RESUMO
Bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity, is characterized by impaired lung development with sustained functional abnormalities due to alterations of airways and the distal lung. Although clinical studies have shown striking associations between antenatal stress and BPD, little is known about the underlying pathogenetic mechanisms. Whether dysanapsis, the concept of discordant growth of the airways and parenchyma, contributes to late respiratory disease as a result of antenatal stress is unknown. We hypothesized that antenatal endotoxin (ETX) impairs juvenile lung function as a result of altered central airway and distal lung structure, suggesting the presence of dysanapsis in this preclinical BPD model. Fetal rats were exposed to intraamniotic ETX (10 µg) or saline solution (control) 2 days before term. We performed extensive structural and functional evaluation of the proximal airways and distal lung in 2-week-old rats. Distal lung structure was quantified by stereology. Conducting airway diameters were measured using micro-computed tomography. Lung function was assessed during invasive ventilation to quantify baseline mechanics, response to methacholine challenge, and spirometry. ETX-exposed pups exhibited distal lung simplification, decreased alveolar surface area, and decreased parenchyma-airway attachments. ETX-exposed pups exhibited decreased tracheal and second- and third-generation airway diameters. ETX increased respiratory system resistance and decreased lung compliance at baseline. Only Newtonian resistance, specific to large airways, exhibited increased methacholine reactivity in ETX-exposed pups compared with controls. ETX-exposed pups had a decreased ratio of FEV in 0.1 second to FVC and a normal FEV in 0.1 second, paralleling the clinical definition of dysanapsis. Antenatal ETX causes abnormalities of the central airways and distal lung growth, suggesting that dysanapsis contributes to abnormal lung function in juvenile rats.
Assuntos
Displasia Broncopulmonar , Ratos , Animais , Feminino , Gravidez , Displasia Broncopulmonar/patologia , Endotoxinas , Cloreto de Metacolina/farmacologia , Microtomografia por Raio-X , Ratos Sprague-Dawley , Animais Recém-Nascidos , Pulmão/patologiaRESUMO
BACKGROUND: Acute kidney injury (AKI) is common in sick neonates and associated with poor pulmonary outcomes, however, the mechanisms responsible remain unknown. We present two novel neonatal rodent models of AKI to investigate the pulmonary effects of AKI. METHODS: In rat pups, AKI was induced surgically via bilateral ischemia-reperfusion injury (bIRI) or pharmacologically using aristolochic acid (AA). AKI was confirmed with plasma blood urea nitrogen and creatinine measurements and kidney injury molecule-1 staining on renal immunohistochemistry. Lung morphometrics were quantified with radial alveolar count and mean linear intercept, and angiogenesis investigated by pulmonary vessel density (PVD) and vascular endothelial growth factor (VEGF) protein expression. For the surgical model, bIRI, sham, and non-surgical pups were compared. For the pharmacologic model, AA pups were compared to vehicle controls. RESULTS: AKI occurred in bIRI and AA pups, and they demonstrated decreased alveolarization, PVD, and VEGF protein expression compared controls. Sham pups did not experience AKI, however, demonstrated decreased alveolarization, PVD, and VEGF protein expression compared to controls. CONCLUSION: Pharmacologic AKI and surgery in neonatal rat pups, with or without AKI, decreased alveolarization and angiogenesis, producing a bronchopulmonary dysplasia phenotype. These models provide a framework for elucidating the relationship between AKI and adverse pulmonary outcomes. IMPACT: There are no published neonatal rodent models investigating the pulmonary effects after neonatal acute kidney injury, despite known clinical associations. We present two novel neonatal rodent models of acute kidney injury to study the impact of acute kidney injury on the developing lung. We demonstrate the pulmonary effects of both ischemia-reperfusion injury and nephrotoxin-induced AKI on the developing lung, with decreased alveolarization and angiogenesis, mimicking the lung phenotype of bronchopulmonary dysplasia. Neonatal rodent models of acute kidney injury provide opportunities to study mechanisms of kidney-lung crosstalk and novel therapeutics in the context of acute kidney injury in a premature infant.
Assuntos
Injúria Renal Aguda , Displasia Broncopulmonar , Traumatismo por Reperfusão , Humanos , Recém-Nascido , Animais , Ratos , Animais Recém-Nascidos , Displasia Broncopulmonar/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Pulmão , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/metabolismoRESUMO
The associations between bronchopulmonary dysplasia (BPD) and the gestational pathologies of chorioamnionitis (CA) and hypertensive disorders of pregnancy (HDP) have become increasingly well recognized. However, the mechanisms through which these antenatal conditions cause increased risk of BPD remain less well characterized. The objective of this review is to discuss the role of the placenta in BPD predisposition as a primary driver of intrauterine alterations adversely impacting fetal lung development. We hypothesize that due to similarities in structure and function, placental disorders during pregnancy can uniquely impact the developing fetal lung, creating a unique placental-pulmonary connection. In the current review, we explore this hypothesis through analysis of clinical literature and preclinical model systems evaluating BPD predisposition, discussion of BPD phenotypes, and an overview on strategies to incorporate placental investigation into research on fetal lung development. We also discuss important concepts learned from research on antenatal steroids as a modulator fetal lung development. Finally, we propose that the appropriate selection of animal models and establishment of in vitro lung developmental model systems incorporating primary human placental components are key in continuing to understand and address antenatal predisposition to BPD.
