RESUMO
Rationale: Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice that were missing critical morphogenetic genes. Epithelial cell lineages were efficiently generated from ESC, but other cell types were mosaic. A complete contribution of donor ESCs to lung tissue has never been achieved. The mouse lung has never been generated in a rat. Objective: We sought to generate the mouse lung in a rat. Methods: Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat one-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESCs into Nkx2-1-deficient rat embryos with lung agenesis. The contribution of mouse ESCs to the lung tissue was examined by immunostaining, flow cytometry, and single-cell RNA sequencing. Measurements and Main Results: Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR-Cas9-mediated disruption of the Nkx2-1 gene. Complementation of rat Nkx2-1-/- blastocysts with mouse ESCs restored pulmonary and thyroid structures in mouse-rat chimeras, leading to a near-99% contribution of ESCs to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to those of respiratory cells from the normal mouse lung. Analysis of receptor-ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat. Conclusions: A combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as "bioreactors."
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Pulmão , Animais , Ratos , Edição de Genes/métodos , Pulmão/embriologia , Camundongos , Fator Nuclear 1 de Tireoide/genética , Células-Tronco EmbrionáriasRESUMO
Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal developmental disorder of lung morphogenesis caused by insufficiency of FOXF1 (forkhead box F1) transcription factor function. The cellular and transcriptional mechanisms by which FOXF1 deficiency disrupts human lung formation are unknown. Objectives: To identify cell types, gene networks, and cell-cell interactions underlying the pathogenesis of ACDMPV. Methods: We used single-nucleus RNA and assay for transposase-accessible chromatin sequencing, immunofluorescence confocal microscopy, and RNA in situ hybridization to identify cell types and molecular networks influenced by FOXF1 in ACDMPV lungs. Measurements and Main Results: Pathogenic single-nucleotide variants and copy-number variant deletions involving the FOXF1 gene locus in all subjects with ACDMPV (n = 6) were accompanied by marked changes in lung structure, including deficient alveolar development and a paucity of pulmonary microvasculature. Single-nucleus RNA and assay for transposase-accessible chromatin sequencing identified alterations in cell number and gene expression in endothelial cells (ECs), pericytes, fibroblasts, and epithelial cells in ACDMPV lungs. Distinct cell-autonomous roles for FOXF1 in capillary ECs and pericytes were identified. Pathogenic variants involving the FOXF1 gene locus disrupt gene expression in EC progenitors, inhibiting the differentiation or survival of capillary 2 ECs and cell-cell interactions necessary for both pulmonary vasculogenesis and alveolar type 1 cell differentiation. Loss of the pulmonary microvasculature was associated with increased VEGFA (vascular endothelial growth factor A) signaling and marked expansion of systemic bronchial ECs expressing COL15A1 (collagen type XV α 1 chain). Conclusions: Distinct FOXF1 gene regulatory networks were identified in subsets of pulmonary endothelial and fibroblast progenitors, providing both cellular and molecular targets for the development of therapies for ACDMPV and other diffuse lung diseases of infancy.
Assuntos
Síndrome da Persistência do Padrão de Circulação Fetal , Recém-Nascido , Humanos , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Redes Reguladoras de Genes/genética , Fator A de Crescimento do Endotélio Vascular/genética , Células Endoteliais/patologia , Multiômica , Pulmão/patologia , RNA , Fatores de Transcrição Forkhead/genéticaRESUMO
BACKGROUND: Pulmonary hypertension (PH) is a common complication in patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV), a severe congenital disorder associated with mutations in the FOXF1 gene. Although the loss of alveolar microvasculature causes PH in patients with ACDMPV, it is unknown whether increasing neonatal lung angiogenesis could prevent PH and right ventricular (RV) hypertrophy. METHODS: We used echocardiography, RV catheterization, immunostaining, and biochemical methods to examine lung and heart remodeling and RV output in Foxf1WT/S52F mice carrying the S52F Foxf1 mutation (identified in patients with ACDMPV). The ability of Foxf1WT/S52F mutant embryonic stem cells to differentiate into respiratory cell lineages in vivo was examined using blastocyst complementation. Intravascular delivery of nanoparticles with a nonintegrating Stat3 expression vector was used to improve neonatal pulmonary angiogenesis in Foxf1WT/S52F mice and determine its effects on PH and RV hypertrophy. RESULTS: Foxf1WT/S52F mice developed PH and RV hypertrophy after birth. The severity of PH in Foxf1WT/S52F mice directly correlated with mortality, low body weight, pulmonary artery muscularization, and increased collagen deposition in the lung tissue. Increased fibrotic remodeling was found in human ACDMPV lungs. Mouse embryonic stem cells carrying the S52F Foxf1 mutation were used to produce chimeras through blastocyst complementation and to demonstrate that Foxf1WT/S52F embryonic stem cells have a propensity to differentiate into pulmonary myofibroblasts. Intravascular delivery of nanoparticles carrying Stat3 cDNA protected Foxf1WT/S52F mice from RV hypertrophy and PH, improved survival, and decreased fibrotic lung remodeling. CONCLUSIONS: Nanoparticle therapies increasing neonatal pulmonary angiogenesis may be considered to prevent PH in ACDMPV.
