RESUMEN
Bronchopulmonary dysplasia (BPD) is the most common lung disease of extreme prematurity, yet mechanisms that associate with or identify neonates with increased susceptibility for BPD are largely unknown. Combining artificial intelligence with gene expression data is a novel approach that may assist in better understanding mechanisms underpinning chronic lung disease and in stratifying patients at greater risk for BPD. The objective of this study is to develop an early peripheral blood transcriptomic signature that can predict preterm neonates at risk for developing BPD. Secondary analysis of whole blood microarray data from 97 very low birth weight neonates on day of life 5 was performed. BPD was defined as positive pressure ventilation or oxygen requirement at 28 days of age. Participants were randomly assigned to a training (70%) and testing cohort (30%). Four gene-centric machine learning models were built, and their discriminatory abilities were compared with gestational age or birth weight. This study adheres to the transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD) statement. Neonates with BPD (n = 62 subjects) exhibited a lower median gestational age (26.0 wk vs. 30.0 wk, P < 0.01) and birth weight (800 g vs. 1,280 g, P < 0.01) compared with non-BPD neonates. From an initial pool (33,252 genes/patient), 4,523 genes exhibited a false discovery rate (FDR) <1%. The area under the receiver operating characteristic curve (AUC) for predicting BPD utilizing gestational age or birth weight was 87.8% and 87.2%, respectively. The machine learning models, using a combination of five genes, revealed AUCs ranging between 85.8% and 96.1%. Pathways integral to T cell development and differentiation were associated with BPD. A derived five-gene whole blood signature can accurately predict BPD in the first week of life.
Asunto(s)
Displasia Broncopulmonar , Recién Nacido , Humanos , Displasia Broncopulmonar/diagnóstico , Displasia Broncopulmonar/genética , Peso al Nacer , Transcriptoma/genética , Inteligencia Artificial , Recien Nacido Prematuro , Edad GestacionalRESUMEN
BACKGROUND: Although studies involving preterm infants ≤34 weeks gestation report a decreased incidence of patent ductus arteriosus after antenatal betamethasone, studies involving younger gestation infants report conflicting results. METHODS: We used preterm baboons, mice, and humans (≤276/7 weeks gestation) to examine betamethasone's effects on ductus gene expression and constriction both in vitro and in vivo. RESULTS: In mice, betamethasone increased the sensitivity of the premature ductus to the contractile effects of oxygen without altering the effects of other contractile or vasodilatory stimuli. Betamethasone's effects on oxygen sensitivity could be eliminated by inhibiting endogenous prostaglandin/nitric oxide signaling. In mice and baboons, betamethasone increased the expression of several developmentally regulated genes that mediate oxygen-induced constriction (K+ channels) and inhibit vasodilator signaling (phosphodiesterases). In human infants, betamethasone increased the rate of ductus constriction at all gestational ages. However, in infants born ≤256/7 weeks gestation, betamethasone's contractile effects were only apparent when prostaglandin signaling was inhibited, whereas at 26-27 weeks gestation, betamethasone's contractile effects were apparent even in the absence of prostaglandin inhibitors. CONCLUSIONS: We speculate that betamethasone's contractile effects may be mediated through genes that are developmentally regulated. This could explain why betamethasone's effects vary according to the infant's developmental age at birth.
