Vascular mediators in chronic lung disease of infancy: Role of endothelial monocyte activating polypeptide II (EMAP II)

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of prematurity. Over the years, the BPD phenotype has evolved, but despite various advances in neonatal management approaches, the reduction in the BPD burden is minimal. With the advent of surfactant, glucocorticoids, and new ventilation strategies, BPD has evolved from a disease of structural injury into a new BPD, marked by an arrest in alveolar growth in the lungs of extremely premature infants. This deficient alveolar growth has been associated with a diminution of pulmonary vasculature. Several investigators have described the epithelial / vascular co‐dependency and the significant role of crosstalk between vessel formation, alveologenesis, and lung dysplasia's; hence identification and study of factors that regulate pulmonary vascular emergence and inflammation has become crucial in devising effective therapeutic approaches for this debilitating condition. The potent antiangiogenic and proinflammatory protein Endothelial Monocyte Activating Polypeptide II (EMAP II) has been described as a mediator of pulmonary vascular and alveolar formation and its expression is inversely related to the periods of vascularization and alveolarization in the developing lung. Hence the study of EMAP II could play a vital role in studying and devising appropriate therapeutics for diseases of aberrant lung development, such as BPD. Herein, we review the vascular contribution to lung development and the implications that vascular mediators such as EMAP II have in distal lung formation during the vulnerable stage of alveolar genesis. Birth Defects Research (Part A) 100:180–188, 2014. © 2014 Wiley Periodicals, Inc.     

TGF-beta-neutralizing antibodies improve pulmonary alveologenesis and vasculogenesis in the injured newborn lung

Pulmonary injury is associated with the disruption of alveologenesis in the developing lung and causes bronchopulmonary dysplasia (BPD) in prematurely born infants. Transforming growth factor (TGF)-beta is an important regulator of cellular differentiation and early lung development, and its levels are increased in newborn lung injury. Although overexpression of TGF-beta in the lungs of newborn animals causes pathological features that are consistent with BPD, the role of endogenous TGF-beta in the inhibition of the terminal stage of lung development is incompletely understood. In this investigation, the hypothesis that O(2)-induced injury of the maturing lung is associated with TGF-beta-mediated disruption of alveologenesis and microvascular development was tested using a murine model of BPD. Here we report that treatment of developing mouse lungs with TGF-beta-neutralizing antibodies attenuates the increase in pulmonary cell phospho-Smad2 nuclear localization, which is indicative of augmented TGF-beta signaling, associated with pulmonary injury induced by chronic inhalation of 85% oxygen. Importantly, the neutralization of the abnormal TGF-beta activity improves quantitative morphometric indicators of alveologenesis, extracellular matrix assembly, and microvascular development in the injured developing lung. Furthermore, exposure to anti-TGF-beta antibodies is associated with improved somatic growth in hyperoxic mouse pups and not with an increase in pulmonary inflammation. These studies indicate that excessive pulmonary TGF-beta signaling in the injured newborn lung has an important role in the disruption of the terminal stage of lung development. In addition, they suggest that anti-TGF-beta antibodies may be an effective therapy for preventing some important developmental diseases of the newborn lung.     

Phosphodiesterase 4 inhibition attenuates persistent heart and lung injury by neonatal hyperoxia in rats

Phosphodiesterase (PDE) 4 inhibitors are potent anti-inflammatory drugs with antihypertensive properties, and their therapeutic role in bronchopulmonary dysplasia (BPD) is still controversial. We studied the role of PDE4 inhibition with piclamilast on normal lung development and its therapeutic value on pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) in neonatal rats with hyperoxia-induced lung injury, a valuable model for premature infants with severe BPD. The cardiopulmonary effects of piclamilast treatment (5 mg·kg(-1)·day(-1)) were investigated in two models of experimental BPD: 1) daily treatment during continuous exposure to hyperoxia for 10 days; and 2) late treatment and injury-recovery in which pups were exposed to hyperoxia or room air for 9 days, followed by 9 or 42 days of recovery in room air combined with treatment started on day 6 of oxygen exposure until day 18. Prophylactic piclamilast treatment reduced pulmonary fibrin deposition, septum thickness, arteriolar wall thickness, arteriolar vascular smooth muscle cell proliferation and RVH, and prolonged survival. In the late treatment and injury-recovery model, hyperoxia caused persistent aberrant alveolar and vascular development, PH, and RVH. Treatment with piclamilast in both models reduced arteriolar wall thickness, attenuated RVH, and improved right ventricular function in the injury recovery model, but did not restore alveolarization or angiogenesis. Treatment with piclamilast did not show adverse cardiopulmonary effects in room air controls in both models. In conclusion, PDE4 inhibition attenuated and partially reversed PH and RVH, but did not advance alveolar development in neonatal rats with hyperoxic lung injury or affect normal lung and heart development.     

