RESUMO
Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analog misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.
Assuntos
Proteínas 14-3-3/metabolismo , Cardiopatias/tratamento farmacológico , Proteínas de Membrana/metabolismo , Misoprostol/uso terapêutico , Proteínas Mitocondriais/metabolismo , Ocitócicos/uso terapêutico , Animais , Modelos Animais de Doenças , Humanos , Misoprostol/farmacologia , Ocitócicos/farmacologia , Ratos , TransfecçãoRESUMO
OBJECTIVE: We aimed to evaluate the use of miR-200b as a prenatal transplacental therapy in the nitrofen rat model of abnormal lung development and congenital diaphragmatic hernia (CDH). BACKGROUND: Pulmonary hypoplasia (PH) and pulmonary hypertension determine mortality and morbidity in CDH babies. There is no safe medical prenatal treatment available. We previously discovered that higher miR-200b is associated with better survival in CDH babies. Here, we investigate the role of miR-200b in the nitrofen rat model of PH and CDH and evaluate its use as an in vivo prenatal therapy. METHODS: We profiled miR-200b expression during nitrofen-induced PH using RT-qPCR and in situ hybridization in the nitrofen rat model of PH and CDH. The effects of nitrofen on downstream miR-200b targets were studied in bronchial lung epithelial cells using a SMAD luciferase assay, Western blotting and Immunohistochemistry. We evaluated miR-200b as a lung growth promoting therapy ex vivo and in vivo using lung explant culture and transplacental prenatal therapy in the nitrofen rat model. RESULTS: We show that late lung hypoplasia in CDH is associated with (compensatory) upregulation of miR-200b in less hypoplastic lungs. Increasing miR-200b abundance with mimics early after nitrofen treatment decreases SMAD-driven TGF-ß signaling and rescues lung hypoplasia both in vitro and in vivo. Also, prenatal miR-200b therapy decreases the observed incidence of CDH. CONCLUSIONS: Our data indicate that miR-200b improves PH and decreases the incidence of CDH. Future studies will further exploit this newly discovered prenatal therapy for lung hypoplasia and CDH.
Assuntos
Anormalidades Múltiplas/terapia , Terapias Fetais/métodos , Hérnias Diafragmáticas Congênitas/terapia , Pneumopatias/terapia , Pulmão/anormalidades , MicroRNAs/uso terapêutico , 2,4-Dinitrofenol/administração & dosagem , Anormalidades Múltiplas/genética , Animais , Modelos Animais de Doenças , Hérnias Diafragmáticas Congênitas/induzido quimicamente , Hérnias Diafragmáticas Congênitas/complicações , Hérnias Diafragmáticas Congênitas/genética , Pneumopatias/complicações , Pneumopatias/genética , Ratos , Ratos Sprague-DawleyRESUMO
miR-200b plays a role in epithelial-to-mesenchymal transition (EMT) in cancer. We recently reported abnormal expression of miR-200b in the context of human pulmonary hypoplasia in congenital diaphragmatic hernia (CDH). Smaller lung size, a lower number of airway generations, and a thicker mesenchyme characterize pulmonary hypoplasia in CDH. The aim of this study was to define the role of miR-200b during lung development. Here we show that miR-200b-/- mice have abnormal lung function due to dysfunctional surfactant, increased fibroblast-like cells and thicker mesenchyme in between the alveolar walls. We profiled the lung transcriptome in miR-200b-/- mice, and, using Gene Ontology analysis, we determined that the most affected biological processes include cell cycle, apoptosis and protein transport. Our results demonstrate that miR-200b regulates distal airway development through maintaining an epithelial cell phenotype. The lung abnormalities observed in miR-200b-/- mice recapitulate lung hypoplasia in CDH.