cysteine of Hb.
Assuntos
Plaquetas/metabolismo , Eritrócitos/metabolismo , Hemoglobinas/metabolismo , Traumatismo por Reperfusão Miocárdica/sangue , Óxido Nítrico/sangue , Pele/irrigação sanguínea , Globinas beta/metabolismo , Alanina , Substituição de Aminoácidos , Animais , Transporte Biológico , Cisteína , Modelos Animais de Doenças , Hemoglobinas/genética , Humanos , Hipóxia/sangue , Hipóxia/fisiopatologia , Preparação de Coração Isolado , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ativação Plaquetária , Ratos Sprague-Dawley , Vasodilatação , Função Ventricular Esquerda , Pressão Ventricular , Globinas beta/genéticaRESUMO
Background We aim to generate a line of "universal donor" human induced pluripotent stem cells (hi PSC s) that are nonimmunogenic and, therefore, can be used to derive cell products suitable for allogeneic transplantation. Methods and Results hi PSC s carrying knockout mutations for 2 key components (ß2 microglobulin and class II major histocompatibility class transactivator) of major histocompatibility complexes I and II (ie, human leukocyte antigen [HLA] I/ II knockout hi PSC s) were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene-editing system and differentiated into cardiomyocytes. Pluripotency-gene expression and telomerase activity in wild-type ( WT ) and HLAI / II knockout hi PSC s, cardiomyocyte marker expression in WT and HLAI / II knockout hi PSC -derived cardiomyocytes, and assessments of electrophysiological properties (eg, conduction velocity, action-potential and calcium transient half-decay times, and calcium transient increase times) in spheroid-fusions composed of WT and HLAI / II knockout cardiomyocytes, were similar. However, the rates of T-cell activation before (≈21%) and after (≈24%) exposure to HLAI / II knockout hi PSC -derived cardiomyocytes were nearly indistinguishable and dramatically lower than after exposure to WT hi PSC -derived cardiomyocytes (≈75%), and when WT and HLAI / II knockout hi PSC -derived cardiomyocyte spheroids were cultured with human peripheral blood mononuclear cells, the WT hi PSC -derived cardiomyocyte spheroids were smaller and displayed contractile irregularities. Finally, expression of HLA -E and HLA -F was inhibited in HLAI / II knockout cardiomyocyte spheroids after coculture with human peripheral blood mononuclear cells, although HLA -G was not inhibited; these results are consistent with the essential role of class II major histocompatibility class transactivator in transcriptional activation of the HLA -E and HLA-F genes, but not the HLA -G gene. Expression of HLA -G is known to inhibit natural killer cell recognition and killing of cells that lack other HLAs. Conclusions HLAI / II knockout hi PSC s can be differentiated into cardiomyocytes that induce little or no activity in human immune cells and, consequently, are suitable for allogeneic transplantation.
Assuntos
Genes MHC da Classe II/genética , Genes MHC Classe I/genética , Células-Tronco Pluripotentes Induzidas/transplante , Transplante de Células-Tronco/métodos , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Técnicas de Inativação de Genes , Humanos , Masculino , Miócitos Cardíacos/transplante , Transplante Homólogo/métodosRESUMO
Dynamic regulation of histone modification enzymes such as PRMT1 (protein arginine methyltransferase 1) determines the ordered epigenetic transitions in hematopoiesis. Sorting cells according to the expression levels of histone modification enzymes may further define subpopulations in hematopoietic lineages with unique differentiation potentials that are presently defined by surface markers. We discovered a vital near infrared dye, E84, that fluoresces brightly following binding to PRMT1 and excitation with a red laser. The staining intensity as measured by flow cytometry is correlated with the PRMT1 expression level. Importantly, E84 staining has no apparent negative effect on the proliferation of the labeled cells. Given that long-term hematopoietic stem cells (LT-HSCs) produce low levels of PRMT1, we used E84 to sort LT-HSCs from mouse bone marrow. We found that SLAM (the signalling lymphocyte activation molecule family) marker-positive LT-HSCs were enriched in the E84low cell fraction. We then performed bone marrow transplantations with E84high or E84low Lin-Sca1+Kit+ (LSK) cells and showed that whole blood cell lineages were successfully reconstituted 16 weeks after transplanting 200 E84low LSK cells. Thus, E84 is a useful new tool to probe the role of PRMT1 in hematopoiesis and leukemogenesis. Developing E84 and other small molecules to label histone modification enzymes provides a convenient approach without modifying gene loci to study the interaction between hematopoietic stem/progenitor cell epigenetic status and differentiation state.