Assuntos
Displasia Broncopulmonar , Corioamnionite , Recém-Nascido , Animais , Feminino , Gravidez , Humanos , Displasia Broncopulmonar/patologia , Placenta/patologia , Corioamnionite/patologia , Pulmão/patologia , Desenvolvimento FetalRESUMO
Antenatal stressors such as chorioamnionitis (CA) increase the risk for bronchopulmonary dysplasia (BPD). Studies have shown that experimental BPD can be ameliorated by postnatal treatment with mesenchymal stromal cell-derived extracellular vesicles (MEx). However, the antenatal efficacy of MEx to prevent BPD is unknown. To determine whether antenatal MEx therapy attenuates intrauterine inflammation and preserves lung growth in a rat model of CA-induced BPD. At embryonic day (E)20, rat litters were treated with intra-amniotic injections of saline, endotoxin (ETX) to model chorioamnionitis, MEx, or ETX plus MEx followed by cesarean section delivery with placental harvest at E22. Placental and lung evaluations were conducted at day 0 and day 14, respectively. To assess the effects of ETX and MEx on lung growth in vitro, E15 lung explants were imaged for distal branching. Placental tissues from ETX-exposed pregnancies showed increased expression of inflammatory markers NLRP-3 and IL-1ß and altered spiral artery morphology. In addition, infant rats exposed to intrauterine ETX had reduced alveolarization and pulmonary vessel density (PVD), increased right ventricular hypertrophy (RVH), and decreased lung mechanics. Intrauterine MEx therapy of ETX-exposed pups reduced inflammatory cytokines, normalized spiral artery architecture, and preserved distal lung growth and mechanics. In vitro studies showed that MEx treatment enhanced distal lung branching and increased VEGF and SPC gene expression. Antenatal MEx treatment preserved distal lung growth and reduced intrauterine inflammation in a model of CA-induced BPD. We speculate that MEx may provide a novel therapeutic strategy to prevent BPD due to antenatal inflammation.
Assuntos
Displasia Broncopulmonar/etiologia , Corioamnionite/patologia , Vesículas Extracelulares/metabolismo , Pulmão/crescimento & desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Animais , Modelos Animais de Doenças , Endotoxinas , Feminino , Inflamação/patologia , Pulmão/irrigação sanguínea , Pulmão/patologia , Placenta/patologia , Gravidez , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Rationale: Antenatal factors, such as chorioamnionitis, preeclampsia, and postnatal injury, are associated with an increased risk for bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH) after preterm birth. IGF-1 (insulin-like growth factor-1) is markedly decreased in normal preterm infants, but whether IGF-1 treatment can prevent BPD or PH is unknown.Objectives: To evaluate whether postnatal treatment with rhIGF-1 (recombinant human IGF-1)/BP3 (binding peptide 3) improves lung growth and prevents PH in two antenatal models of BPD induced by intraamniotic exposure to endotoxin (ETX) or sFlt-1 (soluble fms-like tyrosine kinase 1), and in a postnatal model due to prolonged hyperoxia.Methods: ETX or sFlt-1 were administered into the amniotic sac of pregnant rats at Embryonic Day 20 to simulate antenatal models of chorioamnionitis and preeclampsia, respectively. Pups were delivered by cesarean section at Embryonic Day 22 and treated with rhIGF-1/BP3 (0.02-20 mg/kg/d intraperitoneal) or buffer for 2 weeks. Study endpoints included radial alveolar counts (RACs), vessel density, and right ventricular hypertrophy (RVH). Direct effects of rhIGF-1/BP3 (250 ng/ml) on fetal lung endothelial cell proliferation and tube formation and alveolar type 2 cell proliferation were studied by standard methods in vitro.Measurements and Main Results: Antenatal ETX and antenatal sFlt-1 reduced RAC and decreased RVH in infant rats. In both models, postnatal rhIGF-1/BP3 treatment restored RAC and RVH to normal values when compared with placebo injections. rhIGF-1/BP3 treatment also preserved lung structure and prevented RVH after postnatal hyperoxia. In vitro studies showed that rhIGF-1/BP3 treatment increased lung endothelial cell and alveolar type 2 cell proliferation.Conclusions: Postnatal rhIGF-1/BP3 treatment preserved lung structure and prevented RVH in antenatal and postnatal BPD models. rhIGF-1/BP3 treatment may provide a novel strategy for the prevention of BPD in preterm infants.
