Abnormal Alveolarization Research Articles

TAZ deficiency impairs the autophagy-lysosomal pathway through NRF2 dysregulation and lysosomal dysfunction.

Transcriptional coactivator with a PDZ-binding motif (TAZ) plays a key role in normal tissue homeostasis and tumorigenesis through interaction with several transcription factors. In particular, TAZ deficiency causes abnormal alveolarization and emphysema, and persistent TAZ overexpression contributes to lung cancer and pulmonary fibrosis, suggesting the possibility of a complex mechanism of TAZ function. Recent studies suggest that nuclear factor erythroid 2-related factor 2 (NRF2), an antioxidant defense system, induces TAZ expression during tumorigenesis and that TAZ also activates the NRF2-mediated antioxidant pathway. We thus thought to elucidate the cross-regulation of TAZ and NRF2 and the underlying molecular mechanisms and functions. TAZ directly interacted with NRF2 through the N-terminal domain and suppressed the transcriptional activity of NRF2 by preventing NRF2 from binding to DNA. In addition, the return of NRF2 to basal levels after signaling was inhibited in TAZ deficiency, resulting in sustained nuclear NRF2 levels and aberrantly increased expression of NRF2 targets. TAZ deficiency failed to modulate optimal NRF2 signaling and concomitantly impaired lysosomal acidification and lysosomal enzyme function, accumulating the abnormal autophagy vesicles and reactive oxygen species and causing protein oxidation and cellular damage in the lungs. TAZ restoration to TAZ deficiency normalized dysregulated NRF2 signaling and aberrant lysosomal function and triggered the normal autophagy-lysosomal pathway. Therefore, TAZ is indispensable for the optimal regulation of NRF2-mediated autophagy-lysosomal pathways and for preventing pulmonary damage caused by oxidative stress and oxidized proteins.

Remarkable sex-specific differences at single-cell resolution in neonatal hyperoxic lung injury.

Exposure to supraphysiological concentrations of oxygen (hyperoxia) predisposes to bronchopulmonary dysplasia (BPD), which is characterized by abnormal alveolarization and pulmonary vascular development, in preterm neonates. Neonatal hyperoxia exposure is used to recapitulate the phenotype of human BPD in murine models. Male sex is considered an independent predictor for the development of BPD, but the main mechanisms underlying sexually dimorphic outcomes are unknown. Our objective was to investigate sex-specific and cell-type specific transcriptional changes that drive injury in the neonatal lung exposed to hyperoxia at single-cell resolution and delineate the changes in cell-cell communication networks in the developing lung. We used single-cell RNA sequencing (scRNAseq) to generate transcriptional profiles of >35,000 cells isolated from the lungs of neonatal male and female C57BL/6 mice exposed to 95% [Formula: see text] between PND1-5 (saccular stage of lung development) or normoxia and euthanized at PND7 (alveolar stage of lung development). ScRNAseq identified 22 cell clusters with distinct populations of endothelial, epithelial, mesenchymal, and immune cells. Our data identified that the distal lung vascular endothelium (composed of aerocytes and general capillary endothelial cells) is exquisitely sensitive to hyperoxia exposure with the emergence of an intermediate capillary endothelial population with both general capillaries (gCap) and aerocytes or alveolar capillaries (aCap) markers. We also identified a myeloid-derived suppressor cell population from the lung neutrophils. Sex-specific differences were evident in all lung cell subpopulations but were striking among the lung immune cells. Finally, we identified that the specific intercellular communication networks and the ligand-receptor pairs that are impacted by neonatal hyperoxia exposure.

Nebulized curcumin protects neonatal lungs from antenatal insult in rats.