Assuntos
Células Sanguíneas/metabolismo , Carbocianinas/química , Epigênese Genética , Corantes Fluorescentes/química , Proteína-Arginina N-Metiltransferases/genética , Animais , Ataxina-1/metabolismo , Células Sanguíneas/patologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Transplante de Medula Óssea , Linhagem da Célula , Citometria de Fluxo/métodos , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative for patients with sickle cell disease (SCD). However, morbidity associated with myeloablative conditioning and graft-versus-host disease has limited its utility. To this end, autologous HSCT for SCD using lentiviral gene-modified bone marrow (BM) or peripheral blood stem cells has been undertaken, although toxicities of fully ablative conditioning with busulfan and incomplete engraftment have been encountered. Treosulfan, a busulfan analog with a low extramedullary toxicity profile, has been used successfully as part of a myeloablative conditioning regimen in the allogeneic setting in SCD. To further minimize toxicity of conditioning, noncytotoxic monoclonal antibodies that clear stem cells from the marrow niche, such as anti-c-Kit (ACK2), have been considered. Using a murine model of SCD, we sought to determine whether nonmyeloablative conditioning followed by transplantation with syngeneic BM cells could ameliorate the disease phenotype. Treosulfan and ACK2, in a dose-dependent manner, decreased BM cellularity and induced cytopenia in SCD mice. Conditioning with treosulfan alone at nonmyeloablative dosing (3.6 g/kg), followed by transplantation with syngeneic BM donor cells, permitted long-term mixed-donor chimerism. Level of chimerism correlated with improvement in hematologic parameters, normalization of urine osmolality, and improvement in liver and spleen pathology. Addition of ACK2 to treosulfan conditioning did not enhance engraftment. Our data suggests that pretransplant conditioning with treosulfan alone may allow sufficient erythroid engraftment to reverse manifestations of SCD, with clinical application as a preparative regimen in SCD patients undergoing gene-modified autologous HSCT.
Assuntos
Anemia Falciforme/terapia , Transplante de Medula Óssea/métodos , Bussulfano/análogos & derivados , Condicionamento Pré-Transplante/métodos , Animais , Anticorpos/uso terapêutico , Antineoplásicos Alquilantes/uso terapêutico , Bussulfano/uso terapêutico , Modelos Animais de Doenças , Sobrevivência de Enxerto , Camundongos , Proteínas Proto-Oncogênicas c-kit/imunologia , Resultado do TratamentoRESUMO
Pluripotent stem cells (PSCs) deficient for microRNAs (miRNAs), such as Dgcr8-/- or Dicer-/- embryonic stem cells (ESCs), contain no mature miRNA and cannot differentiate into somatic cells. How miRNA deficiency causes differentiation defects remains poorly understood. Here, we report that miR-302 is sufficient to enable neural differentiation of differentiation-incompetent Dgcr8-/- ESCs. Our data showed that miR-302 directly suppresses the tumor suppressor p53, which is modestly upregulated in Dgcr8-/- ESCs and serves as a barrier restricting neural differentiation. We demonstrated that direct inactivation of p53 by SV40 large T antigen, a short hairpin RNA against Trp53, or genetic ablation of Trp53 in Dgcr8-/- PSCs enables neural differentiation, while activation of p53 by the MDM2 inhibitor nutlin-3a in wild-type ESCs inhibits neural differentiation. Together, we demonstrate that a major function of miRNAs in neural differentiation is suppression of p53 and that modest activation of p53 blocks neural differentiation of miRNA-deficient PSCs.