Assuntos
Técnicas de Transferência de Genes , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/terapia , Nanopartículas , Síndrome da Persistência do Padrão de Circulação Fetal/complicações , Alvéolos Pulmonares/anormalidades , Fator de Transcrição STAT3/genética , Remodelação das Vias Aéreas/genética , Animais , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Ecocardiografia , Fibrose , Fatores de Transcrição Forkhead/deficiência , Terapia Genética , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/metabolismo , Hipertrofia Ventricular Direita/diagnóstico , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/metabolismo , Camundongos , Camundongos Transgênicos , Densidade Microvascular/genética , Miofibroblastos/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Fator de Transcrição STAT3/administração & dosagem , Nanomedicina Teranóstica/métodos , Resultado do Tratamento , Remodelação Vascular/genéticaRESUMO
Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1+cKIT+ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1+cKIT+ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1+cKIT+ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1+cKIT+ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1+cKIT+ endothelial progenitors may be considered for treatment of human ACDMPV.
Assuntos
Células-Tronco Embrionárias/citologia , Células Progenitoras Endoteliais/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Síndrome da Persistência do Padrão de Circulação Fetal/terapia , Transplante de Células-Tronco , Animais , Animais Recém-Nascidos , Sistemas CRISPR-Cas , Quimera , Modelos Animais de Doenças , Células-Tronco Embrionárias/metabolismo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/transplante , Fatores de Transcrição Forkhead/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Recém-Nascido , Camundongos , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Células-Tronco Pluripotentes , RNA-Seq , Ratos , Análise de Célula ÚnicaRESUMO
Rationale: The regeneration and replacement of lung cells or tissues from induced pluripotent stem cell- or embryonic stem cell-derived cells represent future therapies for life-threatening pulmonary disorders but are limited by technical challenges to produce highly differentiated cells able to maintain lung function. Functional lung tissue-containing airways, alveoli, vasculature, and stroma have never been produced via directed differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells. We sought to produce all tissue components of the lung from bronchi to alveoli by embryo complementation.Objectives: To determine whether ESCs are capable of generating lung tissue in Nkx2-1-/- mouse embryos with lung agenesis.Methods: Blastocyst complementation was used to produce chimeras from normal mouse ESCs and Nkx2-1-/- embryos, which lack pulmonary tissues. Nkx2-1-/- chimeras were examined using immunostaining, transmission electronic microscopy, fluorescence-activated cell sorter analysis, and single-cell RNA sequencing.Measurements and Main Results: Although peripheral pulmonary and thyroid tissues are entirely lacking in Nkx2-1 gene-deleted embryos, pulmonary and thyroid structures in Nkx2-1-/- chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of Nkx2-1-/- chimeras were derived almost entirely from ESCs, whereas endothelial, immune, and stromal cells were mosaic. ESC-derived cells from multiple respiratory cell lineages were highly differentiated and indistinguishable from endogenous cells based on morphology, ultrastructure, gene expression signatures, and cell surface proteins used to identify cell types by fluorescence-activated cell sorter.Conclusions: Lung and thyroid tissues were generated in vivo from ESCs by blastocyst complementation. Nkx2-1-/- chimeras can be used as "bioreactors" for in vivo differentiation and functional studies of ESC-derived progenitor cells.