Pulmonary hypertension impairs alveolarization and reduces lung growth in the ovine fetus

Persistent pulmonary hypertension of the newborn (PPHN) is a clinical disorder characterized by abnormal vascular structure, growth, and reactivity. Disruption of vascular growth during early postnatal lung development impairs alveolarization, and newborns with lung hypoplasia often have severe pulmonary hypertension. To determine whether pulmonary hypertension can directly impair vascular growth and alveolarization in the fetus, we studied the effects of chronic intrauterine pulmonary hypertension on lung growth in fetal lambs. We performed surgery, which included partial constriction of the ductus arteriosus (DA) to induce pulmonary hypertension (PH, n = 14) or sham surgery (controls, n = 13) in fetal lambs at 112-125 days (term = 147 days). Tissues were harvested near term for measurement of right ventricular hypertrophy (RVH), radial alveolar counts (RAC), mean linear intercepts (MLI), wall thickness, and vessel density of small pulmonary arteries. Chronic DA constriction caused RVH (P < 0.0001), increased wall thickness of small pulmonary arteries (P < 0.002), and reduced small pulmonary artery density (P < 0.005). PH also reduced alveolarization, causing a 27% reduction in RAC and 20% increase in MLI. Furthermore, prolonged DA constriction (21 days) not only decreased RAC and increased MLI by 30% but also caused a 25% reduction of lung-body weight ratio. We conclude that chronic PH reduces pulmonary arterial growth, decreases alveolar complexity, and impairs lung growth. We speculate that chronic hypertension impairs vascular growth, which disrupts critical signaling pathways regulating lung vascular and alveolar development, thereby interfering with alveolarization and ultimately resulting in lung hypoplasia.     

ANG-1 TIE-2 and BMPR signalling defects are not seen in the nitrofen model of pulmonary hypertension and congenital diaphragmatic hernia

Background

Pulmonary hypertension (PH) is a lethal disease that is associated with characteristic histological abnormalities of the lung vasculature and defects of angiopoetin-1 (ANG-1), TIE-2 and bone morphogenetic protein receptor (BMPR)-related signalling. We hypothesized that if these signalling defects cause PH generically, they will be readily identifiable perinatally in congenital diaphragmatic hernia (CDH), where the typical pulmonary vascular changes are present before birth and are accompanied by PH after birth.

Methods

CDH (predominantly left-sided, LCDH) was created in Sprague-Dawley rat pups by e9.5 maternal nitrofen administration. Left lungs from normal and LCDH pups were compared at fetal and postnatal time points for ANG-1, TIE-2, phosphorylated-TIE-2, phosphorylated-SMAD1/5/8 and phosphorylated-ERK1/2 by immunoprecipitation and Western blotting of lung protein extracts and by immunohistochemistry on lung sections.

Results

In normal lung, pulmonary ANG-1 protein levels fall between fetal and postnatal life, while TIE-2 levels increase. Over the corresponding time period, LCDH lung retained normal expression of ANG-1, TIE-2, phosphorylated-TIE-2 and, downstream of BMPR, phosphorylated-SMAD1/5/8 and phosphorylated-p44/42.

Conclusion

In PH and CDH defects of ANG-1/TIE-2/BMPR-related signalling are not essential for the lethal vasculopathy.     

Congenital diaphragmatic hernia: A retinoid‐signaling pathway disruption during lung development?