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Betametasona/uso terapéutico , Conducto Arterioso Permeable/tratamiento farmacológico , Conducto Arterial/efectos de los fármacos , Animales , Ecocardiografía , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Recien Nacido Prematuro , Exposición Materna , Ratones , Oxígeno/metabolismo , Papio , Reacción en Cadena de la Polimerasa , Prostaglandinas/metabolismoRESUMEN
ABSTRACT During fetal development physiological stretching helps drive lung growth and maturation. At birth, the α-subunit of the alveolar epithelial sodium channel (α-ENaC) is a critical factor in helping to facilitate clearance of lung fluid during the perinatal period. The effects of stretch, however, on α-ENaC expression in the fetal lung have yet to be elucidated. In an effort to explore this question, we used both an in vitro cell culture model that exposes cells to repetitive cyclic stretch (CS) as well as an in vivo preterm animal model of mechanical ventilation (MV). We found that murine lung epithelial (MLE-12) cells exposed to repetitive CS showed a significant rise in α-ENaC mRNA expression. Total and cell-surface protein abundance of α-ENaC were also elevated after 24 h of CS. Stretch-induced increases in α-ENaC expression were suppressed in the presence of either actinomycin D or cycloheximide. Pharmacological inhibition of the extracellular signal-regulated protein kinase (ERK1/2) did not attenuate stretch-induced increases in α-ENaC protein, whereas inhibition of p38 MAPK or c-Jun NH2-terminal kinase (JNK) did. In 29-day preterm rabbits, alveolar stretching secondary to postnatal MV markedly elevated fetal lung α-ENaC expression compared to spontaneously breathing counterparts. In summary, our findings indicate that mechanical stretch promotes α-ENaC expression.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Pulmón/embriología , Mucosa Respiratoria/metabolismo , Estrés Mecánico , Animales , Células Cultivadas , Femenino , Pulmón/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Embarazo , Conejos , Distribución AleatoriaRESUMEN
OBJECTIVE: To compare the ability to successfully intubate extremely preterm baboons using conventional direct laryngoscopy (DL) vs videolaryngoscopy. METHODS: A prospective randomized crossover study using experienced and inexperienced neonatal intubators. All participants were shown an educational video on intubation with each device, followed by attempt of the procedure. The time for successful intubation was the primary outcome. RESULTS: Seven subjects comprised the experienced group, while 10 individuals were in the inexperienced group. The overall intubation success rate was comparable between both devices (53% vs 26%, P = 0.09); however, mean time to intubate with the conventional laryngoscope was faster (25.5 vs 39.4 s, P = 0.02). Although both groups intubated faster with DL, it only reached statistical significance in the inexperienced group (27.0 vs 48.7 s, P < 0.05). CONCLUSION: Conventional DL and videolaryngoscopy are suitable modes for intubating extremely preterm baboons. Although experienced intubators prefer DL, intubation success rate and time to intubate with both devices were comparable. In inexperienced intubators, participants preferred and intubated faster with DL.
Asunto(s)
Intubación Intratraqueal/métodos , Intubación Intratraqueal/estadística & datos numéricos , Laringoscopía/métodos , Laringoscopía/estadística & datos numéricos , Animales , Animales Recién Nacidos , Estudios Cruzados , Humanos , Recién Nacido , Intubación Intratraqueal/veterinaria , Modelos Animales , Papio , Competencia Profesional , Estudios Prospectivos , Factores de Tiempo , Grabación en VideoRESUMEN
Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-ß 2 (TGF-ß(2)) in the developing intestine. We hypothesized that low epithelial TGF-ß(2) expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-ß(2) in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-ß(2), Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-ß(2) than term intestine. TGF-ß(2) expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-ß(2) promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-ß(2). Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-ß(2) in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.
Asunto(s)
Comunicación Autocrina , Colon/metabolismo , Enterocolitis Necrotizante/metabolismo , Mucosa Intestinal/metabolismo , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Animales , Western Blotting , Línea Celular , Colon/patología , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Enterocolitis Necrotizante/genética , Enterocolitis Necrotizante/patología , Edad Gestacional , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Papio anubis , Papio cynocephalus , Nacimiento Prematuro , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína smad7/genética , Transfección , Factor de Crecimiento Transformador beta2/genéticaRESUMEN
Premature birth is a leading cause of childhood morbidity and mortality and often followed by an arrest of postnatal lung development called bronchopulmonary dysplasia. Therapies using exogenous mesenchymal stromal cells (MSC) have proven highly efficacious in term-born rodent models of this disease, but effects of MSC in actual premature-born lungs are largely unknown. Here, we investigated thirteen non-human primates (baboons; Papio spp.) that were born at the limit of viability and given a single, intravenous dose of ten million human umbilical cord tissue-derived MSC per kilogram or placebo immediately after birth. Following two weeks of human-equivalent neonatal intensive care including mechanical ventilation, lung function testing and echocardiographic studies, lung tissues were analyzed using unbiased stereology. We noted that therapy with MSC was feasible, safe and without signs of engraftment when administered as controlled infusion over 15 minutes, but linked to adverse events when given faster. Administration of cells was associated with improved cardiovascular stability, but neither benefited lung structure, nor lung function after two weeks of extrauterine life. We concluded that a single, intravenous administration of MSC had no short- to mid-term lung-protective effects in extremely premature-born baboons, sharply contrasting data from term-born rodent models of arrested postnatal lung development and urging for investigations on the mechanisms of cell-based therapies for diseases of prematurity in actual premature organisms.