Assuntos
Blastocisto/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Pneumopatias/terapia , Pulmão/crescimento & desenvolvimento , Glândula Tireoide/crescimento & desenvolvimento , Engenharia Tecidual/métodos , Animais , Diferenciação Celular/genética , Humanos , Camundongos , Modelos AnimaisRESUMO
Rationale: Advances in neonatal critical care have greatly improved the survival of preterm infants, but the long-term complications of prematurity, including bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. Although VEGF (vascular endothelial growth factor) improves lung structure and function in rodent BPD models, severe side effects of VEGF therapy prevent its use in patients with BPD.Objectives: To test whether nanoparticle delivery of proangiogenic transcription factor FOXM1 (forkhead box M1) or FOXF1 (forkhead box F1), both downstream targets of VEGF, can improve lung structure and function after neonatal hyperoxic injury.Methods: Newborn mice were exposed to 75% O2 for the first 7 days of life before being returned to a room air environment. On Postnatal Day 2, polyethylenimine-(5) myristic acid/polyethylene glycol-oleic acid/cholesterol nanoparticles containing nonintegrating expression plasmids with Foxm1 or Foxf1 cDNAs were injected intravenously. The effects of the nanoparticles on lung structure and function were evaluated using confocal microscopy, flow cytometry, and the flexiVent small-animal ventilator.Measurements and Main Results: The nanoparticles efficiently targeted endothelial cells and myofibroblasts in the alveolar region. Nanoparticle delivery of either FOXM1 or FOXF1 did not protect endothelial cells from apoptosis caused by hyperoxia but increased endothelial proliferation and lung angiogenesis after the injury. FOXM1 and FOXF1 improved elastin fiber organization, decreased alveolar simplification, and preserved lung function in mice reaching adulthood.Conclusions: Nanoparticle delivery of FOXM1 or FOXF1 stimulates lung angiogenesis and alveolarization during recovery from neonatal hyperoxic injury. Delivery of proangiogenic transcription factors has promise as a therapy for BPD in preterm infants.
Assuntos
Indutores da Angiogênese/administração & dosagem , Sistemas de Liberação de Medicamentos , Proteína Forkhead Box M1/administração & dosagem , Fatores de Transcrição Forkhead/administração & dosagem , Hiperóxia/tratamento farmacológico , Nanopartículas , Alvéolos Pulmonares/efeitos dos fármacos , Indutores da Angiogênese/farmacologia , Indutores da Angiogênese/uso terapêutico , Animais , Animais Recém-Nascidos , Western Blotting , Feminino , Citometria de Fluxo , Proteína Forkhead Box M1/farmacologia , Proteína Forkhead Box M1/uso terapêutico , Fatores de Transcrição Forkhead/farmacologia , Fatores de Transcrição Forkhead/uso terapêutico , Hiperóxia/patologia , Hiperóxia/fisiopatologia , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Alvéolos Pulmonares/irrigação sanguínea , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Resultado do TratamentoRESUMO
Rationale: Disruption of alveologenesis is associated with severe pediatric lung disorders, including bronchopulmonary dysplasia (BPD). Although c-KIT+ endothelial cell (EC) progenitors are abundant in embryonic and neonatal lungs, their role in alveolar septation and the therapeutic potential of these cells remain unknown.Objectives: To determine whether c-KIT+ EC progenitors stimulate alveologenesis in the neonatal lung.Methods: We used single-cell RNA sequencing of neonatal human and mouse lung tissues, immunostaining, and FACS analysis to identify transcriptional and signaling networks shared by human and mouse pulmonary c-KIT+ EC progenitors. A mouse model of perinatal hyperoxia-induced lung injury was used to identify molecular mechanisms that are critical for the survival, proliferation, and engraftment of c-KIT+ EC progenitors in the neonatal lung.Measurements and Main Results: Pulmonary c-KIT+ EC progenitors expressing PECAM-1, CD34, VE-Cadherin, FLK1, and TIE2 lacked mature arterial, venal, and lymphatic cell-surface markers. The transcriptomic signature of c-KIT+ ECs was conserved in mouse and human lungs and enriched in FOXF1-regulated transcriptional targets. Expression of FOXF1 and c-KIT was decreased in the lungs of infants with BPD. In the mouse, neonatal hyperoxia decreased the number of c-KIT+ EC progenitors. Haploinsufficiency or endothelial-specific deletion of Foxf1 in mice increased apoptosis and decreased proliferation of c-KIT+ ECs. Inactivation of either Foxf1 or c-Kit caused alveolar simplification. Adoptive transfer of c-KIT+ ECs into the neonatal circulation increased lung angiogenesis and prevented alveolar simplification in neonatal mice exposed to hyperoxia.Conclusions: Cell therapy involving c-KIT+ EC progenitors can be beneficial for the treatment of BPD.