Intrauterine growth restriction (IUGR) increases the risk of bronchopulmonary dysplasia (BPD), one of the major complications of prematurity. Antenatal low-protein diet (LPD) exposure in rats induces IUGR and mimics BPD-related alveolarization disorders. Peroxisome proliferator-activated receptor-γ (PPARγ) plays a key role in normal lung development and was found deregulated following LPD exposure. The objective of this article was to investigate the effects of nebulized curcumin, a natural PPARγ agonist, to prevent IUGR-related abnormal lung development. We studied rat pups antenatally exposed to an LPD or control diet (CTL) and treated with nebulized curcumin (50 mg/kg) or vehicle from postnatal (P) days 1 to 5. The primary readouts were lung morphometric analyses at P21. Immunohistochemistry (P21) and microarrays (P6 and P11) were compared within animals exposed to LPD versus controls, with and without curcumin treatment. Quantitative morphometric analyses revealed that LPD induced abnormal alveolarization as evidenced by a significant increase in mean linear intercept (MLI) observed in P21 LPD-exposed animals. Early curcumin treatment prevented this effect, and two-way ANOVA analysis demonstrated significant interaction between diet and curcumin both for MLI [F(1,39) = 12.67, P = 0.001] and radial alveolar count at P21 [F(1,40) = 6.065, P = 0.0182]. Immunohistochemistry for fatty acid binding protein 4 (FABP4), a major regulator of PPARγ pathway, showed a decreased FABP4+ alveolar cell density in LPD-exposed animals treated by curcumin. Transcriptomic analysis showed that early curcumin significantly prevented the activation of profibrotic pathways observed at P11 in LPD-exposed animals. Nebulized curcumin appears to be a promising strategy to prevent alveolarization disorders in IUGR rat pups, targeting pathways involved in lung development.

Involvement of Hdac3-mediated inhibition of microRNA cluster 17-92 in bronchopulmonary dysplasia development

BackgroundThe incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, has been paradoxically rising despite medical advances. Histone deacetylase 3 (Hdac3) has been reported to be a crucial regulator in alveologenesis. Hence, this study aims to investigate the mechanism of Hdac3 in the abnormal pulmonary angiogenesis and alveolarization of BPD.MethodsA hyperoxia-induced BPD model of was developed in newborn mice, and primary lung fibroblasts were isolated from adult mice. Hdac3 was knocked out in vivo and knocked down in vitro, while microRNA (miR)-17 was downregulated in vivo and in vitro to clarify their roles in abnormal pulmonary angiogenesis and alveolarization. Mechanistic investigations were performed on the interplay of Hdac3, miR-17-92 cluster, enhancer of zeste homolog 1 (EZH1), p65 and placental growth factor (Pgf).ResultsHdac3 was involved in abnormal alveolarization and angiogenesis in BPD mice. Further, the expression of the miR-17-92 cluster in BPD mice was downregulated by Hdac3. miR-17 was found to target EZH1, and Hdac3 rescued the inhibited EZH1 expression by miR-17 in lung fibroblasts. Additionally, EZH1 augmented Pgf expression by recruiting p65 thus enhancing the progression of BPD. Hdac3 augmented the recruitment of p65 in the Pgf promoter region through the miR-17/EZH1 axis, thus enhancing the transcription and expression of Pgf, which elicited abnormal angiogenesis and alveolarization of BPD mice.ConclusionsAltogether, the present study revealed that Hdac3 activated the EZH1-p65-Pgf axis through inhibiting miR-17 in the miR-17-92 cluster, leading to accelerated abnormal pulmonary angiogenesis and alveolarization of BPD mice.

Retraction notice for: "miR-184 mediates hyperoxia-induced injury by targeting cell death and angiogenesis signalling pathways in the developing lung." Dilip Shah, Karmyodh Sandhu, Pragnya Das, Zubair H. Aghai, Sture Andersson, Gloria Pryhuber and Vineet Bhandari. Eur Respir J 2020; in press.

MicroRNAs (miRs) have been shown to disrupt normal lung development and function by interrupting alveolarization and vascularisation leading to development of bronchopulmonary dysplasia (BPD). Here we report that miR-184 has a critical role in the induction of BPD phenotype characterised by abnormal alveolarization and pulmonary angiogenesis in the developing lung. We observed an increased expression of miR-184 in BPD clinical specimens: tracheal aspirates (TA), human neonatal lungs with BPD and in fetal human lung Type II alveolar epithelial cells (TIIAECs) exposed to hyperoxia. Consistent with this, we also detected an upregulated miR-184-3p expression in whole lungs, in freshly isolated TIIAECs from lungs of hyperoxia-induced experimental BPD mice and in fetal mice lung TIIAECs exposed to hyperoxia. We demonstrate that overexpression of miR-184-3p exacerbates the BPD pulmonary phenotype, while downregulation of miR-184-3p expression ameliorated the BPD phenotype and also improved respiratory function. We identified miR-184 specific targets: platelet-derived growth factor-beta (Pdgf-β) and friend of Gata 2 (Fog2), also known as zinc finger protein family member (Zfpm2), and show that they are critically involved in pulmonary alveolarization and angiogenesis. Using cell-based luciferase analysis, downregulation of miR-184-3p expression and gene knockdown of miR-184-3p targets Pdgf-β and Fog2 in lung TIIAECs and endothelial cells, we mechanistically show that inhibition of miR-184-3p expression improves pulmonary alveolarization by regulating PDGF-β/AKT/Foxo3/Bax, Bcl2 signalling and enhances angiogenesis by Fog2/VEGF-A/Angiopoietin-1/2 pathway. Collectively, these data suggest that the use of miR-184-3p specific inhibitors may act as novel therapeutic interventions to control the adverse effects of hyperoxia on lung development and function.

