RESUMEN

The lung macrophages play a crucial role in health and disease. Sexual dimorphism significantly impacts the phenotype and function of tissue-resident macrophages. The primary mechanisms responsible for sexually dimorphic outcomes in bronchopulmonary dysplasia (BPD) remain unidentified. We tested the hypothesis that biological sex plays a crucial role in the transcriptional state of alveolar macrophages, using neonatal murine hyperoxia-induced lung injury as a relevant model for human BPD. The effects of neonatal hyperoxia exposure (95 % FiO2, PND1-5: saccular stage) on the lung myeloid cells acutely after injury and during normoxic recovery were measured. Alveolar macrophages (AM) from room air- and hyperoxia exposed from male and female neonatal murine lungs were subjected to bulk-RNA Sequencing. AMs are significantly depleted in the hyperoxia-exposed lung acutely after injury, with subsequent recovery in both sexes. The transcriptome of the alveolar macrophages is impacted by neonatal hyperoxia exposure and by sex as a biological variable. Pathways related to DNA damage and interferon-signaling were positively enriched in female AMs. Metabolic pathways related to glucose and carbohydrate metabolism were positively enriched in the male AMs, while oxidative phosphorylation was negatively enriched. These pathways were shared with monocytes and airway macrophages from intubated male and female human premature neonates.

Asunto(s)

Animales Recién Nacidos , Hiperoxia , Macrófagos Alveolares , Femenino , Animales , Masculino , Macrófagos Alveolares/metabolismo , Ratones , Hiperoxia/metabolismo , Humanos , Transcriptoma , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patología , Displasia Broncopulmonar/etiología , Caracteres Sexuales , Factores Sexuales , Modelos Animales de Enfermedad , Recién Nacido , Pulmón/metabolismo , Pulmón/patología , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Lesión Pulmonar/etiología

RESUMEN

BACKGROUND: Bronchopulmonary dysplasia (BPD) is characterized by an arrest in lung development and is a leading cause of morbidity in premature neonates. It has been well documented that BPD disproportionally affects males compared to females, but the molecular mechanisms behind this sex-dependent bias remain unclear. Female mice show greater preservation of alveolarization and angiogenesis when exposed to hyperoxia, accompanied by increased miR-30a expression. In this investigation, we tested the hypothesis that loss of miR-30a would result in male and female mice experiencing similar impairments in alveolarization and angiogenesis under hyperoxic conditions. METHODS: Wild-type and miR-30a-/- neonatal mice were exposed to hyperoxia [95% FiO2, postnatal day [PND1-5] or room air before being euthanized on PND21. Alveolarization, pulmonary microvascular development, differences in lung transcriptome, and miR-30a expression were assessed in lungs from WT and miR-30a-/- mice of either sex. Blood transcriptomic signatures from preterm newborns (with and without BPD) were correlated with WT and miR-30a-/- male and female lung transcriptome data. RESULTS: Significantly, the sex-specific differences observed in WT mice were abrogated in the miR-30a-/- mice upon exposure to hyperoxia. The loss of miR-30a expression eliminated the protective effect in females, suggesting that miR-30a plays an essential role in regulating alveolarization and angiogenesis. Transcriptome analysis by whole lung RNA-Seq revealed a significant response in the miR-30a-/- female hyperoxia-exposed lung, with enrichment of pathways related to cell cycle and neuroactive ligand-receptor interaction. Gene expression signature in the miR-30a-/- female lung associated with human BPD blood transcriptomes. Finally, we showed the spatial localization of miR-30a transcripts in the bronchiolar epithelium. CONCLUSIONS: miR-30a could be one of the biological factors mediating the resilience of the female preterm lung to neonatal hyperoxic lung injury. A better understanding of the effects of miR-30a on pulmonary angiogenesis and alveolarization may lead to novel therapeutics for treating BPD.