Assuntos
Células Progenitoras Endoteliais/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Pulmão/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Recém-Nascido , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Técnicas de Cultura de TecidosRESUMO
Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital disorder causing respiratory failure and pulmonary hypertension shortly after birth. There are no effective treatments for ACDMPV other than lung transplant, and new therapeutic approaches are urgently needed. Although ACDMPV is linked to mutations in the FOXF1 gene, molecular mechanisms through which FOXF1 mutations cause ACDMPV are unknown.Objectives: To identify molecular mechanisms by which S52F FOXF1 mutations cause ACDMPV.Methods: We generated a clinically relevant mouse model of ACDMPV by introducing the S52F FOXF1 mutation into the mouse Foxf1 gene locus using CRISPR/Cas9 technology. Immunohistochemistry, whole-lung imaging, and biochemical methods were used to examine vasculature in Foxf1WT/S52F lungs and identify molecular mechanisms regulated by FOXF1.Measurements and Main Results: FOXF1 mutations were identified in 28 subjects with ACDMPV. Foxf1WT/S52F knock-in mice recapitulated histopathologic findings in ACDMPV infants. The S52F FOXF1 mutation disrupted STAT3-FOXF1 protein-protein interactions and inhibited transcription of Stat3, a critical transcriptional regulator of angiogenesis. STAT3 signaling and endothelial proliferation were reduced in Foxf1WT/S52F mice and human ACDMPV lungs. S52F FOXF1 mutant protein did not bind chromatin and was transcriptionally inactive. Furthermore, we have developed a novel formulation of highly efficient nanoparticles and demonstrated that nanoparticle delivery of STAT3 cDNA into the neonatal circulation restored endothelial proliferation and stimulated lung angiogenesis in Foxf1WT/S52F mice.Conclusions: FOXF1 acts through STAT3 to stimulate neonatal lung angiogenesis. Nanoparticle delivery of STAT3 is a promising strategy to treat ACDMPV associated with decreased STAT3 signaling.
Assuntos
Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Mutação , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/fisiopatologia , Alvéolos Pulmonares/anormalidades , Transdução de Sinais/genética , Animais , Humanos , Camundongos , Modelos Animais , Alvéolos Pulmonares/fisiopatologiaRESUMO
Hox transcription factors specify distinct cell types along the anterior-posterior axis of metazoans by regulating target genes that modulate signaling pathways. A well-established example is the induction of Epidermal Growth Factor (EGF) signaling by an Abdominal-A (Abd-A) Hox complex during the specification of Drosophila hepatocyte-like cells (oenocytes). Previous studies revealed that Abd-A is non-cell autonomously required to promote oenocyte fate by directly activating a gene (rhomboid) that triggers EGF secretion from sensory organ precursor (SOP) cells. Neighboring cells that receive the EGF signal initiate a largely unknown pathway to promote oenocyte fate. Here, we show that Abd-A also plays a cell autonomous role in inducing oenocyte fate by activating the expression of the Pointed-P1 (PntP1) ETS transcription factor downstream of EGF signaling. Genetic studies demonstrate that both PntP1 and PntP2 are required for oenocyte specification. Moreover, we found that PntP1 contains a conserved enhancer (PntP1OE) that is activated in oenocyte precursor cells by EGF signaling via direct regulation by the Pnt transcription factors as well as a transcription factor complex consisting of Abd-A, Extradenticle, and Homothorax. Our findings demonstrate that the same Abd-A Hox complex required for sending the EGF signal from SOP cells, enhances the competency of receiving cells to select oenocyte cell fate by up-regulating PntP1. Since PntP1 is a downstream effector of EGF signaling, these findings provide insight into how a Hox factor can both trigger and potentiate the EGF signal to promote an essential cell fate along the body plan.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Fator de Crescimento Epidérmico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Drosophila/enzimologia , Proteínas de Drosophila/genética , Elementos Facilitadores Genéticos , Fator de Crescimento Epidérmico/genética , Regulação da Expressão Gênica no Desenvolvimento , Hepatócitos/citologia , Hepatócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Órgãos dos Sentidos/crescimento & desenvolvimento , Fatores de Transcrição/genéticaRESUMO
The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis.