Epithelial Expression of YAP and TAZ Is Sequentially Required in Lung Development.

TAZ (transcriptional coactivator with PDZ-binding motif) and YAP (Yes-associated protein) are key molecules of the Hippo pathway. Recent studies revealed that these molecules are essential in lung development; however, the precise signaling cascade involving these molecules and the differences in their roles during lung development remain unknown. We aimed to investigate YAP and TAZ functions using lung epithelium-specific Taz and Yap conditional knockout mice. We generated lung epithelium-specific Taz and Yap conditional knockout mice and investigated the functions of YAP and TAZ in lung development. Selective TAZ deficiency in mouse lung epithelial cells resulted in abnormal alveolarization, which mimics lung emphysema, in adults, whereas YAP deficiency caused disruption of bronchial morphogenesis during the embryonic stage. We report that TAZ and YAP are sequentially expressed in the lung and that this could explain their different phenotypes. Furthermore, we report that YAP stimulates Shh (Sonic hedgehog) expression and regulates the FGF (fibroblast growth factor)-SHH feedback loop, thereby contributing to normal bronchial morphogenesis. We also found that TGF-β (transforming growth factor-β) stimulation induced Shh expression in the lung epithelial cells, and both TAZ and YAP are essential in this novel pathway. Our results provide a novel insight into the molecular mechanisms underlying lung development and contribute to a better understanding of the characteristics of TAZ and YAP.

Allogeneic human umbilical cord-derived mesenchymal stem cells for severe bronchopulmonary dysplasia in children: study protocol for a randomized controlled trial (MSC-BPD trial)

BackgroundBronchopulmonary dysplasia (BPD) is a complex lung pathological lesion secondary to multiple factors and one of the most common chronic lung diseases. It has a poor prognosis, especially in preterm infants. However, effective therapies for this disease are lacking. Stem-cell therapy is a promising way to improve lung injury and abnormal alveolarization, and the human umbilical cord (hUC) is a good source of mesenchymal stem cells (MSCs), which have demonstrated efficacy in other diseases. We hypothesized that intravenously administered allogeneic hUC-MSCs are safe and effective for severe BPD.MethodsThe MSC-BPD trial is a randomized, single-center, open-label, dose-escalation, phase-II trial designed to investigate the safety and efficacy of hUC-MSCs in children with severe BPD. In this study, 72 patients will be enrolled and randomly divided into two intervention groups and one control group. Patients in the intervention groups will receive a low dose of hUC-MSCs (n = 24; 2.5 million cells/kg) or a high dose of hUC-MSCs (n = 24; 5 million cells/kg) in combination with traditional supportive treatments for BPD. The patients in the control group (n = 24) will be treated with traditional supportive treatments alone without hUC-MSCs. The primary outcome measures will be cumulative duration of oxygen therapy. Follow-up assessments will be performed at 1, 3, 6, 12, and 24 months post intervention, and the key outcome during follow-up will be changes on chest radiography. Statistical analyses will evaluate the efficacy of the hUC-MSC treatment.DiscussionThis will be the first randomized controlled trial to evaluate the safety and efficacy of intravenously administered hUC-MSCs in children with severe BPD. Its results should provide a new evidence-based therapy for severe BPD.Trial registrationClinicalTrials.gov, ID: NCT03601416. Registered on 26 July 2018.

Bronchopulmonary Dysplasia andUreaplasma: What Do We Know So Far?

