RESUMO
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.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Pulmão/embriologia , Mucosa Respiratória/metabolismo , Estresse Mecânico , Animais , Células Cultivadas , Feminino , Pulmão/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Gravidez , Coelhos , Distribuição AleatóriaRESUMO
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.
Assuntos
Comunicação Autócrina , Colo/metabolismo , Enterocolite Necrosante/metabolismo , Mucosa Intestinal/metabolismo , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Animais , Western Blotting , Linhagem Celular , Colo/patologia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Idade Gestacional , Humanos , Imuno-Histoquímica , Mucosa Intestinal/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Papio anubis , Papio cynocephalus , Nascimento Prematuro , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad7/genética , Transfecção , Fator de Crescimento Transformador beta2/genéticaRESUMO
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.
Assuntos
Displasia Broncopulmonar , Células-Tronco Mesenquimais , Recém-Nascido , Animais , Humanos , Pulmão , Displasia Broncopulmonar/terapia , Recém-Nascido Prematuro , PrimatasRESUMO
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.
Assuntos
Nascimento Prematuro , Animais , Humanos , Recém-Nascido , Pulmão , Papio , Qualidade de Vida , Relação Ventilação-PerfusãoRESUMO
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.
Assuntos
Displasia Broncopulmonar , Disfunção Cognitiva , Hiperóxia , Nascimento Prematuro , Recém-Nascido , Feminino , Camundongos , Humanos , Animais , Hiperóxia/complicações , Hiperóxia/metabolismo , Animais Recém-Nascidos , Displasia Broncopulmonar/genética , Neurogênese , Disfunção Cognitiva/etiologia , Cognição , Pulmão/metabolismoRESUMO
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.
Assuntos
Envelhecimento , Animais Recém-Nascidos , Infecções por Chlamydia/patologia , Infecções por Chlamydia/fisiopatologia , Pneumonia Bacteriana/patologia , Pneumonia Bacteriana/fisiopatologia , Sistema Respiratório/patologia , Sistema Respiratório/fisiopatologia , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Antibacterianos/administração & dosagem , Hiper-Reatividade Brônquica/etiologia , Infecções por Chlamydia/complicações , Infecções por Chlamydia/tratamento farmacológico , Esquema de Medicação , Eritromicina/administração & dosagem , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Complacência Pulmonar , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/complicações , Pneumonia Bacteriana/tratamento farmacológico , Alvéolos Pulmonares/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Respiração Artificial/efeitos adversos , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Hiperóxia/metabolismo , Masculino , Células-Tronco Mesenquimais/fisiologia , Oxigênio/metabolismo , Oxigênio/fisiologia , Coelhos , Respiração Artificial/métodosRESUMO
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.
RESUMO
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.
Assuntos
Bucladesina/farmacologia , Canais Epiteliais de Sódio/metabolismo , Proteínas Imediatamente Precoces/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Cicloeximida/farmacologia , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Canais Epiteliais de Sódio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Imediatamente Precoces/genética , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sódio/metabolismoRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Canais Epiteliais de Sódio/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Bucladesina/farmacologia , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Canais Epiteliais de Sódio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Hipoglicemiantes/administração & dosagem , Resistência à Insulina , Insulina/metabolismo , Ribonucleotídeos/administração & dosagem , Administração Intravenosa , Aminoimidazol Carboxamida/administração & dosagem , Aminoimidazol Carboxamida/sangue , Animais , Animais Recém-Nascidos , Ácidos Graxos não Esterificados/sangue , Feminino , Glicogênio/sangue , Hipoglicemiantes/sangue , Fígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Papio , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ribonucleotídeos/sangueRESUMO
BACKGROUND: In the preterm newborn, a patent ductus arteriosus is in large part a result of the increased sensitivity of the immature ductus to prostaglandin E2 (PGE2). PGE2 acts through 3 G protein-coupled receptors (EP2, EP3, and EP4) that activate both adenyl cyclase and K(ATP) channels. We explored these pathways to identify the mechanisms responsible for the increased sensitivity of the immature ductus to PGE2. METHODS AND RESULTS: We measured EP receptor content (mRNA and protein), receptor binding, cAMP production, and isometric tension in rings of ductus taken from immature (65% gestation) and mature (95% gestation) sheep and baboon fetuses. Ductus relaxation and cAMP generation were augmented in response to selective EP receptor agonists in the immature ductus. 8-Br-cAMP, a stable cAMP analogue, produced greater relaxation in the immature ductus. In the presence of a selective protein kinase A inhibitor, Rp-8-CPT cAMPS, the developmental differences in sensitivity to PGE2 could no longer be demonstrated. EP2, EP3, and EP4 receptor densities were higher in immature ductus, despite similar receptor mRNA and protein contents at the 2 gestational ages. In contrast, forskolin and NaF, direct activators of adenyl cyclase and Gs, respectively, elicited comparable increases in cAMP in both age groups. KATP channel inhibition also had similar effects on PGE2-induced relaxation in both age groups. CONCLUSIONS: Two mechanisms explain the increased sensitivity of the immature ductus to PGE2: (1) increased cAMP production because of increased binding of PGE2 to the individual EP receptors and (2) increased potency of cAMP on protein kinase A-regulated pathways.