Assuntos
Diferenciação Celular/genética , Proteínas de Drosophila/genética , Mucosa Intestinal/metabolismo , Janus Quinases/genética , Proteínas de Membrana/genética , Receptores de Interleucina/genética , Animais , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Endocitose/genética , Regulação da Expressão Gênica no Desenvolvimento , Homeostase/genética , Intestinos/crescimento & desenvolvimento , Janus Quinases/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Células-TroncoRESUMO
Transcription of rRNA genes (rDNAs) in the nucleolus is regulated by epigenetic chromatin modifications including histone H3 lysine (de)methylation. Here we show that LegAS4, a Legionella pneumophila type IV secretion system (TFSS) effector, is targeted to specific rDNA chromatin regions in the host nucleolus. LegAS4 promotes rDNA transcription, through its SET-domain (named after Drosophila Su(var)3-9, enhancer of zeste [E(z)], and trithorax [trx]) histone lysine methyltransferase (HKMTase) activity. LegAS4's association with rDNA chromatin is mediated by interaction with host HP1α/γ. L. pneumophila infection potently activates rDNA transcription in a TFSS-dependent manner. Other bacteria, including Bordetella bronchiseptica and Burkholderia thailandensis, also harbour nucleolus-localized LegAS4-like HKMTase effectors. The B. thailandensis type III effector BtSET promotes H3K4 methylation of rDNA chromatin, contributing to infection-induced rDNA transcription and bacterial intracellular replication. Thus, activation of host rDNA transcription might be a general bacterial virulence strategy.
Assuntos
Proteínas Cromossômicas não Histona/genética , DNA Ribossômico/genética , Epigênese Genética , Interações Hospedeiro-Patógeno/genética , Legionella pneumophila/patogenicidade , Transcrição Gênica , Sequência de Aminoácidos , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/patogenicidade , Burkholderia/genética , Burkholderia/patogenicidade , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , DNA Ribossômico/metabolismo , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Legionella pneumophila/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Células U937RESUMO
Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing Hes1, Dntt and Ebf1, along with mesenchymal stromal cells expressing Cpox, Blvrb and Ermap that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.
RESUMO
Mutations in the FOXF1 gene, a key transcriptional regulator of pulmonary vascular development, cause Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins, a lethal lung disease affecting newborns and infants. Identification of new FOXF1 upstream regulatory elements is critical to explain why frequent non-coding FOXF1 deletions are linked to the disease. Herein, we use multiome single-nuclei RNA and ATAC sequencing of mouse and human patient lungs to identify four conserved endothelial and mesenchymal FOXF1 enhancers. We demonstrate that endothelial FOXF1 enhancers are autoactivated, whereas mesenchymal FOXF1 enhancers are regulated by EBF1 and GLI1. The cell-specificity of FOXF1 enhancers is validated by disrupting these enhancers in mouse embryonic stem cells using CRISPR/Cpf1 genome editing followed by lineage-tracing of mutant embryonic stem cells in mouse embryos using blastocyst complementation. This study resolves an important clinical question why frequent non-coding FOXF1 deletions that interfere with endothelial and mesenchymal enhancers can lead to the disease.