Bronchopulmonary dysplasia (BPD) is the most common morbidity of prematurity. BPD is a chronic respiratory disease related to lung-injury during the primary course of critical lung disease such as respiratory distress syndrome or when abnormal development of the preterm lung occurs. Abnormal lung development not only results from primary lung injury in the first days after birth, but also secondary injury through abnormal repair resulting in arrested and abnormal alveolarization, fibrosis and pulmonary vascular dysgenesis. Chorioamnionitis is a risk factor that plays an important role in the development of BPD. Ureaplasma subspecies (spp.) are the most common isolated organisms from chorioamniotic tissue after premature births. Therefore Ureaplasma spp. appear to play an important role in the development of BPD, and treatment or prophylactic treatment of these infections or colonization may reduce the incidence, morbidity and mortality of BPD. Ureaplasma spp. infections are challenging not only to treat, but also to diagnosis in a timely manner. This review summarizes the current state of treatment and new developments in the treatment of Ureaplasma exposure in premature infants.

Conditional overexpression of TGFβ1 promotes pulmonary inflammation, apoptosis and mortality via TGFβR2 in the developing mouse lung.

BackgroundEarlier studies have reported that transforming growth factor beta 1(TGFβ1) is a critical mediator of hyperoxia-induced acute lung injury (HALI) in developing lungs, leading to impaired alveolarization and a pulmonary phenotype of bronchopulmonary dysplasia (BPD). However, the mechanisms responsible for the TGFβ1-induced inflammatory signals that lead to cell death and abnormal alveolarization are poorly understood. We hypothesized that TGFβ1 signaling via TGFβR2 is necessary for the pathogenesis of the BPD pulmonary phenotype resulting from HALI.MethodsWe utilized lung epithelial cell-specific TGFβ1 overexpressing transgenic and TGFβR2 null mutant mice to evaluate the effects on neonatal mortality as well as pulmonary inflammation and apoptosis in developing lungs. Lung morphometry was performed to determine the impaired alveolarization and multicolor flow cytometry studies were performed to detect inflammatory macrophages and monocytes in lungs. Apoptotic cell death was measured with TUNEL assay, immunohistochemistry and western blotting and protein expression of angiogenic mediators were also analyzed.ResultsOur data reveals that increased TGFβ1 expression in newborn mice lungs leads to increased mortality, macrophage and immature monocyte infiltration, apoptotic cell death specifically in Type II alveolar epithelial cells (AECs), impaired alveolarization, and dysregulated angiogenic molecular markers.ConclusionsOur study has demonstrated the potential role of inhibition of TGFβ1 signaling via TGFβR2 for improved survival, reduced inflammation and apoptosis that may provide insights for the development of potential therapeutic strategies targeted against HALI and BPD.

Pulmonary interstitial glycogenosis in a patient ultimately diagnosed with Noonan syndrome

We describe an infant prenatally diagnosed with hydrops fetalis ultimately found to have Noonan syndrome (NS). Prior to genetic confirmation of diagnosis, lung biopsy was performed which revealed widespread pulmonary interstitial glycogenosis (PIG), abnormal alveolarization, and mild inflammation. Although genetic alterations have been identified in NS, the mutations are heterogeneous and the diagnosis remains one of clinical suspicion. The combination of PIG and NS has not yet been documented in the literature. While the underlying pathophysiologic mechanism of PIG is unclear, we suggest that the mitogen-activated protein kinase signal transduction pathway members (PTPN11, KRAS, SOS1, RAF1, SHOC2, NRAS) involved in cellular growth factor signaling, which are affected in NS, can provide clues. In addition, this case demonstrates that empiric corticosteroids can be considered in complicated cases since biopsy did reveal an inflammatory component, not typically noted in PIG.

TGF-β-Smad3 signaling in emphysema and pulmonary fibrosis: an epigenetic aberration of normal development?