Assuntos
Displasia Broncopulmonar/tratamento farmacológico , Hipertensão Pulmonar/prevenção & controle , Recém-Nascido Prematuro/crescimento & desenvolvimento , Fator de Crescimento Insulin-Like I/uso terapêutico , Pulmão/efeitos dos fármacos , Pulmão/crescimento & desenvolvimento , Cuidado Pós-Natal/métodos , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Displasia Broncopulmonar/fisiopatologia , Feminino , Humanos , Hipertensão Pulmonar/fisiopatologia , Lactente , Recém-Nascido , Masculino , Modelos Animais , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Rationale: Antenatal inflammation with placental dysfunction is strongly associated with high bronchopulmonary dysplasia (BPD) risk in preterm infants. Whether antenatal or postnatal HIF (hypoxia-inducible factor) augmentation can preserve lung structure and function and prevent pulmonary hypertension after intrauterine inflammation is controversial.Objectives: To determine whether antenatal or postnatal prolyl-hydroxylase inhibitor (PHi) therapy increases lung HIF expression, preserves lung growth and function, and prevents pulmonary hypertension in a rat model of chorioamnionitis-induced BPD caused by antenatal inflammation.Methods: Endotoxin (ETX) was administered to pregnant rats by intraamniotic injection at Embryonic Day 20, and pups were delivered by cesarean section at Embryonic Day 22. Selective PHi drugs, dimethyloxalylglycine or GSK360A, were administered into the amniotic space at Embryonic Day 20 or after birth by intraperitoneal injection for 2 weeks. Placentas and lung tissue were collected at birth for morphometric and Western blot measurements of HIF-1a, HIF-2a, VEGF (vascular endothelial growth factor), and eNOS (endothelial nitric oxide synthase) protein contents. At Day 14, lung function was assessed, and tissues were harvested to determine alveolarization by radial alveolar counts, pulmonary vessel density, and right ventricle hypertrophy (RVH).Measurements and Main Results: Antenatal PHi therapy preserves lung alveolar and vascular growth and lung function and prevents RVH after intrauterine ETX exposure. Antenatal administration of PHi markedly upregulates lung HIF-1a, HIF-2a, VEGF, and eNOS expression after ETX exposure.Conclusions: HIF augmentation improves lung structure and function, prevents RVH, and improves placental structure following antenatal ETX exposure. We speculate that antenatal or postnatal PHi therapy may provide novel strategies to prevent BPD due to antenatal inflammation.
Assuntos
Displasia Broncopulmonar/tratamento farmacológico , Fator 1 Induzível por Hipóxia/metabolismo , Pulmão/efeitos dos fármacos , Peptídeo PHI/farmacologia , Prenhez , Aminoácidos Dicarboxílicos/farmacologia , Animais , Animais Recém-Nascidos , Western Blotting , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patologia , Modelos Animais de Doenças , Endotoxinas/efeitos adversos , Endotoxinas/farmacologia , Feminino , Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Imuno-Histoquímica , Técnicas In Vitro , Injeções Intralesionais , Pulmão/embriologia , Gravidez , Cuidado Pré-Natal , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/crescimento & desenvolvimento , Circulação Pulmonar/efeitos dos fármacos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Valores de Referência , Testes de Função Respiratória , Técnicas de Cultura de TecidosRESUMO
Vitamin D deficiency (VDD) during pregnancy is associated with increased respiratory morbidities and risk for chronic lung disease after preterm birth. However, the direct effects of maternal VDD on perinatal lung structure and function and whether maternal VDD increases the susceptibility of lung injury due to hyperoxia are uncertain. In the present study, we sought to determine whether maternal VDD is sufficient to impair lung structure and function and whether VDD increases the impact of hyperoxia on the developing rat lung. Four-week-old rats were fed VDD chow and housed in a room shielded from ultraviolet A/B light to achieve 25-hydroxyvitamin D concentrations <10 ng/ml at mating and throughout lactation. Lung structure was assessed at 2 weeks for radial alveolar count, mean linear intercept, pulmonary vessel density, and lung function (lung compliance and resistance). The effects of hyperoxia for 2 weeks after birth were assessed after exposure to fraction of inspired oxygen of 0.95. At 2 weeks, VDD offspring had decreased alveolar and vascular growth and abnormal airway reactivity and lung function. Impaired lung structure and function in VDD offspring were similar to those observed in control rats exposed to postnatal hyperoxia alone. Maternal VDD causes sustained abnormalities of distal lung growth, increases in airway hyperreactivity, and abnormal lung mechanics during infancy. These changes in VDD pups were as severe as those measured after exposure to postnatal hyperoxia alone. We speculate that antenatal disruption of vitamin D signaling increases the risk for late-childhood respiratory disease.
Assuntos
Hiperóxia/complicações , Complacência Pulmonar/fisiologia , Lesão Pulmonar/etiologia , Pulmão/fisiopatologia , Deficiência de Vitamina D/complicações , Vitamina D/análogos & derivados , Animais , Animais Recém-Nascidos , Feminino , Hiperóxia/metabolismo , Pulmão/metabolismo , Lesão Pulmonar/metabolismo , Gravidez , Ratos , Vitamina D/metabolismoRESUMO
RATIONALE: Pregnancies complicated by antenatal stress, including preeclampsia (PE) and chorioamnionitis (CA), increase the risk for bronchopulmonary dysplasia (BPD) in preterm infants, but biologic mechanisms linking prenatal factors with BPD are uncertain. Levels of sFlt-1 (soluble fms-like tyrosine kinase 1), an endogenous antagonist to VEGF (vascular endothelial growth factor), are increased in amniotic fluid and maternal blood in PE and associated with CA. OBJECTIVES: Because impaired VEGF signaling has been implicated in the pathogenesis of BPD, we hypothesized that fetal exposure to sFlt-1 decreases lung growth and causes abnormal lung structure and pulmonary hypertension during infancy. METHODS: To test this hypothesis, we studied the effects of anti-sFlt-1 monoclonal antibody (mAb) treatment on lung growth in two established antenatal models of BPD that mimic PE and CA induced by intraamniotic (i.a.) injections of sFlt-1 or endotoxin, respectively. In experimental PE, mAb was administered by three different approaches, including antenatal treatment by either i.a. instillation or maternal uterine artery infusion, or by postnatal intraperitoneal injections. RESULTS: With each strategy, mAb therapy improved infant lung structure as assessed by radial alveolar count, vessel density, right ventricular hypertrophy, and lung function. As found in the PE model, the adverse lung effects of i.a. endotoxin were also reduced by antenatal or postnatal mAb therapy. CONCLUSIONS: We conclude that treatment with anti-sFlt-1 mAb preserves lung structure and function and prevents right ventricular hypertrophy in two rat models of BPD of antenatal stress and speculate that early mAb therapy may provide a novel strategy for the prevention of BPD.