Asunto(s)
Displasia Broncopulmonar , Células Madre Mesenquimatosas , Recién Nacido , Animales , Humanos , Pulmón , Displasia Broncopulmonar/terapia , Recien Nacido Prematuro , PrimatesRESUMEN
Preterm neonates are commonly exposed postnatally to pharmacological treatments for a patent ductus arteriosus. Exposure of the developing kidney to nephrotoxic medications may adversely impact renal development. This study aimed to determine the effect of early postnatal ibuprofen treatment, both alone and in combination with a nitric oxide synthase inhibitor (NOSi), on renal development and morphology. Baboon neonates were delivered prematurely at 125-day (125d) gestation (term = 185d) and were euthanized at birth or postnatal day 6. Neonates were divided into four groups: 125d gestational controls (n = 8), Untreated (n = 8), Ibuprofen (n = 6), and ibuprofen (Ibu)+NOSi (n = 4). Animals in the Ibuprofen and Ibu+NOSi groups received five doses of ibuprofen, with the Ibuprofen+NOSi animals additionally administered a NOS inhibitor (N(G)-monomethyl-l-arginine). There was no difference among groups in body weight, kidney weight, or glomerular generation number. Nephrogenic zone width was significantly reduced in the Ibuprofen group (123.5 ± 7.4 µm) compared with the 125d gestational control (176.1 ± 6.9 µm) and Untreated animals (169.7 ± 78.8 µm). In the Ibu+NOSi group, nephrogenic zone width averaged 152.7 ± 3.9 µm, which was not significantly different from any other group. Morphologically abnormal glomeruli were present at a range of 0.0-22.9% in the Untreated group, 0.0-6.1% in the Ibuprofen group, and 0.0-1.4% in the Ibu+NOSi group. In conclusion, early postnatal ibuprofen exposure is associated with a reduced nephrogenic zone width, which may suggest the early cessation of nephrogenesis following treatment. Ultimately, this may impact the number of nephrons formed in the preterm kidney.
Asunto(s)
Antiinflamatorios no Esteroideos/toxicidad , Ibuprofeno/toxicidad , Riñón/efectos de los fármacos , Riñón/crecimiento & desarrollo , Nefronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Presión Sanguínea/fisiología , Peso Corporal/efectos de los fármacos , Ingestión de Líquidos/fisiología , Interacciones Farmacológicas , Conducto Arterioso Permeable/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Femenino , Riñón/citología , Glomérulos Renales/citología , Glomérulos Renales/efectos de los fármacos , Nefronas/citología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Tamaño de los Órganos/efectos de los fármacos , Papio , Embarazo , Nacimiento Prematuro , Orina , omega-N-Metilarginina/farmacologíaRESUMEN
The main respiratory pathophysiological process following premature birth is the delayed or arrested alveolar development that translates to a smaller alveolar surface area (SA). Histological morphometry is the gold standard method to measure the SA but requires invasive tissue sampling or the removal of the whole organ for analysis. Alternatively, the SA could be measured in living subjects by "functional morphometry" using Fick's first law of diffusion and noninvasive measurements of the ventilation to perfusion ratio (VÌa/QÌ). We herein aim to describe a novel functional morphometric method to measure SA using a premature baboon model. We used both functional morphometry and postmortem histological morphometry to measure SA in 11 premature baboons born at 135 days who received intensive care treatment for 14 days. For the calculation of the SA by functional morphology, we measured the septal wall thickness using microscopy, the alveolar arterial oxygen gradient using concurrent measurements of arterial pressure of O2 and CO2, and pulmonary perfusion using echocardiography and integrated Doppler signals. The median [interquartile range (IQR)] SA using functional morphometry was 3,100 (2,080-3,640) cm2 and using histological morphometry was 1,034 (634-1,210) cm2 (left lung only). The SA measured by functional morphometry was not related to the SA measured by histological morphometry. Following linear regression analysis, the VÌa/QÌ significantly predicted the histologically measured SA (R2 = 0.659, P = 0.002). In conclusion, functional measurements of ventilation to perfusion ratio could be used to estimate the alveolar surface area in prematurely born baboons and the ventilation perfusion ratio was the main determinant of the alveolar surface area.NEW & NOTEWORTHY The main morphological characteristic of chronic respiratory disease in prematurely born infants is the impaired/arrested alveolar growth that corresponds to a smaller aggregated alveolar surface area (SA). This decreased SA might be the limiting factor later in life affecting exercise capacity and quality of life. There is paucity of sensitive, noninvasive biomarkers to monitor the evolution of neonatal respiratory disease. Our noninvasive functional morphometric SA might help to bridge the gap between pathophysiology and clinical monitoring.