It is well accepted that TGF-β signaling has critical functional roles in lung development, injury, and repair. We showed previously that null mutation of Smad3, a critical node in the TGF-β pathway, protects mice against fibrosis induced by bleomycin. However, more recently we noticed that abnormal alveolarization also occurs in Smad3-deficient mice and that this is followed by progressive emphysema-like alveolar wall destruction mediated by MMP9. We now know that Smad3 cooperates with c-Jun to synergistically regulate a protein deacetylase SIRT1, by binding to an AP-1 site in the SIRT1 promoter. Consistently, Smad3 knockout lung at postnatal day 28 had reduced SIRT1 expression, which in turn resulted in increased histone acetylation at the binding sites of the transcription factors AP-1, NF-κB, and Pea3 on the MMP9 promoter, as well as increased acetylation of NF-κB. Thus, upon TGF-β activation, phosphorylated Smad3 can be translocated into the nucleus with Smad4, whereat Smad3 in turn collaborates with c-Jun to activate SIRT1 transcription. SIRT1 can deacetylate NF-κB at lysine 30, as well as histones adjacent to the transcription factor AP-1, NF-κB, and Pea3 binding sites of the MMP9 promoter, thereby suppressing MMP9 transcription, hence fixing MMP9 in the OFF mode. Conversely, when Smad3 is missing, this regulatory pathway is inactivated so that MMP9 is epigenetically turned ON. We postulate that these developmental epigenetic mechanisms by which Smad3 regulates MMP9 transcription cell autonomously may be important in modulating both emphysema and pulmonary fibrosis and that this could explain why both pathologies can appear within the same lung specimen.

Maternal smoking and the retinoid pathway in the developing lung

BackgroundMaternal smoking is a risk factor for pediatric lung disease, including asthma. Animal models suggest that maternal smoking causes defective alveolarization in the offspring. Retinoic acid signaling modulates both lung development and postnatal immune function. Thus, abnormalities in this pathway could mediate maternal smoking effects. We tested whether maternal smoking disrupts retinoic acid pathway expression and functioning in a murine model.MethodsFemale C57Bl/6 mice with/without mainstream cigarette smoke exposure (3 research cigarettes a day, 5 days a week) were mated to nonsmoking males. Cigarette smoke exposure continued throughout the pregnancy and after parturition. Lung tissue from the offspring was examined by mean linear intercept analysis and by quantitative PCR. Cell culture experiments using the type II cell-like cell line, A549, tested whether lipid-soluble cigarette smoke components affected binding and activation of retinoic acid response elements in vitro.ResultsCompared to tobacco-naïve mice, juvenile mice with tobacco toxin exposure had significantly (P < 0.05) increased mean linear intercepts, consistent with an alveolarization defect. Tobacco toxin exposure significantly (P < 0.05) decreased mRNA and protein expression of retinoic acid signaling pathway elements, including retinoic acid receptor alpha and retinoic acid receptor beta, with the greatest number of changes observed between postnatal days 3–5. Lipid-soluble cigarette smoke components significantly (P < 0.05) decreased retinoic acid-induced binding and activation of the retinoic acid receptor response element in A549 cells.ConclusionsA murine model of maternal cigarette smoking causes abnormal alveolarization in association with altered retinoic acid pathway element expression in the offspring. An in vitro cell culture model shows that lipid-soluble components of cigarette smoke decrease retinoic acid response element activation. It is feasible that disruption of retinoic acid signaling contributes to the pediatric lung dysfunction caused by maternal smoking.

Endotoxin inhalation alters lung development in neonatal mice.

Childhood asthma is a significant public health problem. Epidemiologic evidence suggests an association between childhood asthma exacerbations and early life exposure to environmental endotoxin. Although the pathogenesis of endotoxin-induced adult asthma is well studied, questions remain about the impact of environmental endotoxin on pulmonary responsiveness in early life. We developed a murine model of neonatal/juvenile endotoxin exposures approximating those in young children and evaluated the lungs inflammatory and remodeling responses. Persistent lung inflammation induced by the inhalation of endotoxin in early life was demonstrated by the influx of inflammatory cells and pro-inflammatory mediators to the airways and resulted in abnormal alveolarization. Results of this study advance the understanding of the impact early life endotoxin inhalation has on the lower airways, and demonstrates the importance of an experimental design that approximates environmental exposures as they occur in young children.