Assuntos
Displasia Broncopulmonar/fisiopatologia , Endotélio Vascular/crescimento & desenvolvimento , Pulmão/crescimento & desenvolvimento , Alvéolos Pulmonares/crescimento & desenvolvimento , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/embriologia , Modelos Animais de Doenças , Endotélio Vascular/embriologia , Feminino , Humanos , Pulmão/embriologia , Gravidez , Alvéolos Pulmonares/embriologia , Ratos , Ratos Sprague-DawleyRESUMO
Intrauterine growth restriction (IUGR) in premature newborns increases the risk for bronchopulmonary dysplasia, a chronic lung disease characterized by disrupted pulmonary angiogenesis and alveolarization. We previously showed that experimental IUGR impairs angiogenesis; however, mechanisms that impair pulmonary artery endothelial cell (PAEC) function are uncertain. The NF-κB pathway promotes vascular growth in the developing mouse lung, and we hypothesized that IUGR disrupts NF-κB-regulated proangiogenic targets in fetal PAEC. PAECs were isolated from the lungs of control fetal sheep and sheep with experimental IUGR from an established model of chronic placental insufficiency. Microarray analysis identified suppression of NF-κB signaling and significant alterations in extracellular matrix (ECM) pathways in IUGR PAEC, including decreases in collagen 4α1 and laminin α4, components of the basement membrane and putative NF-κB targets. In comparison with controls, immunostaining of active NF-κB complexes, NF-κB-DNA binding, baseline expression of NF-κB subunits p65 and p50, and LPS-mediated inducible activation of NF-κB signaling were decreased in IUGR PAEC. Although pharmacological NF-κB inhibition did not affect angiogenic function in IUGR PAEC, angiogenic function of control PAEC was reduced to a similar degree as that observed in IUGR PAEC. These data identify reductions in endothelial NF-κB signaling as central to the disrupted angiogenesis observed in IUGR, likely by impairing both intrinsic PAEC angiogenic function and NF-κB-mediated regulation of ECM components necessary for vascular development. These data further suggest that strategies that preserve endothelial NF-κB activation may be useful in lung diseases marked by disrupted angiogenesis such as IUGR.
Assuntos
Displasia Broncopulmonar , Células Endoteliais , Retardo do Crescimento Fetal , Subunidade p50 de NF-kappa B/metabolismo , Artéria Pulmonar , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Animais , Displasia Broncopulmonar/induzido quimicamente , Displasia Broncopulmonar/embriologia , Displasia Broncopulmonar/patologia , Displasia Broncopulmonar/fisiopatologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Retardo do Crescimento Fetal/induzido quimicamente , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Retardo do Crescimento Fetal/fisiopatologia , Lipopolissacarídeos/toxicidade , Gravidez , Artéria Pulmonar/embriologia , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , OvinosRESUMO
BACKGROUND: Chorioamnionitis (CA) is associated with a high risk for the development of bronchopulmonary dysplasia (BPD) after preterm birth, but mechanisms that increase susceptibility for BPD and strategies to prevent BPD are uncertain. As a model of CA, antenatal intra-amniotic (IA) endotoxin (ETX) exposure alters placental structure, causes fetal growth restriction, increases perinatal mortality, and causes sustained cardiorespiratory abnormalities throughout infancy. Vitamin D (Vit D) has been shown to have both anti-inflammatory and proangiogenic properties. Antenatal IA treatment with Vit D (1,25-(OH)2D3) during IA ETX exposure improves survival and increases vascular and alveolar growth in infant rats. Whether IA ETX causes decreased placental vascular development and if the protective effects of prenatal Vit D treatment are due to direct effects on the fetus or to improved placental vascular development remain unknown. OBJECTIVE: The objective of this study was to determine if IA ETX impairs placental vascular development and Vit D metabolism, and whether 1,25-(OH)2D3 treatment improves placental vascularity after IA ETX exposure during late gestation in pregnant rats. DESIGN/METHODS: Fetal rats were exposed to ETX (10 mg), ETX + 1,25-(OH)2D3 (1 ng/mL), 1,25-(OH)2D3 (1 ng/mL), or saline (control) via IA injection at E20 and delivered 2 days later. To assess placental vascular development, histologic sections from the placenta were stained for CD31 and vessel density per high power field (HPF) was determined and analyzed using Matlab software. To determine the effects of ETX on placental Vit D metabolism, Vit D receptor (VDR) and activity of the Vit D conversion enzyme, CYP27B1, were assayed from placental homogenates. Angiogenic mediators were measured by reverse transcription polymerase chain reaction by RNA extracted from placental tissue. RESULTS: IA ETX reduced placenta and newborn birth weights by 22 and 20%, respectively, when compared with controls (placental weight: 0.60 vs. 0.47 g; p < 0.0001; birth weight: 4.68 vs. 5.88 g; p < 0.0001). IA 1,25-(OH)2D3 treatment increased birth weight by 12% in ETX-exposed pups (5.25 vs. 4.68 g; p < 0.001). IA ETX decreased placental vessel density by 24% in comparison with controls (1,114 vs. 848 vessels per HPF; p < 0.05). Treatment with IA 1,25-(OH)2D3 increased placenta vessel density twofold after ETX exposure (1,739 vs. 848); p < 0.0001), and increased vessel density compared with saline controls by 56% (1,739 vs. 1,114; p < 0.0001). IA ETX decreased both VDR and CYP27B1 expression by 83 and 35%, respectively (p < 0.01). CONCLUSION: IA ETX decreases placental growth and vessel density and decreases placental VDR and CYP27B1 protein expression, and that antenatal 1,25-(OH)2D3 restores placental weight and vessel density, as well as birth weight. We speculate that 1,25-(OH)2D3 treatment preserves placental function in experimental CA and that these effects may be mediated by increased vascular growth.