Assuntos
Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead , Mesoderma , Síndrome da Persistência do Padrão de Circulação Fetal , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Animais , Humanos , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/patologia , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Camundongos , Elementos Facilitadores Genéticos/genética , Mesoderma/metabolismo , Mesoderma/embriologia , Pulmão/patologia , Células Endoteliais/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Células-Tronco Embrionárias/metabolismo , Alvéolos Pulmonares/anormalidadesRESUMO
Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/ß-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/ß-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/ß-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.
Assuntos
Células Endoteliais , Fatores de Transcrição Forkhead , Receptores Frizzled , Neoplasias Pulmonares , Animais , Humanos , Camundongos , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Progressão da Doença , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Receptores Frizzled/metabolismo , Receptores Frizzled/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Neovascularização Patológica/genética , Via de Sinalização WntRESUMO
Pathogenic bacteria deliver effector proteins into host cells through the type III secretion apparatus to modulate the host function. We identify a family of proteins, homologous to the type III effector Cif from enteropathogenic Escherichia coli, in pathogens including Yersinia, Photorhabdus, and Burkholderia that contain functional type III secretion systems. Like Cif, this family of proteins is capable of arresting the host cell cycle at G(2)/M. Structure of one of the family members, Cif homolog in Burkholderia pseudomallei (CHBP), reveals a papain-like fold and a conserved Cys-His-Gln catalytic triad despite the lack of primary sequence identity. For CHBP and Cif, only the putative catalytic Cys is susceptible to covalent modification by E-64, a specific inhibitor of papain-like cysteine proteases. Unlike papain-like enzymes where the S2 site is the major determinant of cleavage-site specificity, CHBP has a characteristic negatively charged pocket occupying surface areas corresponding to the S1/S1' site in papain-like proteases. The negative charge is provided by a conserved aspartate, and the pocket best fits an arginine, as revealed by molecular docking analysis. Mutation analysis establishes the essential role of the catalytic triad and the negatively charged pocket in inducing cell cycle arrest in host cells. Our results demonstrate that bacterial pathogens have evolved a unique papain-like hydrolytic activity to block the normal host cell cycle progression.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/enzimologia , Ciclo Celular , Papaína/metabolismo , Sequência de Aminoácidos , Ácido Aspártico , Sítios de Ligação , Biocatálise , Cisteína Endopeptidases , Células HeLa , Humanos , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Pulmonary endothelial progenitor cells (EPCs) are critical for neonatal lung angiogenesis and represent a subset of general capillary cells (gCAPs). Molecular mechanisms through which EPCs stimulate lung angiogenesis are unknown. Herein, we used single-cell RNA sequencing to identify the BMP9/ACVRL1/SMAD1 pathway signature in pulmonary EPCs. BMP9 receptor, ACVRL1, and its downstream target genes were inhibited in EPCs from Foxf1WT/S52F mutant mice, a model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Expression of ACVRL1 and its targets were reduced in lungs of ACDMPV subjects. Inhibition of FOXF1 transcription factor reduced BMP9/ACVRL1 signaling and decreased angiogenesis in vitro. FOXF1 synergized with ETS transcription factor FLI1 to activate ACVRL1 promoter. Nanoparticle-mediated silencing of ACVRL1 in newborn mice decreased neonatal lung angiogenesis and alveolarization. Treatment with BMP9 restored lung angiogenesis and alveolarization in ACVRL1-deficient and Foxf1WT/S52F mice. Altogether, EPCs promote neonatal lung angiogenesis and alveolarization through FOXF1-mediated activation of BMP9/ACVRL1 signaling.
Assuntos
Células Progenitoras Endoteliais , Síndrome da Persistência do Padrão de Circulação Fetal , Pneumonia , Animais , Camundongos , Receptores de Activinas Tipo II/metabolismo , Células Progenitoras Endoteliais/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Pulmão/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/genética , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Pneumonia/metabolismo , Alvéolos Pulmonares/anormalidadesRESUMO
Generation of bone marrow (BM) from embryonic stem cells (ESCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce BM cell lineages from mouse ESCs in a rat. Based on fluorescence-activated cell sorting analysis and single-cell RNA sequencing, mouse ESCs differentiated into multiple hematopoietic and stromal cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Receptor-ligand interactions identified Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44, and App-Il18rap as major signaling pathways between hematopoietic progenitors and stromal cells. Multiple hematopoietic progenitors, including hematopoietic stem cells (HSCs) in mouse-rat chimeras derived more efficiently from mouse ESCs, whereas chondrocytes predominantly derived from rat cells. In the dorsal aorta and fetal liver of mouse-rat chimeras, mouse HSCs emerged and expanded faster compared to endogenous rat cells. Sequential BM transplantation of ESC-derived cells from mouse-rat chimeras rescued lethally irradiated syngeneic mice and demonstrated long-term reconstitution potential of donor HSCs. Altogether, a fully functional BM was generated from mouse ESCs using rat embryos as 'bioreactors'.