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Bronchopulmonary dysplasia (BPD) is a lung condition that affects babies born prematurely, causing problems with their lung development. Interestingly, BPD tends to affect boys more than girls, but we do not fully understand why. To investigate this, we conducted a study using mice. Female mice had better lung development and blood vessel formation when exposed to high oxygen levels. We found higher expression of a molecule called miR-30a in the female mice and seemed to be protective. So, we wanted to see if removing miR-30a would have the same effect on both male and female mice. To test this, we exposed newborn mice without miR-30a and normal mice to high oxygen levels or regular room air. Interestingly, the differences between normal males and females were no longer present in the mice without miR-30a. This suggested that miR-30a plays an important role in lung development. We also identified that the female mice without miR-30a, when exposed to high oxygen, had the greatest number of genes affected, and these gene changes were like those seen in blood samples from premature babies with BPD. Finally, we report that miR-30a was in a specific part of the lung called the bronchiolar epithelium. Overall, this study suggests that miR-30a is crucial in protecting premature lungs from damage caused by high oxygen levels. By understanding how miR-30a affects lung development, we may be able to develop new treatments for BPD in the future.

Asunto(s)

Displasia Broncopulmonar , Hiperoxia , Lesión Pulmonar , MicroARNs , Animales , Femenino , Masculino , Ratones , Animales Recién Nacidos , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Hiperoxia/complicaciones , Hiperoxia/metabolismo , Pulmón/metabolismo , Lesión Pulmonar/genética , Lesión Pulmonar/complicaciones , Lesión Pulmonar/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Factores Sexuales

RESUMEN

Recovery from lung injury during the neonatal period requires the orchestration of many biological pathways. The modulation of such pathways can drive the developing lung towards proper repair or persistent maldevelopment that can lead to a disease phenotype. Sex as a biological variable can regulate these pathways differently in the male and female lung exposed to neonatal hyperoxia. In this study, we assessed the contribution of cellular diversity in the male and female neonatal lung following injury. Our objective was to investigate sex and cell-type specific transcriptional changes that drive repair or persistent injury in the neonatal lung and delineate the alterations in the immune-endothelial cell communication networks using single cell RNA sequencing (sc-RNAseq) in a murine model of hyperoxic injury. We generated transcriptional profiles of >55,000 cells isolated from the lungs of postnatal day 1 (PND 1) and postnatal day 21 (PND 21) neonatal male and female C57BL/6 mice exposed to 95% FiO 2 between PND 1-5 (saccular stage of lung development). We show the presence of sex-based differences in the transcriptional states of lung endothelial and immune cells at PND 1 and PND 21. Furthermore, we demonstrate that biological sex significantly influences the response to injury, with a greater number of differentially expressed genes showing sex-specific patterns than those shared between male and female lungs. Pseudotime trajectory analysis highlighted genes needed for lung development that were altered by hyperoxia. Finally, we show intercellular communication between endothelial and immune cells at saccular and alveolar stages of lung development with sex-based biases in the crosstalk and identify novel ligand-receptor pairs. Our findings provide valuable insights into the cell diversity, transcriptional state, developmental trajectory, and cell-cell communication underlying neonatal lung injury, with implications for understanding lung development and possible therapeutic interventions while highlighting the crucial role of sex as a biological variable.

RESUMEN

Recovery from lung injury during the neonatal period requires the orchestration of many biological pathways. The modulation of such pathways can drive the developing lung towards proper repair or persistent maldevelopment that can lead to a disease phenotype. Sex as a biological variable can regulate these pathways differently in the male and female lung exposed to neonatal hyperoxia. In this study, we assessed the contribution of cellular diversity in the male and female neonatal lung following injury. Our objective was to investigate sex and cell-type specific transcriptional changes that drive repair or persistent injury in the neonatal lung and delineate the alterations in the immune-endothelial cell communication networks using single cell RNA sequencing (sc-RNAseq) in a murine model of hyperoxic injury. We generated transcriptional profiles of >55,000 cells isolated from the lungs of postnatal day 1 (PND 1; pre-exposure), PND 7, and PND 21neonatal male and female C57BL/6 mice exposed to 95 % FiO2 between PND 1-5 (saccular stage of lung development). We show the presence of sex-based differences in the transcriptional states of lung endothelial and immune cells at PND 1 and PND 21. Furthermore, we demonstrate that biological sex significantly influences the response to injury, with a greater number of differentially expressed genes showing sex-specific patterns than those shared between male and female lungs. Pseudotime trajectory analysis highlighted genes needed for lung development that were altered by hyperoxia. Finally, we show intercellular communication between endothelial and immune cells at saccular and alveolar stages of lung development with sex-based biases in the crosstalk and identify novel ligand-receptor pairs. Our findings provide valuable insights into the cell diversity, transcriptional state, developmental trajectory, and cell-cell communication underlying neonatal lung injury, with implications for understanding lung development and possible therapeutic interventions while highlighting the crucial role of sex as a biological variable.