Assuntos
Adenosina/análogos & derivados , Alprostadil/análogos & derivados , AMP Cíclico/análogos & derivados , Dinoprostona/farmacologia , Canal Arterial/efeitos dos fármacos , Receptores de Prostaglandina E/efeitos dos fármacos , Sistema Vasomotor/efeitos dos fármacos , 16,16-Dimetilprostaglandina E2/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Adenilil Ciclases/metabolismo , Alprostadil/farmacologia , Animais , Compostos de Bifenilo/farmacologia , Colforsina/farmacologia , AMP Cíclico/biossíntese , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Idade Gestacional , Glibureto/farmacologia , Indometacina/farmacologia , Contração Isométrica , NG-Nitroarginina Metil Éster/farmacologia , Nitroprussiato/farmacologia , Papio , Canais de Potássio/efeitos dos fármacos , Gravidez , Receptores de Prostaglandina E/fisiologia , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4 , Ovinos , Transdução de Sinais , Fluoreto de Sódio/farmacologia , Tionucleotídeos/farmacologia , Sistema Vasomotor/fisiologiaRESUMO
Premature infants develop hyperglycemia shortly after birth, increasing their morbidity and death. Surviving infants have increased incidence of diabetes as young adults. Our understanding of the biological basis for the insulin resistance of prematurity and developmental regulation of glucose production remains fragmentary. The objective of this study was to examine maturational differences in insulin sensitivity and the insulin-signaling pathway in skeletal muscle and adipose tissue of 30 neonatal baboons using the euglycemic hyperinsulinemic clamp. Preterm baboons (67% gestation) had reduced peripheral insulin sensitivity shortly after birth (M value 12.5 ± 1.5 vs 21.8 ± 4.4 mg/kg · min in term baboons) and at 2 weeks of age (M value 12.8 ± 2.6 vs 16.3 ± 4.2, respectively). Insulin increased Akt phosphorylation, but these responses were significantly lower in preterm baboons during the first week of life (3.2-fold vs 9.8-fold). Preterm baboons had lower glucose transporter-1 protein content throughout the first 2 weeks of life (8%-12% of term). In preterm baboons, serum free fatty acids (FFAs) did not decrease in response to insulin, whereas FFAs decreased by greater than 80% in term baboons; the impaired suppression of FFAs in the preterm animals was paired with a decreased glucose transporter-4 protein content in adipose tissue. In conclusion, peripheral insulin resistance and impaired non-insulin-dependent glucose uptake play an important role in hyperglycemia of prematurity. Impaired insulin signaling (reduced Akt) contributes to the defect in insulin-stimulated glucose disposal. Counterregulatory hormones are not major contributors.