Assuntos
Indutores da Angiogênese/farmacologia , Displasia Broncopulmonar/prevenção & controle , Corioamnionite/prevenção & controle , Desenvolvimento Fetal/efeitos dos fármacos , Placenta , Vitamina D , Animais , Endotoxinas/antagonistas & inibidores , Feminino , Retardo do Crescimento Fetal/prevenção & controle , Placenta/irrigação sanguínea , Placenta/efeitos dos fármacos , Placenta/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Vitamina D/farmacologia , Vitaminas/farmacologiaRESUMO
Bronchopulmonary dysplasia (BPD) is the chronic lung disease associated with premature birth, characterized by impaired vascular and alveolar growth. In neonatal rats bleomycin decreases lung growth and causes pulmonary hypertension (PH), which is poorly responsive to nitric oxide. In the developing lung, through Rho kinase (ROCK) activation, ET-1 impairs endothelial cell function; however, whether ET-1-ROCK interactions contribute to impaired vascular and alveolar growth in experimental BPD is unknown. Neonatal rats were treated daily with intraperitoneal bleomycin with and without selective ETA (BQ123/BQ610) and ETB (BQ788) receptor blockers, nonselective ET receptor blocker (ETRB) (bosentan), or fasudil (ROCK inhibitor). At day 14, lungs were harvested for morphometrics, and measurements of Fulton's index (RV/LV+S), medial wall thickness (MWT), and vessel density. Lung ET-1 protein and ROCK activity (phospho-MYPT-1:total MYPT-1 ratio) were also measured by Western blot analysis. Bleomycin increased lung ET-1 protein expression by 65%, RV/LV+S by 60%, mean linear intercept (MLI) by 212%, and MWT by 140% and decreased radial alveolar count (RAC) and vessel density by 40 and 44%, respectively (P < 0.01 for each comparison). After bleomycin treatment, fasudil and bosentan partially restored RAC and vessel density and decreased MLI, RV/LV+S, and MWT to normal values. Bleomycin increased ROCK activity by 120%, which was restored to normal values by bosentan but not selective ETRB. We conclude that ET-1-ROCK interactions contribute to decreased alveolar and vascular growth and PH in experimental BPD. We speculate that nonselective ETRB and ROCK inhibitors may be effective in the treatment of infants with BPD and PH.
Assuntos
Endotelina-1/metabolismo , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Pulmão/patologia , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Animais Recém-Nascidos , Bleomicina , Vasos Sanguíneos/efeitos dos fármacos , Bosentana , Imunofluorescência , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/patologia , Imuno-Histoquímica , Injeções Intraperitoneais , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Piperidinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de Endotelina/metabolismo , Sulfonamidas/farmacologiaRESUMO
Prevention or treatment of lung diseases caused by the failure to form, or destruction of, existing alveoli, as observed in infants with bronchopulmonary dysplasia and adults with emphysema, requires understanding of the molecular mechanisms of alveolar development. In addition to its critical role in gas exchange, the pulmonary circulation also contributes to alveolar morphogenesis and maintenance by the production of paracrine factors, termed "angiocrines," that impact the development of surrounding tissue. To identify lung angiocrines that contribute to alveolar formation, we disrupted pulmonary vascular development by conditional inactivation of the Vegf-A gene during alveologenesis. This resulted in decreased pulmonary capillary and alveolar development and altered lung elastin and retinoic acid (RA) expression. We determined that RA is produced by pulmonary endothelial cells and regulates pulmonary angiogenesis and elastin synthesis by induction of VEGF-A and fibroblast growth factor (FGF)-18, respectively. Inhibition of RA synthesis in newborn mice decreased FGF-18 and elastin expression and impaired alveolarization. Treatment with RA and vitamin A partially reversed the impaired vascular and alveolar development induced by VEGF inhibition. Thus we identified RA as a lung angiocrine that regulates alveolarization through autocrine regulation of endothelial development and paracrine regulation of elastin synthesis via induction of FGF-18 in mesenchymal cells.