Asunto(s)
Nacimiento Prematuro , Animales , Humanos , Recién Nacido , Pulmón , Papio , Calidad de Vida , Relación Ventilacion-PerfusiónRESUMEN
Preterm birth is the leading cause of death in children under 5 years of age. Premature infants who receive life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD), a chronic lung disease. Infants with BPD are at a high risk of abnormal neurodevelopment, including motor and cognitive difficulties. While neural progenitor cells (NPCs) are crucial for proper brain development, it is unclear whether they play a role in BPD-associated neurodevelopmental deficits. Here, we show that hyperoxia-induced experimental BPD in newborn mice led to lifelong impairments in cerebrovascular structure and function as well as impairments in NPC self-renewal and neurogenesis. A neurosphere assay utilizing nonhuman primate preterm baboon NPCs confirmed impairment in NPC function. Moreover, gene expression profiling revealed that genes involved in cell proliferation, angiogenesis, vascular autoregulation, neuronal formation, and neurotransmission were dysregulated following neonatal hyperoxia. These impairments were associated with motor and cognitive decline in aging hyperoxia-exposed mice, reminiscent of deficits observed in patients with BPD. Together, our findings establish a relationship between BPD and abnormal neurodevelopmental outcomes and identify molecular and cellular players of neonatal brain injury that persist throughout adulthood that may be targeted for early intervention to aid this vulnerable patient population.
Asunto(s)
Displasia Broncopulmonar , Disfunción Cognitiva , Hiperoxia , Nacimiento Prematuro , Recién Nacido , Femenino , Ratones , Humanos , Animales , Hiperoxia/complicaciones , Hiperoxia/metabolismo , Animales Recién Nacidos , Displasia Broncopulmonar/genética , Neurogénesis , Disfunción Cognitiva/etiología , Cognición , Pulmón/metabolismoRESUMEN
Respiratory dysfunction in adults has been correlated with neonatal Chlamydia trachomatis pneumonia in several studies, but a causal association has not been clearly demonstrated. In this study, we examined radial alveolar counts (RACs) by microscopy, and airway and parenchymal lung function using a small animal ventilator in juvenile (5 weeks age) and adult (8 weeks age) BALB/c mice challenged as neonates with Chlamydia muridarum (C. mur) on day 1 or day 7 after birth, representing saccular (human pre-term neonates) and alveolar (human term neonates) stages of lung development, respectively. Pups challenged with C. mur on either day 1 or 7 after birth demonstrated significantly enhanced airway hyperreactivity and lung compliance, both as juveniles (5 weeks age) and adults (8 weeks age), compared with mock-challenged mice. Moreover, mice challenged neonatally with Chlamydia displayed significantly reduced RACs, suggesting emphysematous changes. Antimicrobial treatment during the neonatal infection induced early bacterial clearance and partially ameliorated the Chlamydia-induced lung dysfunction as adults. These results suggest that neonatal chlamydial pneumonia, especially in pre-term neonates, is a cause of respiratory dysfunction continuing into adulthood, and that antimicrobial administration may be partially effective in preventing the adverse respiratory sequelae in adulthood. The results of our studies also emphasize the importance of prenatal screening and treatment of pregnant women for C. trachomatis in order to prevent the infection of neonates.