Assuntos
Glucose/metabolismo , Resistência à Insulina/fisiologia , Papio/metabolismo , Nascimento Prematuro , Transdução de Sinais/fisiologia , Insuficiência Vertebrobasilar/metabolismo , Animais , Glicemia , Feminino , Regulação da Expressão Gênica , Glucagon , Técnica Clamp de Glucose , Músculo Esquelético/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMO
Surfactant-associated proteins (SP-A, SP-B, and SP-C) are critical for the endogenous function of surfactant. Keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) are key regulators of lung development. The objective of this study was to evaluate the effects of early mechanical ventilation on the expression of these important regulatory proteins in a preterm rabbit model. Premature fetuses were delivered at 29 d of gestation and randomized to necropsy at birth, i.e. no ventilation (NV), spontaneous breathing (SB), or mechanical ventilation (MV) for 16 h. MV animals were further randomized to treatment with dexamethasone (dex). Our findings showed that SB rabbits increased their expression of SP-A mRNA and protein after birth compared with NV controls. MV significantly attenuated this response in the absence of dex. Exposure to dex elevated SP-B mRNA expression in both SB and MV rabbits. KGF protein levels were markedly increased in SB animals compared with MV counterparts. VEGF levels were similar in SB and MV animals, but were significantly increased compared with NV controls. These data suggest that MV alters surfactant-associated protein and growth factor expression, which may contribute to injury in the developing lung.
Assuntos
Fator 7 de Crescimento de Fibroblastos/metabolismo , Idade Gestacional , Proteína A Associada a Surfactante Pulmonar/metabolismo , Respiração Artificial , Animais , Animais Recém-Nascidos , Dexametasona/metabolismo , Regulação para Baixo , Feminino , Fator 7 de Crescimento de Fibroblastos/genética , Glucocorticoides/metabolismo , Gravidez , Proteína A Associada a Surfactante Pulmonar/genética , Proteína B Associada a Surfactante Pulmonar/genética , Proteína B Associada a Surfactante Pulmonar/metabolismo , Proteína C Associada a Surfactante Pulmonar/genética , Proteína C Associada a Surfactante Pulmonar/metabolismo , Coelhos , Distribuição Aleatória , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
After birth, constriction of the full-term ductus arteriosus induces oxygen, glucose and ATP depletion, cell death, and anatomic remodeling of the ductus wall. The immature ductus frequently fails to develop the same degree of constriction or anatomic remodeling after birth. In addition, the immature ductus loses its ability to respond to vasoconstrictive agents, like oxygen or indomethacin, with increasing postnatal age. We examined the effects of premature delivery and postnatal constriction on the immature baboon ductus arteriosus. By 6 days after birth, surrogate markers of hypoxia (HIF1alpha/VEGF mRNA) and cell death [dUTP nick-end labeling (TUNEL)-staining] increased, while glucose and ATP concentrations (bioluminescence imaging) decreased in the immature ductus. TUNEL-staining was significantly related to the degree of glucose and ATP depletion. Glucose and ATP depletion were directly related to the degree of ductus constriction; while TUNEL-staining was logarithmically related to the degree of ductus constriction. Extensive cell death (>15% TUNEL-positive cells) occurred only when there was no Doppler flow through the ductus lumen. In contrast, HIF1alpha/VEGF expression and ATP concentrations were significantly altered even when the immature ductus remained open after birth. Decreased ATP concentrations produced decreased oxygen-induced contractile responses in the immature ductus. We hypothesize that ATP depletion in the persistently patent immature newborn ductus is insufficient to induce cell death and remodeling but sufficient to decrease its ability to constrict after birth. This may explain its decreasing contractile response to oxygen, indomethacin, and other contractile agents with increasing postnatal age.
Assuntos
Trifosfato de Adenosina/metabolismo , Canal Arterial/metabolismo , Canal Arterial/patologia , Animais , Animais Recém-Nascidos , Morte Celular , Canal Arterial/fisiologia , Canal Arterial/fisiopatologia , Regulação da Expressão Gênica , Glucose/metabolismo , Glicogênio/metabolismo , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Papio , Ovinos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cortisol and thyroid hormones are critical to normal fetal development and neonatal transition, and baseline values and stimulation tests are abnormal after preterm birth. To evaluate cortisol and thyroxine (T4) responses that are not influenced by uncontrolled antenatal events associated with human preterm labor, we measured cortisol and T4 after standard-dose adrenocorticotropin (ACTH) and corticotropin-releasing hormone (CRH) stimulation tests, as well as high-dose CRH and thyrotropin-releasing hormone stimulation tests in baboons that were delivered for 3 separate protocols at 125 days of gestation (term is 186 days). The animals were surfactant treated and ventilated for up to 14 days. Some fetuses were exposed to fetal or maternal betamethasone, and some newborns were treated with 10 microg/kg T4 for 9 days after birth. Baseline cortisol levels were in a stress range of 30-60 microg/dl by day 5. Cortisol did not increase consistently until day 11 in response to a high CRH dose or ACTH. T4 treatment for 9 days after birth suppressed the cortisol responses and subsequent baseline T4 levels. The hypothalamic-pituitary-adrenal (HPA) axis was unresponsive to standard dose stimulation tests until 11 days of age in preterm baboons, indicating HPA immaturity.