Assuntos
Células Endoteliais/metabolismo , Endotélio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Pulmão/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Capilares/metabolismo , Células Cultivadas , Camundongos Transgênicos , Neovascularização FisiológicaRESUMO
Impaired vascular endothelial growth factor (VEGF) signaling contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We hypothesized that the effects of VEGF on lung structure during development may be mediated through its downstream effects on both endothelial nitric oxide synthase (eNOS) and hepatocyte growth factor (HGF) activity, and that, in the absence of eNOS, trophic effects of VEGF would be mediated through HGF signaling. To test this hypothesis, we performed an integrative series of in vitro (fetal rat lung explants and isolated fetal alveolar and endothelial cells) and in vivo studies with normal rat pups and eNOS(-/-) mice. Compared with controls, fetal lung explants from eNOS(-/-) mice had decreased terminal lung bud formation, which was restored with recombinant human VEGF (rhVEGF) treatment. Neonatal eNOS(-/-) mice were more susceptible to hyperoxia-induced inhibition of lung growth than controls, which was prevented with rhVEGF treatment. Fetal alveolar type II (AT2) cell proliferation was increased with rhVEGF treatment only with mesenchymal cell (MC) coculture, and these effects were attenuated with anti-HGF antibody treatment. Unlike VEGF, HGF directly stimulated isolated AT2 cells even without MC coculture. HGF directly stimulates fetal pulmonary artery endothelial cell growth and tube formation, which is attenuated by treatment with JNJ-38877605, a c-Met inhibitor. rHGF treatment preserves alveolar and vascular growth after postnatal exposure to SU-5416, a VEGF receptor inhibitor. We conclude that the effects of VEGF on AT2 and endothelial cells during lung development are partly mediated through HGF-c-Met signaling and speculate that reciprocal VEGF-HGF signaling between epithelia and endothelia is disrupted in infants who develop BPD.
Assuntos
Fator de Crescimento de Hepatócito/fisiologia , Pulmão/crescimento & desenvolvimento , Fator A de Crescimento do Endotélio Vascular/fisiologia , Células Epiteliais Alveolares/fisiologia , Animais , Adesão Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Feminino , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/genética , Artéria Pulmonar/citologia , Artéria Pulmonar/metabolismo , OvinosRESUMO
Vitamin D [vit D; 1,25-(OH)2D] treatment improves survival and lung alveolar and vascular growth in an experimental model of bronchopulmonary dysplasia (BPD) after antenatal exposure to endotoxin (ETX). However, little is known about lung-specific 1,25-(OH)2D3 regulation during development, especially regarding maturational changes in lung-specific expression of the vitamin D receptor (VDR), 1α-hydroxylase (1α-OHase), and CYP24A1 during late gestation and the effects of antenatal ETX exposure on 1,25-(OH)2D3 metabolism in the lung. We hypothesized that vit D regulatory proteins undergo maturation regulation in the late fetal and early neonatal lung and that prenatal exposure to ETX impairs lung growth partly through abnormal endogenous vit D metabolism. Normal fetal rat lungs were harvested between embryonic day 15 and postnatal day 14. Lung homogenates were assayed for VDR, 1α-OHase, and CYP24A1 protein contents by Western blot analysis. Fetal rats were injected on embryonic day 20 with intra-amniotic ETX, ETX + 1,25-(OH)2D3, or saline and delivered 2 days later. Pulmonary artery endothelial cells (PAECs) from fetal sheep were assessed for VDR, 1α-OHase, and CYP24A1 expression after treatment with 25-(OH)D3, 1,25-(OH)2D3, ETX, ETX + 25-(OH)D3, or ETX + 1,25-(OH)2D3. We found that lung VDR, 1α-OHase, and CYP2741 protein expression dramatically increase immediately before birth (P < 0.01 vs. early fetal values). Antenatal ETX increases CYP24A1 expression (P < 0.05) and decreases VDR and 1α-OHase expression at birth (P < 0.001), but these changes are prevented with concurrent vit D treatment (P < 0.001). ETX-induced reduction of fetal PAEC growth and tube formation and lung 1α-OHase expression are prevented by vit D treatment (P < 0.001). We conclude that lung VDR, 1α-OHase, and CYP24A1 protein content markedly increase before birth and that antenatal ETX disrupts lung vit D metabolism through downregulation of VDR and increased vit D catabolic enzyme expression, including changes in developing endothelium. We speculate that endogenous vitamin D metabolism modulates normal fetal lung development and that prenatal disruption of vit D signaling may contribute to impaired postnatal lung growth at least partly through altered angiogenic signaling.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/biossíntese , Células Endoteliais/metabolismo , Endotoxinas/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Pulmão/embriologia , Receptores de Calcitriol/biossíntese , Animais , Displasia Broncopulmonar/induzido quimicamente , Displasia Broncopulmonar/embriologia , Displasia Broncopulmonar/patologia , Calcifediol/metabolismo , Células Endoteliais/patologia , Pulmão/patologia , Artéria Pulmonar/embriologia , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Ovinos , Vitamina D3 24-Hidroxilase/biossínteseRESUMO
High pulmonary vascular resistance (PVR), proximal pulmonary artery (PA) impedance, and right ventricular (RV) afterload due to remodeling contribute to the pathogenesis and severity of pulmonary hypertension (PH). Intra-amniotic exposure to endotoxin (ETX) causes sustained PH and high mortality in rat pups at birth, which are associated with impaired vascular growth and RV hypertrophy in survivors. Treatment of ETX-exposed pups with antenatal vitamin D (vit D) improves survival and lung growth, but the effects of ETX exposure on RV-PA coupling in the neonatal lung are unknown. We hypothesized that intrauterine ETX impairs RV-PA coupling through sustained abnormalities of PA stiffening and RV performance that are attenuated with vit D therapy. Fetal rats were exposed to intra-amniotic injections of ETX, ETX+vit D, or saline at 20 days gestation (term = 22 days). At postnatal day 14, pups had pressure-volume measurements of the RV and isolated proximal PA, respectively. Lung homogenates were assayed for extracellular matrix (ECM) composition by Western blot. We found that ETX lungs contain decreased α-elastin, lysyl oxidase, collagen I, and collagen III proteins (P < 0.05) compared control and ETX+vit D lungs. ETX-exposed animals have increased RV mechanical stroke work (P < 0.05 vs. control and ETX+vit D) and elastic potential energy (P < 0.05 vs. control and ETX+vit D). Mechanical stiffness and ECM remodeling are increased in the PA (P < 0.05 vs. control and ETX+vit D). We conclude that intrauterine exposure of fetal rats to ETX during late gestation causes persistent impairment of RV-PA coupling throughout infancy that can be prevented with early vit D treatment.