Asunto(s)
Envejecimiento , Animales Recién Nacidos , Infecciones por Chlamydia/patología , Infecciones por Chlamydia/fisiopatología , Neumonía Bacteriana/patología , Neumonía Bacteriana/fisiopatología , Sistema Respiratorio/patología , Sistema Respiratorio/fisiopatología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Antibacterianos/administración & dosificación , Hiperreactividad Bronquial/etiología , Infecciones por Chlamydia/complicaciones , Infecciones por Chlamydia/tratamiento farmacológico , Esquema de Medicación , Eritromicina/administración & dosificación , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/fisiopatología , Rendimiento Pulmonar , Ratones , Ratones Endogámicos BALB C , Neumonía Bacteriana/complicaciones , Neumonía Bacteriana/tratamiento farmacológico , Alveolos Pulmonares/crecimiento & desarrolloRESUMEN
OBJECTIVE: To determine whether low platelet counts are related to the incidence of patent ductus arteriosus (PDA) after indomethacin treatment in preterm human infants. STUDY DESIGN: Multivariable logistic regression modeling was used for a cohort of 497 infants, who received indomethacin (within 15 hours of birth). RESULTS: Platelet counts were not related to the incidence of permanent closure after indomethacin constriction. There was a relationship between platelet counts and the initial degree of constriction; however, this relationship appeared to be primarily influenced by the high end of the platelet distribution curve. PDA incidence was similar in infants with platelet counts < 50 × 109/L and those with platelet counts above this range. Only when platelet counts were consistently >230 ×109/L was there a decrease in PDA incidence. CONCLUSION: In contrast to the evidence in mice, low circulating platelet counts do not affect permanent ductus closure (or ductus reopening) in human preterm infants.
Asunto(s)
Fármacos Cardiovasculares/farmacología , Conducto Arterioso Permeable/sangre , Conducto Arterioso Permeable/tratamiento farmacológico , Indometacina/farmacología , Recuento de Plaquetas , Estudios de Cohortes , Conducto Arterial/efectos de los fármacos , Ecocardiografía/métodos , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro , Masculino , Análisis de Regresión , Factores de Tiempo , Resultado del TratamientoRESUMEN
Permanent closure of the newborn ductus arteriosus requires the development of neointimal mounds to completely occlude its lumen. VEGF is required for neointimal mound formation. The size of the neointimal mounds (composed of proliferating endothelial and migrating smooth muscle cells) is directly related to the number of VLA4 mononuclear cells that adhere to the ductus lumen after birth. We hypothesized that VEGF plays a crucial role in attracting CD14/CD163 mononuclear cells (expressing VLA4) to the ductus lumen and that CD14/CD163 cell adhesion to the ductus lumen is important for neointimal growth. We used neutralizing antibodies against VEGF and VLA-4 to determine their respective roles in remodeling the ductus of premature newborn baboons. Anti-VEGF treatment blocked CD14/CD163 cell adhesion to the ductus lumen and prevented neointimal growth. Anti-VLA-4 treatment blocked CD14/CD163 cell adhesion to the ductus lumen, decreased the expression of PDGF-B (which promotes smooth muscle migration), and blocked smooth muscle influx into the neointimal subendothelial space (despite the presence of increased VEGF in the ductus wall). We conclude that VEGF is necessary for CD14/CD163 cell adhesion to the ductus lumen and that CD14/CD163 cell adhesion is essential for VEGF-induced expansion of the neointimal subendothelial zone.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Conducto Arterioso Permeable/patología , Leucocitos Mononucleares/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Neointima , Receptores de Superficie Celular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos Neutralizantes/metabolismo , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Conducto Arterioso Permeable/metabolismo , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Recién Nacido , Integrina alfa4beta1/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Papio , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Three independent risk factors (immature gestation, absence of antenatal glucocorticoid exposure, and presence of the rs2817399(A) allele of the gene TFAP2B) are associated with patent ductus arteriosus (PDAs) that fail to close during prostaglandin inhibition. We hypothesized that these three factors may affect a common set of genes that increase the risk of persistent PDA after birth. We studied baboon ductus from term, preterm, and glucocorticoid-treated preterm fetuses and found that both immature gestation and absence of antenatal glucocorticoid exposure decreased RNA expression of calcium- and potassium-channel genes involved in oxygen-induced constriction, and phosphodiesterase genes (that modulate cAMP/cGMP signaling). Ductus obtained from second trimester human pregnancies were genotyped for TFAP2B polymorphisms. When present, the rs2817399(A) allele also was associated with decreased expression of calcium- and potassium-channel genes. In contrast, alleles of two other TFAP2B polymorphisms, rs2817419(G) and rs2635727(T), which are not related to the incidence of PDA after birth, had no effect on RNA expression. In conclusion, three calcium- and potassium-channel genes (CACNA1G/ alpha1G, CACNB 2/CaL-beta2, and KCNA2/ Kv1.2) were similarly affected by each of the PDA risk factors. We speculate that these channels may play a significant role in closing the preterm ductus during prostaglandin inhibition and may be potential targets for future pharmacologic manipulations.