Assuntos
Glândulas Suprarrenais/fisiopatologia , Animais Recém-Nascidos , Pneumopatias/fisiopatologia , Papio , Respiração Artificial/efeitos adversos , Glândula Tireoide/fisiopatologia , Hormônio Adrenocorticotrópico/administração & dosagem , Hormônio Adrenocorticotrópico/farmacologia , Animais , Hormônio Liberador da Corticotropina/administração & dosagem , Hormônio Liberador da Corticotropina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Sangue Fetal , Idade Gestacional , Hidrocortisona/sangue , Pneumopatias/sangue , Pneumopatias/etiologia , Tiroxina/sangueRESUMO
OBJECTIVE: Renal and cardiovascular function is improved during the first 24 hours of life in preterm ventilated baboons exposed to prenatal betamethasone (BETA). We hypothesized BETA-induced effects would be sustained through day 6 of life. Study design Pregnant baboons received saline or BETA (6 mg) 48 and 24 hours before preterm delivery at 125 days' gestation. The newborn baboons were ventilated for 6 days, and assessed for renal, cardiovascular, and endocrine function. RESULTS: Mean arterial blood pressure (MAP) and glomerular filtration rate (GFR) values 24 hours after delivery were higher in the BETA group. Kidney Na, K-ATPase activity was higher in the BETA group by day 6. All other measures were similar in both groups by day 6. CONCLUSION: Prenatal BETA exposure in the premature baboon: (1) increases MAP and GFR on day 1 without measurable effects by day 6 and (2) increases kidney Na, K-ATPase activity.
Assuntos
Adaptação Fisiológica , Animais Recém-Nascidos/fisiologia , Betametasona/farmacologia , Glucocorticoides/farmacologia , Rim/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Animais , Betametasona/administração & dosagem , Pressão Sanguínea/efeitos dos fármacos , Sistema Cardiovascular/efeitos dos fármacos , Sistema Endócrino/efeitos dos fármacos , Feminino , Maturidade dos Órgãos Fetais , Taxa de Filtração Glomerular/efeitos dos fármacos , Glucocorticoides/administração & dosagem , Masculino , Papio , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Respiração Artificial/veterináriaRESUMO
At birth, lung fluid clearance is coupled to Na+ transport through epithelial Na+ channels (ENaC) in the distal lung epithelium. We evaluated the effect of postnatal glucocorticoids (GC) on lung alpha-ENaC expression in preterm 29-day gestational age (GA) fetal rabbits. Postnatal treatment of 29-day GA fetuses with 0.5 mg/kg of dexamethasone (Dex) iv resulted in a 2- and 22-fold increase in lung alpha-ENaC mRNA expression compared with saline-treated fetuses after 8 and 16 h, respectively. Lung alpha-ENaC protein levels in Dex-treated fetuses were also elevated compared with saline-treated counterparts. The extravascular lung water (EVLW)/dry lung tissue weight ratios of 29-day GA fetuses treated with either saline or Dex decreased over 24 h compared with that observed at birth; however, at 24 h, the EVLW/dry lung tissue weight ratios of saline- and Dex-treated fetuses were similar. Dex-induced alpha-ENaC mRNA and protein levels were attenuated by glucocorticoid receptor (GCR) antagonist RU-486 in fetal distal lung epithelial cells isolated from 29-day GA fetuses, indicating that GC-dependent augmentation of lung alpha-ENaC requires the presence of functional GCR. Lung GCR mRNA expression and protein levels were elevated in 29-day GA fetuses compared with fetuses at earlier GA. Exposure of 29-day GA fetuses to Dex for 16 h caused a 2.1-fold increase in lung GCR mRNA expression, but GCR protein levels were decreased in Dex-treated fetuses after 24 h. We conclude that postnatal treatment of preterm 29-day GA fetal rabbits with GC results in an elevation of lung alpha-ENaC accompanied by an autoregulation of pulmonary GCR.