Congenital diaphragmatic hernia (CDH) usually occurs sporadically. The prognosis remains poor, with a 50% perinatal mortality rate. Most deaths result from hypoxemia due to lung hypoplasia and abnormal development of pulmonary vasculature that results in persistent pulmonary hypertension. Our current understanding of the pathogenesis of CDH is based on an assumption linking herniation of abdominal viscera into the thorax with compression of the developing lung. Pulmonary hypoplasia, however, can also result from reduced distension of the developing lung secondary to impaired fetal breathing movements. Moreover, a nitrofen-induced CDH model shows that lung hypoplasia precedes the diaphragmatic defect, leading to a "dual-hit hypothesis." Recent data reveal the role of a retinoid-signaling pathway disruption in the pathogenesis of CDH. We describe the clinical and epidemiological aspects of human CDH, the metabolic and molecular aspects of the retinoid-signaling pathway, and the implications of retinoids in the development of the diaphragm and the lung. Finally, we highlight the existing links between CDH and disruption of the retinoid-signaling pathway, which may suggest an eventual use of retinoids in the treatment of CDH.     

Assessment of inhibited alveolar‐capillary membrane structural development and function in bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of extreme prematurity and is defined clinically by dependence on supplemental oxygen due to impaired gas exchange. Optimal gas exchange is dependent on the development of a sufficient surface area for diffusion. In the mammalian lung, rapid acquisition of distal lung surface area is accomplished in neonatal and early adult life by means of vascularization and secondary septation of distal lung airspaces. Extreme preterm birth interrupts secondary septation and pulmonary capillary development and ultimately reduces the efficiency of the alveolar‐capillary membrane. Although pulmonary health in BPD infants rapidly improves over the first few years, persistent alveolar‐capillary membrane dysfunction continues into adolescence and adulthood. Preventative therapies have been largely ineffective, and therapies aimed at promoting normal development of the air‐blood barrier in infants with established BPD remain largely unexplored. The purpose of this review will be: (1) to summarize the histological evidence of aberrant alveolar‐capillary membrane development associated with extreme preterm birth and BPD, (2) to review the clinical evidence assessing the long‐term impact of BPD on alveolar‐capillary membrane function, and (3) to discuss the need to develop and incorporate direct measurements of functional gas exchange into clinically relevant animal models of inhibited alveolar development. Birth Defects Research (Part A) 100:168–179, 2014. © 2014 Wiley Periodicals, Inc.     

The Axonal Guidance Cue Semaphorin 3C Contributes to Alveolar Growth and Repair

Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD) in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C), contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O₂-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.     

IL-33-ST2 pathway regulates AECII transdifferentiation by targeting alveolar macrophage in a bronchopulmonary dysplasia mouse model

Evidence points to the indispensable function of alveolar macrophages (AMs) in normal lung development and tissue homeostasis. However, the importance of AMs in bronchopulmonary dysplasia (BPD) has not been elucidated. Here, we identified a significant role of abnormal AM proliferation and polarization in alveolar dysplasia during BPD, which is closely related to the activation of the IL-33-ST2 pathway. Compared with the control BPD group, AMs depletion partially abolished the epithelialmesenchymal transition process of AECII and alleviated pulmonary differentiation arrest. In addition, IL-33 or ST2 knockdown has protective effects against lung injury after hyperoxia, which is associated with reduced AM polarization and proliferation. The protective effect disappeared following reconstitution of AMs in injured IL-33 knockdown mice, and the differentiation of lung epithelium was blocked again. In conclusion, the IL-33-ST2 pathway regulates AECII transdifferentiation by targeting AMs proliferation and polarization in BPD, which shows a novel strategy for manipulating the IL-33–ST2-AMs axis for the diagnosis and intervention of BPD.     

Aberrant signaling pathways of the lung mesenchyme and their contributions to the pathogenesis of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in infants born extremely preterm, typically before 28 weeks' gestation, characterized by a prolonged need for supplemental oxygen or positive pressure ventilation beyond 36 weeks postmenstrual age. The limited number of autopsy samples available from infants with BPD in the postsurfactant era has revealed a reduced capacity for gas exchange resulting from simplification of the distal lung structure with fewer, larger alveoli because of a failure of normal lung alveolar septation and pulmonary microvascular development. The mechanisms responsible for alveolar simplification in BPD have not been fully elucidated, but mounting evidence suggests that aberrations in the cross-talk between growth factors of the lung mesenchyme and distal airspace epithelium have a key role. Animal models that recapitulate the human condition have expanded our knowledge of the pathology of BPD and have identified candidate matrix components and growth factors in the developing lung that are disrupted by conditions that predispose infants to BPD and interfere with normal vascular and alveolar morphogenesis. This review focuses on the deviations from normal lung development that define the pathophysiology of BPD and summarizes the various candidate mesenchyme-associated proteins and growth factors that have been identified as being disrupted in animal models of BPD. Finally, future areas of research to identify novel targets affected in arrested lung development and recovery are discussed.     