Asunto(s)
Conducto Arterioso Permeable/etiología , Conducto Arterial/embriología , Regulación del Desarrollo de la Expresión Génica , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo T/genética , Esquema de Medicación , Conducto Arterioso Permeable/genética , Conducto Arterioso Permeable/prevención & control , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Edad Gestacional , Glucocorticoides/administración & dosificación , Glucocorticoides/efectos adversos , Humanos , Canal de Potasio Kv.1.2/genética , Modelos Lineales , Modelos Logísticos , Papio , Polimorfismo de Nucleótido Simple , Embarazo , ARN Mensajero/análisis , Medición de Riesgo , Factores de RiesgoRESUMEN
Resident/endogenous mesenchymal stromal cells function to promote the normal development, growth, and repair of tissues. Following premature birth, the effects of routine neonatal care (e.g. oxygen support and mechanical ventilation) on the biological properties of lung endogenous mesenchymal stromal cells is (L-MSCs) is poorly understood. New Zealand white preterm rabbits were randomized into the following groups: (i) sacrificed at birth (Fetal), (ii) spontaneously breathing with 50% O2 for 4 hours (SB), or (iii) mechanical ventilation with 50% O2 for 4h (MV). At time of necropsy, L-MSCs were isolated, characterized, and compared. L-MSCs isolated from the MV group had decreased differentiation capacity, ability to form stem cell colonies, and expressed less vascular endothelial growth factor mRNA. Compared to Fetal L-MSCs, 98 and 458 genes were differentially expressed in the L-MSCs derived from the SB and MV groups, respectively. Gene ontology analysis revealed these genes were involved in key regulatory processes including cell cycle, cell division, and angiogenesis. Furthermore, the L-MSCs from the SB and MV groups had smaller mitochondria, nuclear changes, and distended endoplasmic reticula. Short-term hyperoxia/mechanical ventilation after birth alters the biological properties of L-MSCs and stimulates genomic changes that may impact their reparative potential.
Asunto(s)
Pulmón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Respiración Artificial/efectos adversos , Animales , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Hiperoxia/metabolismo , Masculino , Células Madre Mesenquimatosas/fisiología , Oxígeno/metabolismo , Oxígeno/fisiología , Conejos , Respiración Artificial/métodosRESUMEN
Intra-amniotic exposure to proinflammatory cytokines such as interleukin-1 (IL-1) correlates with a decreased incidence of respiratory distress syndrome (RDS) in infants following premature birth. At birth, inadequate absorption of fluid from the fetal lung contributes to the onset RDS. Lung fluid clearance is coupled to Na+ transport via epithelial sodium channels (ENaC). In this study, we assessed the effects of IL-1 on the expression of ENaC, particularly the α-subunit which is critical for fetal lung fluid clearance at birth. Cultured mouse lung epithelial (MLE-12) cells were treated with either IL-1α or IL-1ß to determine their effects on α-ENaC expression. Changes in IL-1-induced α-ENaC levels in the presence of IL-1 receptor antagonist (IL-1ra), cycloheximide, NF-κB inhibitor, and MAP kinase inhibitors were investigated. IL-1α and IL-1ß independently induced a significant increase of α-ENaC mRNA and protein after 24 h compared to untreated cells. IL-1-dependent increases in α-ENaC protein were mitigated by IL-1ra and cycloheximide. IL-1 exposure induced NF-κB binding activity. Attenuation of IL-1-induced NF-κB activation by its inhibitor SN50 decreased α-ENaC protein abundance. Inhibition of ERK 1,2 MAPK significantly decreased both IL-1α and ß-induced α-ENaC protein expression whereas inhibition of p38 MAPK only blocked IL-1ß-induced α-ENaC protein levels. In contrast, IL-1-induced α-ENaC protein levels were unaffected by a c-Jun N-terminal kinase (JNK) inhibitor. Our results suggest that in MLE-12 cells, IL-1-induced elevation of α-ENaC is mediated via NF-κB activation and in part involves stimulation of the ERK 1,2 and p38 MAPK signaling pathways.