Assuntos
MicroRNAs/genética , Neurogênese , Células-Tronco Pluripotentes/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Cultivadas , Camundongos , MicroRNAs/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Sickle erythrocytes' (SSRBCs) unique physical adaptation to hypoxic conditions renders them able to home to hypoxic tumor niches in vivo, shut down tumor blood flow and induce tumoricidal responses. SSRBCs are also useful vehicles for transport of encapsulated drugs and oncolytic virus into hypoxic tumors with enhanced anti-tumor effects. In search of additional modes for arming sickle cells with cytotoxics, we turned to a lentiviral ß-globin vector with optimized Locus Control Region/ß-globin coding region/promoter/enhancers. We partially replaced the ß-globin coding region of this vector with genes encoding T cell cytolytics, perforin and granzyme or immune modulating superantigens SEG and SEI. These modified vectors efficiently transduced Sca+ ckit- Lin- hematopoietic stem cells (HSCs) from humanized sickle cell knockin mice. Irradiated mice reconstituted with these HSCs displayed robust expression of transgenic RNAs and proteins in host sickle cells that was sustained for more than 10 months. SSRBCs from reconstituted mice harboring SEG/SEI transgenes induced robust proliferation and a prototypical superantigen-induced cytokine reaction when exposed to human CD4+/CD8+ cells. The ß-globin lentiviral vector therefore produces a high level of functional, erythroid-specific immune modulators and cytotoxics that circulate without toxicity. Coupled with their unique ability to target and occlude hypoxic tumor vessels these armed SSRBCs constitute a potentially useful tool for treatment of solid tumors.
Assuntos
Anemia Falciforme , Citotoxicidade Imunológica , Eritrócitos Anormais/imunologia , Neoplasias Experimentais/imunologia , Neovascularização Patológica/imunologia , Globinas beta/genética , Anemia Falciforme/sangue , Animais , Citotoxicidade Imunológica/genética , Sistemas de Liberação de Medicamentos , Eritrócitos Anormais/metabolismo , Eritrócitos Anormais/transplante , Técnicas de Introdução de Genes , Vetores Genéticos , Transplante de Células-Tronco Hematopoéticas , Hipóxia , Lentivirus/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/terapia , Neovascularização Patológica/patologia , Neovascularização Patológica/terapiaRESUMO
Sickle cell disease (SCD)-associated nephropathy is a major source of morbidity and mortality in patients because of the lack of efficacious treatments targeting renal manifestations of the disease. Here, we describe a long-term treatment strategy with the selective endothelin-A receptor (ETA) antagonist, ambrisentan, designed to interfere with the development of nephropathy in a humanized mouse model of SCD. Ambrisentan preserved GFR at the level of nondisease controls and prevented the development of proteinuria, albuminuria, and nephrinuria. Microscopy studies demonstrated prevention of podocyte loss and structural alterations, the absence of vascular congestion, and attenuation of glomerulosclerosis in treated mice. Studies in isolated glomeruli showed that treatment reduced inflammation and oxidative stress. At the level of renal tubules, ambrisentan treatment prevented the increased excretion of urinary tubular injury biomarkers. Additionally, the treatment strategy prevented tubular brush border loss, diminished tubular iron deposition, blocked the development of interstitial fibrosis, and prevented immune cell infiltration. Furthermore, the prevention of albuminuria in treated mice was associated with preservation of cortical megalin expression. In a separate series of identical experiments, combined ETA and ETB receptor antagonism provided only some of the protection observed with ambrisentan, highlighting the importance of exclusively targeting the ETA receptor in SCD. Our results demonstrate that ambrisentan treatment provides robust protection from diverse renal pathologies in SCD mice, and suggest that long-term ETA receptor antagonism may provide a strategy for the prevention of renal complications of SCD.
Assuntos
Anemia Falciforme/complicações , Antagonistas do Receptor de Endotelina A/uso terapêutico , Nefropatias/etiologia , Nefropatias/prevenção & controle , Fenilpropionatos/uso terapêutico , Piridazinas/uso terapêutico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Fatores de TempoRESUMO
Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated by binding of chemoattractants to interleukin (IL) 8 receptors RA and RB (IL8RA/B) C-C chemokine receptors (CCR) 2 and 5 expressed on neutrophil and monocyte/macrophage membranes. Endothelial cells (ECs) derived from rat-induced pluripotent stem cells (RiPS) were transduced with adenovirus containing cDNA of IL8RA/B and/or CCR2/5. We hypothesized that RiPS-ECs overexpressing IL8RA/B (RiPS-IL8RA/B-ECs), CCR2/5 (RiPS-CCR2/5-ECs), or both receptors (RiPS-IL8RA/B+CCR2/5-ECs) will inhibit inflammatory responses and neointima formation in balloon-injured rat carotid artery. Twelve-week-old male Sprague-Dawley rats underwent balloon injury of the right carotid artery and intravenous infusion of (a) saline vehicle, (b) control RiPS-Null-ECs (ECs transduced with empty virus), (c) RiPS-IL8RA/B-ECs, (d) RiPS-CCR2/5-ECs, or (e) RiPS-IL8RA/B+CCR2/5-ECs. Inflammatory mediator expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 hours postinjury by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Neointima formation was assessed at 14 days postinjury. RiPS-ECs expressing the IL8RA/B or CCR2/5 homing device targeted the injured arteries and decreased injury-induced inflammatory cytokine expression, neutrophil/macrophage infiltration, and neointima formation. Transfused RiPS-ECs overexpressing IL8RA/B and/or CCR2/5 prevented inflammatory responses and neointima formation after vascular injury. Targeted delivery of iPS-ECs with a homing device to inflammatory mediators in injured arteries provides a novel strategy for the treatment of cardiovascular diseases. Stem Cells Translational Medicine 2017;6:1168-1177.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Receptores de Interleucina-8/metabolismo , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/terapia , Animais , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Neutrófilos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
In the failing heart, iNOS is expressed by both macrophages and cardiomyocytes. We hypothesized that inflammatory cell-localized iNOS exacerbates left ventricular (LV) remodeling. Wild-type (WT) C57BL/6 mice underwent total body irradiation and reconstitution with bone marrow from iNOS-/- mice (iNOS-/-c) or WT mice (WTc). Chimeric mice underwent coronary ligation to induce large infarction and ischemic heart failure (HF), or sham surgery. After 28 days, as compared with WTc sham mice, WTc HF mice exhibited significant (p < 0.05) mortality, LV dysfunction, hypertrophy, fibrosis, oxidative/nitrative stress, inflammatory activation, and iNOS upregulation. These mice also exhibited a ~twofold increase in circulating Ly6Chi pro-inflammatory monocytes, and ~sevenfold higher cardiac M1 macrophages, which were primarily CCR2- cells. In contrast, as compared with WTc HF mice, iNOS-/-c HF mice exhibited significantly improved survival, LV function, hypertrophy, fibrosis, oxidative/nitrative stress, and inflammatory activation, without differences in overall cardiac iNOS expression. Moreover, iNOS-/-c HF mice exhibited lower circulating Ly6Chi monocytes, and augmented cardiac M2 macrophages, but with greater infiltrating monocyte-derived CCR2+ macrophages vs. WTc HF mice. Lastly, upon cell-to-cell contact with naïve cardiomyocytes, peritoneal macrophages from WT HF mice depressed contraction, and augmented cardiomyocyte oxygen free radicals and peroxynitrite. These effects were not observed upon contact with macrophages from iNOS-/- HF mice. We conclude that leukocyte iNOS is obligatory for local and systemic inflammatory activation and cardiac remodeling in ischemic HF. Activated macrophages in HF may directly induce cardiomyocyte contractile dysfunction and oxidant stress upon cell-to-cell contact; this juxtacrine response requires macrophage-localized iNOS.
Assuntos
Insuficiência Cardíaca/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Remodelação Ventricular/fisiologia , Animais , Western Blotting , Ecocardiografia , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Imuno-Histoquímica , Isquemia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
One critical event in reprogramming to pluripotency is erasure of the somatic transcriptional program of starting cells. Here, we present the proof of principle of a strategy for reprogramming to pluripotency facilitated by small molecules that interfere with the somatic transcriptional memory. We show that mild chemical targeting of the acetyllysine-binding pockets of the BET bromodomains, the transcriptional bookmarking domains, robustly enhances reprogramming. Furthermore, we show that chemical targeting of the transcriptional bookmarking BET bromodomains downregulates or turns off the expression of somatic genes in both naive and reprogramming fibroblasts. Chemical blocking of the BET bromodomains also results in loss of fibroblast morphology early in reprogramming. We therefore experimentally demonstrate that cell fate conversion can be achieved by chemically targeting the transcriptional bookmarking BET bromodomains responsible for transcriptional memory.