Lung interstitial cells during alveolarization

Recent progress in neonatal medicine has enabled survival of many extremely low-birth-weight infants. Prenatal steroids, surfactants, and non-invasive ventilation have helped reduce the incidence of the classical form of bronchopulmonary dysplasia characterized by marked fibrosis and emphysema. However, a new form of bronchopulmonary dysplasia marked by arrest of alveolarization remains a complication in the postnatal course of extremely low-birth-weight infants. To better understand this challenging complication, detailed alveolarization mechanisms should be delineated. Proper alveolarization involves the temporal and spatial coordination of a number of cells, mediators, and genes. Cross-talk between the mesenchyme and the epithelium through soluble and diffusible factors are key processes of alveolarization. Lung interstitial cells derived from the mesenchyme play a crucial role in alveolarization. Peak alveolar formation coincides with intense lung interstitial cell proliferation. Myofibroblasts are essential for secondary septation, a critical process of alveolarization, and localize to the front lines of alveologenesis. The differentiation and migration of myofibroblasts are strictly controlled by various mediators and genes. Disruption of this finely controlled mechanism leads to abnormal alveolarization. Since arrest in alveolarization is a hallmark of a new form of bronchopulmonary dysplasia, knowledge regarding the role of lung interstitial cells during alveolarization and their control mechanism will enable us to find more specific therapeutic strategies for bronchopulmonary dysplasia. In this review, the role of lung interstitial cells during alveolarization and control mechanisms of their differentiation and migration will be discussed.

Transcriptional Coactivator with PDZ-binding Motif Is Essential for Normal Alveolarization in Mice

Transcriptional coactivator with PDZ-binding motif (TAZ) is assumed to act as a coactivator of several transcription factors including smad2/3. In the lung, surfactant protein C (Sftpc) is known to be a downstream target of thyroid transcription factor-1 (TTF-1)-TAZ transcriptional coactivation. The lung phenotype of Taz-deficient mice was explored. Taz-deficient mice were analyzed pathologically and physiologically. Next, we performed microarray analysis to determine the genes closely related to abnormal lung development. Finally, Taz-heterozygous mice were injected with bleomycin. Taz-deficient homozygotes showed abnormal alveolarization during lung development, which caused in adult mice airspace enlargement mimicking emphysema. There was no significant difference in the expression of Sftpc between wild-type and Taz-deficient lungs. Instead, microarray analysis identified some candidate downstream genes related to the pathogenesis, including the connective tissue growth factor (Ctgf) gene, which is required for normal lung development. In vitro studies showed that TAZ up-regulated Ctgf expression not only by reinforcing transforming growth factor-beta/smad signals, but also by interfering in the more proximal Ctgf promoter region (from bp -123 to -76), defined as the TAZ response element. Furthermore, Taz-heterozygous mice were resistant to bleomycin-induced lung fibrosis. The results indicate the importance of TAZ in lung alveolarization and its involvement in the pathogenesis of lung fibrosis.

Altered expression of key growth factors (TGFα, TGFβ 1 , PDGF‐A) and flawed formation of alveoli and elastin (Eln) in lungs of preterm (PT) lambs with chronic lung disease (CLD)

Mechanical ventilation (MV) with O2-rich gas for 3 wks leads to CLD in PT lambs, as it does in PT infants. Reduced numbers of enlarged airspaces and excess, disordered lung Eln are key features of CLD. Studies in mice show that modified expression of specific growth factors (overexpressed TGFα or TGFβ1; deleted PDGF-A) and of genes that impact Eln assembly (deleted fibulin 5, fbn 5, or lysyl oxidase-like 1, LOXL 1) yield lung defects resembling CLD. Our goal was to define changes in gene expression of key growth factors and proteins that regulate Eln assembly in lungs of PT lambs with evolving CLD. We used quantitative RT-PCR to measure mRNA for TGFα, TGFβ1, PDGF-A, and Eln-related genes (tropoelastin, TE; lysyl oxidase, LOX; fibrillin 1, fib 1; fbn 5; LOXL 1) in lungs of PT lambs that received MV with O2 for 1 d, 3 d or 3 wks. Controls included PT fetal lungs and unventilated lungs of term lambs (same post-conception age as PT lambs that had MV for 3 wks). Lung mRNA for TGFα and TGFβ1 was 2- to 3-fold greater in PT lambs given MV for 1 d compared to control fetuses. PDGF-A mRNA decreased to <50% of fetal control values after 3 wks of MV. Fbn 5 mRNA did not change during 3 wks of MV (in contrast to increased lung mRNA of TE, LOX, fib 1, LOXL 1). We conclude that early postnatal increases in lung TGFα and TGFβ1, which can impact Eln fiber formation, coupled with later reduction in lung PDGF-A, which directs septal localization of Eln, could induce abnormal alveolarization in lambs with CLD. Increased lung expression of genes that regulate Eln synthesis and assembly, with no increase in fbn 5 mRNA, could impair lung Eln assembly in lambs with CLD. Funded by NIH RO1 HL62512, P50 HL56401