Assuntos
Endotoxinas/efeitos adversos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Pulmão/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Vitamina D/administração & dosagem , Animais , Animais Recém-Nascidos , Elastina/metabolismo , Feminino , Ventrículos do Coração/metabolismo , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Hipertrofia Ventricular Direita/induzido quimicamente , Hipertrofia Ventricular Direita/metabolismo , Hipertrofia Ventricular Direita/patologia , Pulmão/metabolismo , Pulmão/patologia , Gravidez , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Fenômenos Fisiológicos Respiratórios/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologiaRESUMO
BACKGROUND: Pulmonary hypertension (PH) secondary to vascular remodeling contributes to poor outcomes in congenital diaphragmatic hernia (CDH), however mechanisms responsible are unknown. We hypothesized that pulmonary artery endothelial cell (PAEC) dysfunction contributes to smooth muscle cell (SMC) hyperplasia in experimental CDH. METHODS: PAEC and SMC were isolated from fetal sheep with experimental CDH and controls. SMC growth was assessed alone and with SOD plus catalase and during coculture with control or CDH PAEC with and without ET-1 siRNA transfection. ET-1 protein was measured in PAEC and SMC lysates and supernatant. ROS production was measured in normal and CDH PAECs with and without ET-1 siRNA. PAEC growth and tube formation were measured with SOD plus catalase. RESULTS: CDH SMC growth was decreased and increased with coculture with CDH PAEC more than control PAEC. Treatment of CDH PAEC with SOD plus catalase or ET-1 siRNA prevented the increase in SMC growth seen with coculture. ET-1 protein was increased in CDH PAEC and SMC. ROS production was increased in CDH PAEC and decreased with ET-1 SiRNA. SOD plus catalase restored CDH PAEC growth and tube formation. CONCLUSION: PAEC dysfunction in experimental CDH increases SMC proliferation via ET-1 induced ROS production by PAEC.
Assuntos
Células Endoteliais/citologia , Hérnias Diafragmáticas Congênitas/patologia , Miócitos de Músculo Liso/citologia , Artéria Pulmonar/embriologia , Artéria Pulmonar/patologia , Animais , Bosentana , Catalase/metabolismo , Proliferação de Células , Técnicas de Cocultura , Modelos Animais de Doenças , Endotelina-1/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ovinos , Transdução de Sinais , Sulfonamidas/química , Superóxido Dismutase/metabolismoRESUMO
Exposure to intrauterine inflammation impairs lung growth but paradoxically protects the neonatal pulmonary vasculature from hyperoxic injury. The mechanisms mediating these contradictory effects are unknown. The objective is to identify the role of NF-κB in mediating cytoprotective and proinflammatory responses to inflammation in the fetal pulmonary endothelium. In newborn rats exposed to intra-amniotic LPS, we found increased expression of the NF-κB target gene manganese superoxide dismutase (MnSOD) in the pulmonary endothelium. Supporting these in vivo findings, LPS induced NF-κB activation and MnSOD expression in isolated fetal pulmonary arterial endothelial cells. In addition, LPS exposure caused apoptosis and suppressed cellular growth and induced P-selectin expression. LPS-induced NF-κB activation that proceeded through specific isoforms of the inhibitory protein IκB mediated these diverse responses; NF-κB signaling through IκBα degradation resulted in MnSOD upregulation and preserved cell growth, whereas NF-κB signaling through IκBß degradation mediated apoptosis and P-selectin expression. These findings suggest that selective inhibition of NF-κB activation that results from IκBß degradation preserves the enhanced antioxidant defense and protects the developing pulmonary vascular endothelium from ongoing inflammatory injury.
Assuntos
Endotélio Vascular/imunologia , Feto/imunologia , Quinase I-kappa B/fisiologia , Proteínas I-kappa B/fisiologia , Mediadores da Inflamação/fisiologia , Pulmão/imunologia , NF-kappa B/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Antioxidantes/metabolismo , Apoptose/imunologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Feto/irrigação sanguínea , Feto/patologia , Pulmão/irrigação sanguínea , Pulmão/enzimologia , Inibidor de NF-kappaB alfa , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase/biossíntese , Superóxido Dismutase/metabolismoRESUMO
Vitamin D (vit D) has anti-inflammatory properties and modulates lung growth, but whether vit D can prevent lung injury after exposure to antenatal inflammation is unknown. We hypothesized that early and sustained vit D treatment could improve survival and preserve lung growth in an experimental model of bronchopulmonary dysplasia induced by antenatal exposure to endotoxin (ETX). Fetal rats (E20) were exposed to ETX (10 µg), ETX + Vit D (1 ng/ml), or saline (control) via intra-amniotic (IA) injections and delivered 2 days later. Newborn pups exposed to IA ETX received daily intraperitoneal injections of vit D (1 ng/g) or saline for 14 days. Vit D treatment improved oxygen saturations (78 vs. 87%; P < 0.001) and postnatal survival (84% vs. 57%; P < 0.001) after exposure to IA ETX compared with IA ETX alone. Postnatal vit D treatment improved alveolar and vascular growth at 14 days by 45% and 25%, respectively (P < 0.05). Vit D increased fetal sheep pulmonary artery endothelial cell (PAEC) growth and tube formation by 64% and 44%, respectively (P < 0.001), and prevented ETX-induced reductions of PAEC growth and tube formation. Vit D directly increased fetal alveolar type II cell (ATIIC) growth by 26% (P < 0.001) and enhanced ATIIC growth in the presence of ETX-induced growth suppression by 73% (P < 0.001). We conclude that antenatal vit D therapy improved oxygenation and survival in newborn rat pups and enhanced late lung structure after exposure to IA ETX in vivo, which may partly be due to direct effects on vascular and alveolar growth.