Assuntos
Medula Óssea , Transplante de Células-Tronco Hematopoéticas , Camundongos , Animais , Ratos , Medula Óssea/fisiologia , Antígeno CD47 , Quimera , Ligantes , Células-Tronco Embrionárias , Células da Medula ÓsseaRESUMO
Isolate of Amanita spissa was obtained from basidiome stipe material collected from environment. It could utilize a broad range of carbon and nitrogen resources. Study on the influence of different conditions for solid culture was carried out. Optimal culture conditions were at 28 degrees C, pH6, in the dark. A. spissa was then fermentated in liquid culture for more mycelia. In flask and Airlift/ff bioreactor, maximum dry mycelia weight of A. spissa reached 0.893 g/L and 2.33 g/L, respectively. Mycelia obtained from solid culture and Airlift/ff bioreactor were then analyzed by HPLC. The results showed that mycelia from both cultures contained amatoxins but no phallotoxins. alpha-Amanitin in mycelia reached 26.02 microg/DWg under solid culture condition, and 15.25 microg/DWg under liquid culture condition. The amanitins were also confirmed by bud-inhibited assay. The results revealed that the effect of amanitin on mung bean cell was identical to that of authentic amanitins. This work suggests that it is possible to produce amatoxin by liquid culturing of A. spissa.
Assuntos
Amanita/crescimento & desenvolvimento , Amanitinas/análise , Técnicas de Cultura/métodos , Amanita/química , Amanita/efeitos dos fármacos , Amanitinas/isolamento & purificação , Amanitinas/toxicidade , Reatores Biológicos , Carbono/farmacologia , Cromatografia Líquida de Alta Pressão , Escuridão , Fabaceae/efeitos dos fármacos , Fabaceae/crescimento & desenvolvimento , Fermentação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Micélio/química , Micélio/efeitos dos fármacos , Micélio/crescimento & desenvolvimento , Nitrogênio/farmacologia , TemperaturaRESUMO
The liquid culture of Laetiporus sulphureus var. sulphureus was lethal against fruit fly. It was found that extracellular metabolites were primary causation of the lethal effect against fruit fly, which was influenced by pH value. Isolation and analysis with ion-exchange resin column chromatography and HPLC demonstrated that oxalic acid was present in supernatant of Laetiporus sulphureus var. sulphureus, and it was one of the contributing factors to lethal effect against fruit fly and decrease of pH value of culture system. When cultured in airlift reactor, concentration of oxalic acid, quantity of mycelia and pH value was correlated with each other. Further analysis on elution revealed that a kind of oligidic pigment of amaranth in alkaline condition were also lethal against fruit fly.
Assuntos
Polyporaceae/metabolismo , Animais , Drosophila melanogaster/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Ácido Oxálico/análise , Ácido Oxálico/farmacologia , Polyporaceae/crescimento & desenvolvimento , Polyporaceae/patogenicidadeRESUMO
Hedgehog (Hh) acts as a morphogen to activate the transcription of diverse target genes via its downstream effector Cubitus interruptus (Ci). Currently, it is less understood how Ci recruits co-factors to activate transcription. Here we report that hyperplastic discs (hyd), an E3 ubiquitin ligase, can differentially regulate the transcriptional outputs of Hh signaling. We show that loss of Hyd activity caused upregulation of some, but not all of Hh target genes. Importantly, Hyd does not affect the stability of Ci. Our data suggest that Hyd differentially restrains the transcriptional activity of Ci via selective association with respective promoters.