Impaired Developmental Expression of Lung Endothelial Nitric Oxide Synthase and Abnormal Alveolarization in the Fawn-Hooded Rat : Implications for the Risk of Developing Pulmonary Hypertension 1689

The fawn-hooded rat (FHR) strain is characterized by platelet abnormalities and systemic hypertension, and has been used to study genetic risk factors for pulmonary hypertension (PH). Unlike other strains, FHR develop progressive PH when exposed to slight decreases in alveolar PaO2. We hypothesized that developmental abnormalities in lung vasoreactivity or growth may predispose the FHR for PH. Since endogenous nitric oxide (NO) modulates pulmonary vasoreactivity and growth, we further hypothesized that early decreases in lung endothelial NO synthase (eNOS) content may contribute to the development of PH in FHR. We measured lung eNOS content and growth in FHR and control rat strains (Fischer and Sprague-Dawley (SDR)). Lungs were harvested from late fetal and postnatal ages (days 1 & 7) and eNOS protein content measured by western blot analysis. Immunostaining for eNOS was performed with paraffin sections and the eNOS monoclonal antibody. Histology was assessed after tracheal installation of formalin at constant pressure (10cm water) and radial alveolar counts performed. Lung to body weight ratios were reduced in fetal and newborn FHR by 20-46% compared to controls (p<0.05). In comparison with SDR, FHR lung eNOS protein was decreased to 69±8%, 56±8%, and 68±7% at fetal, and postnatal days 1 and 7, respectively (p<0.05 at each age). Immunostaining studies demonstrated selective staining for eNOS only in vascular endothelium and no difference in the pattern of eNOS expression between strains. Histology revealed striking changes in lung maturation and growth in FHR at all ages, including reduced alveolarization(FHR 58±2% of SDR at day 7, p<0.05). We conclude that in comparison with other rat strains, lung eNOS content is decreased in the FHR fetus and newborn. In addition, the FHR lung has decreased alveolarization and histologic signs of delayed lung maturation. We speculate that reduced vascular surface area and eNOS increase the risk for developing PH, and that decreased NO production may be associated with abnormal lung growth and development.

Nitric oxide and lung surfactant

Inhalation of nitric oxide (NO) is an experimental treatment for severe pulmonary hypertension. Being rapidly metabolized by hemoglobin, inhaled NO causes selective vasodilation in the pulmonary vascular bed. In addition to the vascular smooth muscle, other pulmonary structures are exposed to inhaled NO, resulting in suppression of NO synthesis in a variety of pulmonary cells and in potential toxicity. NO is a free radical that interacts with a number of proteins, particularly metalloproteins. Together with superoxide radical, it rapidly forms highly toxic peroxynitrite. Peroxynitrite is involved in the killing of microbes by activated phagocytosing macrophages. In severe inflammation, peroxynitrite may be responsible for damaging proteins, lipids, and DNA. Peroxynitrite added to surfactant in vitro is capable of decreasing the surface activity, inducing lipid peroxidation, decreasing the function of surfactant proteins, SP-A and SP-B, and inducing protein-associated nitro-tyrosine. Exposure of animals for prolonged periods (48 to 72 hours) to inhaled NO (80 to 120 ppm) has been associated with a decrease in surface activity. This is caused by binding of surfactant to iron-proteins that are modified by NO (particularly methemoglobin), or by peroxynitrite induced damage of surfactant. In contrast, exposure of isolated surfactant complex to NO during surface cycling strikingly decreases the inactivation of surfactant, preventing the conversion of surfactant to small vesicles that are no longer surface-active, and preventing lipid peroxidation. This finding is consistent with the function of NO as a lipid-soluble chain-braking antioxidant. It is possible that this lipophilic gas has as yet undefined roles in regulation of surfactant metabolism and maintenance of surface activity. Deficiency in pulmonary NO may be present during the early neonatal period in respiratory distress syndrome and in persistent fetal circulation. The premature lung is likely to be sensitive to NO toxicity that may include lung damage, abnormal alveolarization, and mutagenicity. Defining of the indications, the dosage, and the toxicity of inhaled NO therapy remains the challenge for experimental and clinical research.