Assuntos
Azepinas/farmacologia , Técnicas de Reprogramação Celular/métodos , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas/antagonistas & inibidores , Triazóis/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Insights from the study of cancer resistance in animals have led to the discovery of novel anticancer pathways and opened new venues for cancer prevention and treatment. Sickle cells (SSRBCs) from subjects with homozygous sickle cell anemia (SCA) have been shown to target hypoxic tumor niches, induce diffuse vaso-occlusion, and potentiate a tumoricidal response in a heme- and oxidant-dependent manner. These findings spawned the hypothesis that SSRBCs and the vasculopathic microenvironment of subjects with SCA might be inimical to tumor outgrowth and thereby constitute a natural antitumor defense. We therefore implanted the B16F10 melanoma into humanized hemoglobin SS knockin mice which exhibit the hematologic and vasculopathic sequelae of human SCA. Over the 31-day observation period, hemoglobin SS mice showed no significant melanoma outgrowth. By contrast, 68-100% of melanomas implanted in background and hemoglobin AA knockin control mice reached the tumor growth end point (p < 0.0001). SS knockin mice also exhibited established markers of underlying vasculopathy, e.g., chronic hemolysis (anemia, reticulocytosis) and vascular inflammation (leukocytosis) that differed significantly from all control groups. Genetic differences or normal AA gene knockin do not explain the impaired tumor outgrowth in SS knockin mice. These data point instead to the chronic pro-oxidative vasculopathic network in these mice as the predominant cause. In related studies, we demonstrate the ability of the sickle cell component of this system to function as a therapeutic vehicle in potentiating the oncolytic/vasculopathic effect of RNA reovirus. Sickle cells were shown to efficiently adsorb and transfer the virus to melanoma cells where it induced apoptosis even in the presence of anti-reovirus neutralizing antibodies. In vivo, SSRBCs along with their viral cargo rapidly targeted the tumor and initiated a tumoricidal response exceeding that of free virus and similarly loaded normal RBCs without toxicity. Collectively, these data unveil two hitherto unrecognized findings: hemoglobin SS knockin mice appear to present a natural barrier to melanoma tumorigenesis while SSRBCs demonstrate therapeutic function as a vehicle for enhancing the oncolytic effect of free reovirus against established melanoma.
RESUMO
CRISPR/Cas enhanced correction of the sickle cell disease (SCD) genetic defect in patient-specific induced Pluripotent Stem Cells (iPSCs) provides a potential gene therapy for this debilitating disease. An advantage of this approach is that corrected iPSCs that are free of off-target modifications can be identified before differentiating the cells into hematopoietic progenitors for transplantation. In order for this approach to be practical, iPSC generation must be rapid and efficient. Therefore, we developed a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector, rCLAE-R6, that delivers six reprogramming factors episomally. HDAd/EBV transduction of keratinocytes from SCD patients resulted in footprint-free iPSCs with high efficiency. Subsequently, the sickle mutation was corrected by delivering CRISPR/Cas9 with adenovirus followed by nucleoporation with a 70 nt single-stranded oligodeoxynucleotide (ssODN) correction template. Correction efficiencies of up to 67.9% (ß(A)/[ß(S)+ß(A)]) were obtained. Whole-genome sequencing (WGS) of corrected iPSC lines demonstrated no CRISPR/Cas modifications in 1467 potential off-target sites and no modifications in tumor suppressor genes or other genes associated with pathologies. These results demonstrate that adenoviral delivery of reprogramming factors and CRISPR/Cas provides a rapid and efficient method of deriving gene-corrected, patient-specific iPSCs for therapeutic applications.
Assuntos
Adenoviridae/metabolismo , Anemia Falciforme/genética , Anemia Falciforme/terapia , Sistemas CRISPR-Cas/genética , Terapia Genética , Vetores Genéticos/metabolismo , Vírus Auxiliares/metabolismo , Sequência de Bases , Linhagem Celular , Herpesvirus Humano 4 , Homozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismoRESUMO
Antisense RNAs regulate the transcription and translation of the corresponding sense genes. Here, we report that an antisense RNA, AS-RBM15, is transcribed in the opposite direction within exon 1 of RBM15 RBM15 is a regulator of megakaryocyte (MK) differentiation and is also involved in a chromosome translocation t(1;22) in acute megakaryocytic leukemia. MK terminal differentiation is enhanced by up-regulation of AS-RBM15 expression and attenuated by AS-RBM15 knockdown. At the molecular level, AS-RBM15 enhances RBM15 protein translation in a CAP-dependent manner. The region of the antisense AS-RBM15 RNA, which overlaps with the 5'UTR of RBM15, is sufficient for the up-regulation of RBM15 protein translation. In addition, we find that transcription of both RBM15 and AS-RBM15 is activated by the transcription factor RUNX1 and repressed by RUNX1-ETO, a leukemic fusion protein. Therefore, AS-RBM15 is a regulator of megakaryocyte differentiation and may play a regulatory role in leukemogenesis.