RESUMEN
Compared with the full-term ductus arteriosus, the premature ductus is less likely to constrict when exposed to postnatal oxygen concentrations. We used isolated fetal sheep ductus arteriosus (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine whether changes in K+ - and CaL-channel activity could account for the developmental differences in oxygen-induced tension. In the mature ductus, KV-channels appear to be the only K+-channels that oppose ductus tension. Oxygen concentrations between (2% and 15%) inhibit KV-channel activity, which increases the CaL-channel-mediated increase in tension. Low oxygen concentrations have a direct inhibitory effect on CaL-channel activity in the immature ductus; this is not the case in the mature ductus. In the immature ductus, three different K+-channel activities (KV, KCa, and KATP) oppose ductus tension and contribute to its decreased tone. Oxygen inhibits the activities of all three K+ -channels. The inhibitory effects of the three K+-channel activities decline with advancing gestation. The decline in K+ -channel activity is not due to decreased K+ -channel expression. Super-physiologic oxygen concentrations (>or=30% O2) constrict the ductus by using calcium-dependent pathways that are independent of K+- and CaL-channel activities. Super-physiologic oxygen concentrations eliminate the difference in tensions between the two age groups.
Asunto(s)
Canales de Calcio/metabolismo , Conducto Arterial/fisiología , Feto/fisiología , Regulación de la Expresión Génica/fisiología , Oxígeno/metabolismo , Canales de Potasio/metabolismo , Vasoconstricción/fisiología , Animales , Edad Gestacional , Reacción en Cadena de la Polimerasa , Ovinos , Estadísticas no ParamétricasRESUMEN
Alpha-ENaC expression and activity is regulated by a variety of hormones including beta-adrenergic agonists via the second messenger cAMP. We evaluated the early intermediate pathways involved in the up-regulation of SGK1 by DbcAMP and whether SGK1 is a prerequisite for induction of alpha-ENaC expression. Submandibular gland epithelial (SMG-C6) cells treated with DbcAMP (1 mM) induced both SGK1 mRNA and protein expression. DbcAMP-stimulated SGK1 mRNA expression was decreased by actinomycin D and mRNA and protein expressions were attenuated by PKA inhibitors (H-89 and KT5720). Inhibition of PI3-K with either LY294002 or dominant negative PI3-K reduced DbcAMP-stimulated SGK1 protein and mRNA levels, attenuated the phosphorylation of CREB (a cAMP-activated transcription factor) and decreased alpha-ENaC protein levels and Na(+) transport. In addition, the combination of PKA inhibitors with dominant negative PI3-K synergistically inhibited DbcAMP-induced Na(+) transport. Inhibition of SGK1 expression by siRNA decreased but did not obliterate DbcAMP-induced alpha-ENaC expression. Thus, in a cell line which endogenously exhibits minimal alpha-ENaC expression, induction of SGK1 by DbcAMP occurs via the PI3-K and PKA pathways. Increased alpha-ENaC levels and function are partly dependent upon the early induction of SGK1 expression.