Vasculoprotective effects of heme oxygenase-1 in a murine model of hyperoxia-induced bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is characterized by simplified alveolarization and arrested vascular development of the lung with associated evidence of endothelial dysfunction, inflammation, increased oxidative damage, and iron deposition. Heme oxygenase-1 (HO-1) has been reported to be protective in the pathogenesis of diseases of inflammatory and oxidative etiology. Because HO-1 is involved in the response to oxidative stress produced by hyperoxia and is critical for cellular heme and iron homeostasis, it could play a protective role in BPD. Therefore, we investigated the effect of HO-1 in hyperoxia-induced lung injury using a neonatal transgenic mouse model with constitutive lung-specific HO-1 overexpression. Hyperoxia triggered an increase in pulmonary inflammation, arterial remodeling, and right ventricular hypertrophy that was attenuated by HO-1 overexpression. In addition, hyperoxia led to pulmonary edema, hemosiderosis, and a decrease in blood vessel number, all of which were markedly improved in HO-1 overexpressing mice. The protective vascular response may be mediated at least in part by carbon monoxide, due to its anti-inflammatory, antiproliferative, and antiapoptotic properties. HO-1 overexpression, however, did not prevent alveolar simplification nor altered the levels of ferritin and lactoferrin, proteins involved in iron binding and transport. Thus the protective mechanisms elicited by HO-1 overexpression primarily preserve vascular growth and barrier function through iron-independent, antioxidant, and anti-inflammatory pathways.     

CTGF disrupts alveolarization and induces pulmonary hypertension in neonatal mice: implication in the pathogenesis of severe bronchopulmonary dysplasia

The pathological hallmarks of bronchopulmonary dysplasia (BPD), one of the most common long-term pulmonary complications associated with preterm birth, include arrested alveolarization, abnormal vascular growth, and variable interstitial fibrosis. Severe BPD is often complicated by pulmonary hypertension characterized by excessive pulmonary vascular remodeling and right ventricular hypertrophy that significantly contributes to the mortality and morbidity of these infants. Connective tissue growth factor (CTGF) is a multifunctional protein that coordinates complex biological processes during tissue development and remodeling. We have previously shown that conditional overexpression of CTGF in airway epithelium under the control of the Clara cell secretory protein promoter results in BPD-like architecture in neonatal mice. In this study, we have generated a doxycycline-inducible double transgenic mouse model with overexpression of CTGF in alveolar type II epithelial (AT II) cells under the control of the surfactant protein C promoter. Overexpression of CTGF in neonatal mice caused dramatic macrophage and neutrophil infiltration in alveolar air spaces and perivascular regions. Overexpression of CTGF also significantly decreased alveolarization and vascular development. Furthermore, overexpression of CTGF induced pulmonary vascular remodeling and pulmonary hypertension. Most importantly, we have also demonstrated that these pathological changes are associated with activation of integrin-linked kinase (ILK)/glucose synthesis kinase-3β (GSK-3β)/β-catenin signaling. These data indicate that overexpression of CTGF in AT II cells results in lung pathology similar to those observed in infants with severe BPD and that ILK/GSK-3β/β-catenin signaling may play an important role in the pathogenesis of severe BPD.     