Asunto(s)

Animales Recién Nacidos , Hiperoxia , Lesión Pulmonar , Animales , Femenino , Masculino , Ratones , Hiperoxia/metabolismo , Hiperoxia/genética , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Lesión Pulmonar/genética , Lesión Pulmonar/etiología , Pulmón/metabolismo , Pulmón/patología , Factores Sexuales , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Transcriptoma , Perfilación de la Expresión Génica , Análisis de la Célula Individual

RESUMEN

While changes in MeCP2 dosage cause Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), its transcriptional regulation is poorly understood. Here, we identified six putative noncoding regulatory elements of Mecp2, two of which are conserved in humans. Upon deletion in mice and human iPSC-derived neurons, these elements altered RNA and protein levels in opposite directions and resulted in a subset of RTT- and MDS-like behavioral deficits in mice. Our discovery provides insight into transcriptional regulation of Mecp2/MECP2 and highlights genomic sites that could serve as diagnostic and therapeutic targets in RTT or MDS.

Asunto(s)

Regulación de la Expresión Génica/genética , Discapacidad Intelectual Ligada al Cromosoma X/genética , Proteína 2 de Unión a Metil-CpG/genética , Neuronas/patología , Elementos Reguladores de la Transcripción/genética , Síndrome de Rett/genética , Animales , Conducta Animal/fisiología , Secuencia Conservada/genética , Eliminación de Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados

RESUMEN

RATIONALE: We previously identified somatic activating mutations in the KRAS (Kirsten rat sarcoma viral oncogene homologue) gene in the endothelium of the majority of human sporadic brain arteriovenous malformations; a disorder characterized by direct connections between arteries and veins. However, whether this genetic abnormality alone is sufficient for lesion formation, as well as how active KRAS signaling contributes to arteriovenous malformations, remains unknown. OBJECTIVE: To establish the first in vivo models of somatic KRAS gain of function in the endothelium in both mice and zebrafish to directly observe the phenotypic consequences of constitutive KRAS activity at a cellular level in vivo, and to test potential therapeutic interventions for arteriovenous malformations. METHODS AND RESULTS: Using both postnatal and adult mice, as well as embryonic zebrafish, we demonstrate that endothelial-specific gain of function mutations in Kras (G12D or G12V) are sufficient to induce brain arteriovenous malformations. Active KRAS signaling leads to altered endothelial cell morphogenesis and increased cell size, ectopic sprouting, expanded vessel lumen diameter, and direct connections between arteries and veins. Furthermore, we show that these lesions are not associated with altered endothelial growth dynamics or a lack of proper arteriovenous identity but instead seem to feature exuberant angiogenic signaling. Finally, we demonstrate that KRAS-dependent arteriovenous malformations in zebrafish are refractory to inhibition of the downstream effector PI3K but instead require active MEK (mitogen-activated protein kinase kinase 1) signaling. CONCLUSIONS: We demonstrate that active KRAS expression in the endothelium is sufficient for brain arteriovenous malformations, even in the setting of uninjured adult vasculature. Furthermore, the finding that KRAS-dependent lesions are reversible in zebrafish suggests that MEK inhibition may represent a promising therapeutic treatment for arteriovenous malformation patients. Graphical Abstract: A graphical abstract is available for this article.

Asunto(s)

Células Endoteliales/enzimología , Mutación con Ganancia de Función , Malformaciones Arteriovenosas Intracraneales/genética , MAP Quinasa Quinasa 1/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Predisposición Genética a la Enfermedad , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Malformaciones Arteriovenosas Intracraneales/enzimología , Malformaciones Arteriovenosas Intracraneales/patología , Hemorragias Intracraneales/enzimología , Hemorragias Intracraneales/genética , Hemorragias Intracraneales/patología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Masculino , Ratones Transgénicos , Permeabilidad , Fenotipo , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra

RESUMEN

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

Asunto(s)

Elementos Transponibles de ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Biblioteca de Genes , Mutagénesis Insercional , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Western Blotting , Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiología , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Larva/genética , Larva/metabolismo , Aprendizaje/fisiología , Microscopía Confocal , Factores de Tiempo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , alfa Catenina/genética , alfa Catenina/metabolismo