Asunto(s)
Bucladesina/farmacología , Canales Epiteliales de Sodio/metabolismo , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Cicloheximida/farmacología , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Canales Epiteliales de Sodio/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Sodio/metabolismoRESUMEN
A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (alpha, beta, and gamma). The alpha-subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG-C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of alpha-ENaC mRNA and protein expression and amiloride-sensitive short-circuit current (I(SC)). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H-89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP-induced alpha-ENaC protein formation and I(SC). Exposure of SMG-C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H-89. Dominant-negative CREB decreased DbcAMP-induced alpha-ENaC expression. Suppression of the extracellular signal-regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen-activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP-induced alpha-ENaC protein levels in SMG-C6 cells. DbcAMP-induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP-induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H-89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of alpha-ENaC observed after 24 h of treatment in SMG-C6 epithelial cells.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Canales Epiteliales de Sodio/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Bucladesina/farmacología , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Canales Epiteliales de Sodio/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Activación del Canal Iónico/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Premature baboons exhibit peripheral insulin resistance and impaired insulin signaling. 5' AMP-activated protein kinase (AMPK) activation improves insulin sensitivity by enhancing glucose uptake (via increased glucose transporter type 4 [GLUT4] translocation and activation of the extracellular signal-regulated kinase [ERK]/ atypical protein kinase C [aPKC] pathway), and increasing fatty acid oxidation (via inhibition of acetyl-CoA carboxylase 1 [ACC]), while downregulating gluconeogenesis (via induction of small heterodimer partner [SHP] and subsequent downregulation of the gluconeogenic enzymes: phosphoenolpyruvate carboxykinase [PEPCK], glucose 6-phosphatase [G6PASE], fructose- 1,6-bisphosphatase 1 [FBP1], and forkhead box protein 1 [FOXO1]). The purpose of this study was to investigate whether pharmacologic activation of AMPK with AICAR (5-aminoimidazole-4-carboximide riboside) administration improves peripheral insulin sensitivity in preterm baboons. 11 baboons were delivered prematurely at 125±2 days (67%) gestation. 5 animals were randomized to receive 5 days of continuous AICAR infusion at a dose of 0.5 mg·g-1·day-1. 6 animals were in the placebo group. Euglycemic hyperinsulinemic clamps were performed at 5±2 and 14±2 days of life. Key molecules potentially altered by AICAR (AMPK, GLUT4, ACC, PEPCK, G6PASE, FBP1, and FOXO1), and the insulin signaling molecules: insulin receptor (INSR), insulin receptor substrate 1 (IRS-1), protein kinase B (AKT), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were measured using RT-PCR and western blotting. AICAR infusion did not improve whole body insulin-stimulated glucose disposal in preterm baboons (12.8±2.4 vs 12.4±2.0 mg/(kg·min), p = 0.8, placebo vs AICAR). One animal developed complications during treatment. In skeletal muscle, AICAR infusion did not increase phosphorylation of ACC, AKT, or AMPK whereas it increased mRNA expression of ACACA (ACC), AKT, and PPARGC1A (PGC1α). In the liver, INSR, IRS1, G6PC3, AKT, PCK1, FOXO1, and FBP1 were unchanged, whereas PPARGC1A mRNA expression increased after AICAR infusion. This study provides evidence that AICAR does not improve insulin sensitivity in premature euglycemic baboons, and may have adverse effects.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Hipoglucemiantes/administración & dosificación , Resistencia a la Insulina , Insulina/metabolismo , Ribonucleótidos/administración & dosificación , Administración Intravenosa , Aminoimidazol Carboxamida/administración & dosificación , Aminoimidazol Carboxamida/sangre , Animales , Animales Recién Nacidos , Ácidos Grasos no Esterificados/sangre , Femenino , Glucógeno/sangre , Hipoglucemiantes/sangre , Hígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Papio , ARN Mensajero/metabolismo , Distribución Aleatoria , Ribonucleótidos/sangreRESUMEN
Premature infants have altered glucose regulation early in life and increased risk for diabetes in adulthood. Although prematurity leads to an increased risk of diabetes and metabolic syndrome in adult life, the role of hepatic glucose regulation and adaptation to an early extrauterine environment in preterm infants remain unknown. The purpose of this study was to investigate developmental differences in glucose metabolism, hepatic protein content, and gene expression of key insulin-signaling/gluconeogenic molecules. Fetal baboons were delivered at 67%, 75%, and term gestational age and euthanized at birth. Neonatal baboons were delivered prematurely (67% gestation), survived for two weeks, and compared with similar postnatal term animals and underwent serial hyperinsulinemic-euglycemic clamp studies. Premature baboons had decreased endogenous glucose production (EGP) compared with term animals. Consistent with these results, the gluconeogenic molecule, phosphoenolpyruvate carboxykinase messenger RNA, was decreased in preterm baboons compared with terms. Hepatic insulin signaling was altered by preterm birth as evidenced by decreased insulin receptor-ß, p85 subunit of phosphoinositide 3-kinase, phosphorylated insulin receptor substrate 1, and Akt-1 under insulin-stimulated conditions. Furthermore, preterm baboons failed to have the normal increase in glycogen synthase kinase-α from fetal to postnatal life. The blunted responses in hepatic insulin signaling may contribute to the hyperglycemia of prematurity, while impaired EGP leads to hypoglycemia of prematurity.