Pulmonary hypertension in bronchopulmonary dysplasia

Pulmonary hypertension is common in bronchopulmonary dysplasia and is associated with increased mortality and morbidity. This pulmonary hypertension is due to abnormal microvascular development and pulmonary vascular remodeling resulting in reduced cross‐sectional area of pulmonary vasculature. The epidemiology, etiology, clinical features, diagnosis, suggested management, and outcomes of pulmonary hypertension in the setting of bronchopulmonary dysplasia are reviewed. In summary, pulmonary hypertension is noted in a fifth of extremely low birth weight infants, primarily those with moderate or severe bronchopulmonary dysplasia, and persists to discharge in many infants. Diagnosis is generally by echocardiography, and some infants require cardiac catheterization to identify associated anatomic cardiac lesions or systemic‐pulmonary collaterals, pulmonary venous obstruction or myocardial dysfunction. Serial echocardiography and B‐type natriuretic peptide measurement may be useful for following the course of pulmonary hypertension. Currently, there is not much evidence to indicate optimal management approaches, but many clinicians maintain oxygen saturation in the range of 91 to 95%, avoiding hypoxia and hyperoxia, and often provide inhaled nitric oxide, sometimes combined with sildenafil, prostacyclin, or its analogs, and occasionally endothelin‐receptor antagonists. Birth Defects Research (Part A) 100:240–246, 2014. © 2014 Wiley Periodicals, Inc.     

Downregulation of type II bone morphogenetic protein receptor in hypoxic pulmonary hypertension

Heterozygous mutations in the type II receptor for bone morphogenetic protein (BMPR-II) and dysfunction of BMPR-II have been implicated in patients with primary pulmonary hypertension (PH). To clarify the possible involvement of BMP and BMPR-II in the development of hypoxic PH, the expression of BMP-2, BMPR-II, and their downstream signals were investigated in rat lung under normal and hypoxic conditions by RT-PCR, immunoblot, and immunohistochemical methods. In rats under normal conditions, BMP-2 is localized in the endothelium of the pulmonary artery, whereas BMPR-II is abundantly expressed in the endothelium, smooth muscle cells, and adventitial fibroblasts. After 0.5 and 3 days of exposure to hypoxia, upregulation of BMP-2 was observed in the intrapulmonary arteries. The change was accompanied by activation of its downstream signaling, p38 MAPK, and Erk1/2 MAPK, and the apoptotic process, measured by caspase-3 activity and TdT-mediated dUTP nick end labeling-positive cells. In contrast, a significant decrease in the expression of BMPR-II and inactivation of p38 MAPK and caspase-3 were observed in the pulmonary vasculature after 7-21 days of hypoxia exposure. Because BMP-2 is known to inhibit proliferation of vascular smooth muscle cells and promote cellular apoptosis, disruption of BMP signaling pathway through downregulation of BMPR-II in chronic hypoxia may result in pulmonary vascular remodeling due to the failure of critical antiproliferative/differentiation programs in the pulmonary vasculature. These results suggest abrogation of BMP signaling may be a common molecular pathogenesis in the development of PH with various pathophysiological events, including primary and hypoxic PH.     

Matrix metalloproteinase gene polymorphisms and bronchopulmonary dysplasia: identification of MMP16 as a new player in lung development

Backgound

Alveolarization requires coordinated extracellular matrix remodeling, a process in which matrix metalloproteinases (MMPs) play an important role. We postulated that polymorphisms in MMP genes might affect MMP function in preterm lungs and thus influence the risk of bronchopulmonary dysplasia (BPD).

Methods and Findings

Two hundred and eighty-four consecutive neonates with a gestational age of <28 weeks were included in this prospective study. Forty-five neonates developed BPD. Nine single-nucleotide polymorphisms (SNPs) were sought in the MMP2, MMP14 and MMP16 genes. After adjustment for birth weight and ethnic origin, the TT genotype of MMP16 C/T (rs2664352) and the GG genotype of MMP16 A/G (rs2664349) were found to protect from BPD. These genotypes were also associated with a smaller active fraction of MMP2 and with a 3-fold-lower MMP16 protein level in tracheal aspirates collected within 3 days after birth. Further evaluation of MMP16 expression during the course of normal human and rat lung development showed relatively low expression during the canalicular and saccular stages and a clear increase in both mRNA and protein levels during the alveolar stage. In two newborn rat models of arrested alveolarization the lung MMP16 mRNA level was less than 50% of normal.

Conclusions

MMP16 may be involved in the development of lung alveoli. MMP16 polymorphisms appear to influence not only the pulmonary expression and function of MMP16 but also the risk of BPD in premature infants.     