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Pulmão/embriologia , Receptores de Glucocorticoides/metabolismo , Canais de Sódio/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Canais Epiteliais de Sódio , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Idade Gestacional , Pulmão/citologia , Pulmão/efeitos dos fármacos , Gravidez , RNA Mensageiro/análise , Coelhos , Canais de Sódio/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Anatomic remodeling and permanent closure of the newborn ductus arteriosus appears to require the development of intense hypoxia within the constricted vessel wall. Hypoxic ductus smooth muscle cells express vascular endothelial cell growth factor (VEGF). We studied premature baboons and sheep to determine the effects of VEGF inhibition (in baboons) and VEGF stimulation (in sheep) on ductus remodeling in vivo. For study of VEGF inhibition, 13 premature newborn baboons (68% gestation) were treated with inhibitors of both prostaglandin and nitric oxide production to constrict the ductus and induce ductus wall hypoxia. Six received a neutralizing monoclonal antibody against VEGF (A.4.6.1, mAbVEGF), while seven did not. Both groups developed the same degree of ductus constriction, tissue hypoxia, and VEGF expression. The mAbVEGF treatment produced a significant (P < 0.05) reduction in ductus vasa vasorum ingrowth and neointima formation (due to both a decrease in luminal endothelial cell proliferation and a decrease in smooth muscle cell migration into the neointima). For study of VEGF stimulation, nine sheep fetuses (70% gestation) had their ductus wall injected with either VEGF (n = 6) or vehicle (n = 4) in vivo. VEGF administration produced a significant (P < 0.05) increase in vasa vasorum ingrowth and neointima formation. We conclude that VEGF plays an important role in the formation of neointimal mounds and vasa vasorum ingrowth during permanent ductus closure.
Assuntos
Canal Arterial/fisiologia , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/farmacologia , Fatores de Crescimento Endotelial/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Expressão Gênica/fisiologia , Idade Gestacional , Hipóxia/fisiopatologia , Linfocinas/imunologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Papio , RNA Mensageiro/análise , Ovinos , Túnica Íntima/citologia , Túnica Íntima/metabolismo , Vasa Vasorum/fisiologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Because minimal information is available about surfactant metabolism in bronchopulmonary dysplasia, we measured half-lives and pool sizes of surfactant phosphatidylcholine in very preterm baboons recovering from respiratory distress syndrome and developing bronchopulmonary dysplasia, using stable isotopes, radioactive isotopes, and direct pool size measurements. Eight ventilated premature baboons received (2)H-DPPC (dipalmitoyl phosphatidylcholine) on d 5 of life, and radioactive (14)C-DPPC with a treatment dose of surfactant on d 8. After 14 d, lung pool sizes of saturated phosphatidylcholine were measured. Half-life of (2)H-DPPC (d 5) in tracheal aspirates was 28 +/- 4 h (mean +/- SEM). Half-life of radioactive DPPC (d 8) was 35 +/- 4 h. Saturated phosphatidylcholine pool size measured with stable isotopes on d 5 was 129 +/- 14 micro mol/kg, and 123 +/- 11 micro mol/kg on d 14 at autopsy. Half-lives were comparable to those obtained at d 0 and d 6 in our previous baboon studies. We conclude that surfactant metabolism does not change during the early development of bronchopulmonary dysplasia, more specifically, the metabolism of exogenous surfactant on d 8 is similar to that on the day of birth. Surfactant pool size is low at birth, increases after surfactant therapy, and is kept constant during the first 2 wk of life by endogenous surfactant synthesis. Measurements with stable isotopes are comparable to measurements with radioactive tracers and measurements at autopsy.