Assuntos
Displasia Broncopulmonar/prevenção & controle , Endotoxinas/toxicidade , Alvéolos Pulmonares/efeitos dos fármacos , Vitamina D/farmacologia , Vitaminas/farmacologia , Líquido Amniótico/metabolismo , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/induzido quimicamente , Displasia Broncopulmonar/mortalidade , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Feminino , Masculino , Oxigênio/sangue , Gravidez , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/crescimento & desenvolvimento , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/crescimento & desenvolvimento , Ratos , Ratos Sprague-Dawley , Ovinos , Taxa de SobrevidaRESUMO
Peroxisome proliferator-activated receptor-γ (PPARγ) and Rho-kinase (ROCK) regulate smooth muscle cell (SMC) proliferation and contribute to vascular remodeling in adult pulmonary hypertension. Whether these pathways interact to contribute to the development of vascular remodeling in persistent pulmonary hypertension of the newborn (PPHN) remains unknown. We hypothesized that ROCK-PPARγ interactions increase SMC proliferation resulting in vascular remodeling in experimental PPHN. Pulmonary artery SMCs (PASMCs) were harvested from fetal sheep after partial ligation of the ductus arteriosus in utero (PPHN) and controls. Cell counts were performed daily for 5 days with or without PPARγ agonists and ROCK inhibition. PPARγ and ROCK protein expression/activity were measured by Western blot in normal and PPHN PASMCs. We assessed PPARγ-ROCK interactions by studying the effect of ROCK activation on PPARγ activity and PPARγ inhibition (siRNA) on ROCK activity and PASMC proliferation. At baseline, PPHN PASMC cell number was increased by 38% above controls on day 5. ROCK protein expression/activity were increased by 25 and 34% and PPARγ protein/activity decreased by 40 and 50% in PPHN PASMC. ROCK inhibition and PPARγ activation restored PPHN PASMC growth to normal values. ROCK inhibition increased PPARγ activity by 50% in PPHN PASMC, restoring PPARγ activity to normal. In normal PASMCs, ROCK activation decreased PPARγ activity and PPARγ inhibition increased ROCK activity and cell proliferation, resulting in a PPHN hyperproliferative PASMC phenotype. PPARγ-ROCK interactions regulate SMC proliferation and contribute to increased PPHN PASMC proliferation and vascular remodeling in PPHN. Restoring normal PPARγ-ROCK signaling may prevent vascular remodeling and improve outcomes in PPHN.
Assuntos
PPAR gama/metabolismo , Síndrome da Persistência do Padrão de Circulação Fetal/metabolismo , Quinases Associadas a rho/metabolismo , Animais , PPAR gama/antagonistas & inibidores , PPAR gama/biossíntese , Ovinos , Transdução de Sinais , Quinases Associadas a rho/biossínteseRESUMO
Increased endothelin-1 (ET-1) disrupts angiogenesis in persistent pulmonary hypertension of the newborn (PPHN), but pathogenic mechanisms are unclear. Peroxisome proliferator activated receptor γ (PPARγ) is decreased in adult pulmonary hypertension, but whether ET-1-PPARγ interactions impair endothelial cell function and angiogenesis in PPHN remains unknown. We hypothesized that increased PPHN pulmonary artery endothelial cell (PAEC) ET-1 production decreases PPARγ signaling and impairs tube formation in vitro. Proximal PAECs were harvested from fetal sheep after partial ligation of the ductus arteriosus in utero (PPHN) and controls. PPARγ and phospho-PPARγ protein were compared between normal and PPHN PAECs ± ET-1 and bosentan (ETA/ETB receptor blocker). Tube formation was assessed in response to PPARγ agonists ± ET-1, N-nitro-l-arginine (LNA) (NOS inhibitor), and PPARγ siRNA. Endothelial NO synthase (eNOS), phospho-eNOS, and NO production were measured after exposure to PPARγ agonists and PPARγ siRNA. At baseline, PPHN PAECs demonstrate decreased tube formation and PPARγ protein expression and activity. PPARγ agonists restored PPHN tube formation to normal. ET-1 decreased normal and PPHN PAEC tube formation, which was rescued by PPARγ agonists. ET-1 decreased PPARγ protein and activity, which was prevented by bosentan. PPARγ agonists increased eNOS protein and activity and NO production in normal and PPHN PAECs. LNA inhibited the effect of PPARγ agonists on tube formation. PPARγ siRNA decreased eNOS protein and tube formation in normal PAECs. We conclude that ET-1 decreases PPARγ signaling and contributes to PAEC dysfunction and impaired angiogenesis in PPHN. We speculate that therapies aimed at decreasing ET-1 production will restore PPARγ signaling, preserve endothelial function, and improve angiogenesis in PPHN.