Heparan sulfate in lung morphogenesis: The elephant in the room

Heparan sulfate (HS) is a structurally complex polysaccharide located on the cell surface and in the extracellular matrix, where it participates in numerous biological processes through interactions with a vast number of regulatory proteins such as growth factors and morphogens. HS is crucial for lung development; disruption of HS synthesis in flies and mice results in a major aberration of airway branching, and in mice, it results in neonatal death as a consequence of malformed lungs and respiratory distress. Epithelial–mesenchymal interactions governing lung morphogenesis are directed by various diffusible proteins, many of which bind to, and are regulated by HS, including fibroblast growth factors, sonic hedgehog, and bone morphogenetic proteins. The majority of research into the molecular mechanisms underlying defective lung morphogenesis and pulmonary pathologies, such as bronchopulmonary dysplasia and pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH), has focused on abnormal protein expression. The potential contribution of HS to abnormalities of lung development has yet to be explored to any significant extent, which is somewhat surprising given the abnormal lung phenotype exhibited by mutant mice synthesizing abnormal HS. This review summarizes our current understanding of the role of HS and HS‐binding proteins in lung morphogenesis and will present in vitro and in vivo evidence for the fundamental importance of HS in airway development. Finally, we will discuss the future possibility of HS‐based therapeutics for ameliorating insufficient lung growth associated with lung diseases such as CDH. Birth Defects Research (Part C) 90:32–44, 2010. © 2010 Wiley‐Liss, Inc.     

Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.     

Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia

Epidemiological studies have shown that maternal preeclampsia (PE) increases the risk of bronchopulmonary dysplasia (BPD), but the underlying mechanism is unknown. Soluble vascular endothelial growth factor receptor-1 (soluble VEGFR1, known as soluble fms-like tyrosine kinase 1, or sFlt-1), an endogenous antagonist of vascular endothelial growth factor (VEGF), is markedly elevated in amniotic fluid and maternal blood in PE. Therefore, we hypothesized that antenatal exposure to excess sFlt-1 disrupts lung development through impaired VEGF signaling in utero, providing a mechanistic link between PE and BPD. To determine whether increased sFlt-1 in amniotic fluid is sufficient to cause sustained abnormalities of lung structure during infancy, sFlt-1 or saline was injected into amniotic sacs of pregnant Sprague-Dawley rats at 20 days of gestation (term, 22 days). After birth, pups were observed through 14 days of age for study. We found that intra-amniotic sFlt-1 treatment decreased alveolar number, reduced pulmonary vessel density, and caused right and left ventricular hypertrophy in 14-day-old rats. In addition, intra-amniotic sFlt-1 treatment suppressed activation of lung VEGF receptor-2 and increased apoptosis in endothelial and mesenchymal cells in the newborn lung. We conclude that exposure to excess sFlt-1 in amniotic fluid during late gestation causes sustained reductions in alveolarization and pulmonary vascular growth during infancy, accompanied by biventricular hypertrophy suggesting pulmonary and systemic hypertension. We speculate that impaired VEGF signaling in utero due to exposure of high amniotic fluid levels of sFlt-1 in PE disrupts lung growth and contributes to the increased risk of BPD in infants born to mothers with PE.     

Defective pulmonary innervation and autonomic imbalance in congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is associated with significant mortality due to lung hypoplasia and pulmonary hypertension. The role of embryonic pulmonary innervation in normal lung development and lung maldevelopment in CDH has not been defined. We hypothesize that developmental defects of intrapulmonary innervation, in particular autonomic innervation, occur in CDH. This abnormal embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. To define patterns of pulmonary innervation in CDH, human CDH and control lung autopsy specimens were stained with the pan-neural marker S-100. To further characterize patterns of overall and autonomic pulmonary innervation during lung development in CDH, the murine nitrofen model of CDH was utilized. Immunostaining for protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (a sympathetic marker), vesicular acetylcholine transporter (a parasympathetic marker), or VIP (a parasympathetic marker) was performed on lung whole mounts and analyzed via confocal microscopy and three-dimensional reconstruction. Peribronchial and perivascular neuronal staining pattern is less complex in human CDH than control lung. In mice, protein gene product 9.5 staining reveals less complex neuronal branching and decreased neural tissue in nitrofen-treated lungs from embryonic day 12.5 to 16.5 compared with controls. Furthermore, nitrofen-treated embryonic lungs exhibited altered autonomic innervation, with a relative increase in sympathetic nerve staining and a decrease in parasympathetic nerve staining compared with controls. These results suggest a primary defect in pulmonary neural developmental in CDH, resulting in less complex neural innervation and autonomic imbalance. Defective embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH.