Curcumin augments lung maturation, preventing neonatal lung injury by inhibiting TGF-β signaling

There is no effective intervention to prevent or treat bronchopulmonary dysplasia (BPD). Curcumin has potent antioxidant and anti-inflammatory properties, and it modulates signaling of peroxisome proliferator-activated receptor-γ (PPARγ), an important molecule in the pathobiology of BPD. However, its role in the prevention of BPD is not known. We determined 1) if curcumin enhances neonatal lung maturation, 2) if curcumin protects against hyperoxia-induced neonatal lung injury, and 3) if this protection is mediated by blocking TGF-β. Embryonic day 19 fetal rat lung fibroblasts were exposed to 21% or 95% O(2) for 24 h following 1 h of treatment with curcumin. Curcumin dose dependently accelerated e19 fibroblast differentiation [increased parathyroid hormone-related protein (PTHrP) receptor, PPARγ, and adipocyte differentiation-related protein (ADRP) levels and triolein uptake] and proliferation (increased thymidine incorporation). Pretreatment with curcumin blocked the hyperoxia-induced decrease (PPARγ and ADRP) and increase (α-smooth muscle actin and fibronectin) in markers of lung injury/repair, as well as the activation of TGF-β signaling. In a separate set of experiments, neonatal Sprague-Dawley rat pups were exposed to 21% or 95% O(2) for 7 days with or without intraperitoneal administration of curcumin. Analysis for markers of lung injury/repair [PTHrP receptor, PPARγ, ADRP, fibronectin, TGF-β receptor (activin receptor-like kinase 5), and Smad3] and lung morphology (radial alveolar count) demonstrated that curcumin effectively blocks TGF-β activation and hyperoxia-induced lung injury. Therefore, curcumin accelerates lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and prevents hyperoxia-induced neonatal lung injury, possibly by blocking TGF-β activation, suggesting that it is a potential intervention against BPD.     

Mechanism of nicotine-induced pulmonary fibroblast transdifferentiation

We tested the hypothesis that in vitro nicotine exposure disrupts specific epithelial-mesenchymal paracrine signaling pathways and results in pulmonary interstitial lipofibroblast (LIF)-to-myofibroblast (MYF) transdifferentiation, resulting in altered pulmonary development and function. Studies were done to determine whether nicotine induces LIF-to-MYF transdifferentiation and to elucidate underlying molecular mechanism(s) involved and to determine whether nicotine-induced LIF-to-MYF transdifferentiation could be prevented by stimulating specific alveolar interstitial fibroblast lipogenic pathway. WI38 cells, a human embryonic pulmonary fibroblast cell line, were treated with nicotine with or without specific agonists of alveolar fibroblast lipogenic pathway, PTHrP, DBcAMP, or the potent PPARgamma stimulant rosiglitazone (RGZ) for 7 days. Expression of key lipogenic and myogenic markers was examined by RT-PCR, Western hybridization, and immunohistochemistry. The effect of nicotine on triglyceride uptake by WI38 cells and PTHrP binding to its receptor was also determined. Finally, the effect of transfecting WI38 cells with a PPARgamma expression vector on nicotine-induced LIF-to-MYF transdifferentiation was determined. Nicotine treatment resulted in significantly decreased expression of lipogenic and increased expression of myogenic markers in a dose-dependent manner, indicating nicotine-induced LIF-to-MYF transdifferentiation. This was accompanied by decreased PTHrP receptor binding to its receptor. The nicotine-induced LIF-to-MYF transdifferentiation was completely prevented by concomitant treatment with PTHrP, DBcAMP, RGZ, and by transiently overexpressing PPARgamma. Our data suggest nicotine induces alveolar LIF-to-MYF transdifferentiation through a mechanism involving downregulation of lipogenic PTHrP-mediated, cAMP-dependent PKA signaling pathway, which can be prevented using specific molecular targets. Potential therapeutic implications of these observations against in utero nicotine-induced lung injury remain to be tested.     

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells

Background

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.

Methods

Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.

Results

The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-21 was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-21 inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.

Conclusions

Our experiments demonstrate that microRNA-21 is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.     

The evolutionary continuum from lung development to homeostasis and repair

A functional, developmental, and comparative biological approach is probably the most effective way for arranging gene regulatory networks (GRNs) in their biological contexts. Evolutionary developmental biology allows comparison of GRNs during development across phyla. For lung evolution, the parathyroid hormone-related protein (PTHrP) GRN exemplifies a continuum from ontogeny to phylogeny, homeostasis, and repair. PTHrP signaling between the lung endoderm and mesoderm stimulates lipofibroblast differentiation by downregulating the myofibroblast Wnt signaling pathway and upregulating the protein kinase A-dependent cAMP signaling pathway, inducing the lipofibroblast phenotype. Leptin secreted by the lipofibroblast, in turn, binds to its receptor on the alveolar type II cell, stimulating surfactant synthesis to ensure alveolar homeostasis. Failure of the PTHrP/PTHrP receptor signaling mechanism causes transdifferentiation of lipofibroblasts to myofibroblasts, which are the hallmark for lung fibrosis. We have shown that by targeting peroxisome proliferator-activated receptor gamma, the downstream target for lipofibroblast PTHrP signaling, we can prevent lung fibrosis. We speculate that the recapitulation of the myofibroblast phenotype during transdifferentiation is consistent with lung injury as lung evolution in reverse. Repair recapitulates ontogeny because it is programmed to express the cross talk between epithelium and mesoderm through evolution. This model demonstrates how epithelial-mesenchymal cross talk, when seen as a recapitulation of ontogeny and phylogeny (in both a forward and reverse direction), predicts novel, effective diagnostic and therapeutic targets.     

Lipopolysaccharide increases alveolar type II cell number in fetal mouse lungs through Toll-like receptor 4 and NF-kappaB

Chorioamnionitis is a major cause of preterm delivery. Infants exposed to inflammation in utero and then born preterm may have improved lung function in the immediate postnatal period. We developed a mouse model of chorioamnionitis to study the inflammatory signaling mechanisms that might influence fetal lung maturation. With this in vivo model, we found that Escherichia coli lipopolysaccharide (LPS) increased the number of alveolar type II cells in the fetal mouse lung. LPS also increased type II cell number in cultured fetal lung explants, suggesting that LPS could directly signal the fetal lung in the absence of maternal influences. Using immunostaining, we localized cells within the fetal mouse lung expressing the LPS receptor molecule Toll-like receptor 4 (TLR4). Similar to the signaling pathways in inflammatory cells, LPS activated NF-kappaB in fetal lung explants. Activation of the TLR4/NF-kappaB pathway appeared to be required, as LPS did not increase the number of type II cells in C.C3H-Tlr4(Lps-d) mice, a congenic strain containing a loss of function mutation in tlr4. In addition, the sesquiterpene lactone parthenolide inhibited NF-kappaB activation following LPS exposure and blocked the LPS-induced increase in type II cells. On the basis of these data from our mouse model of chorioamnionitis, it appears that LPS specifically activated the TLR4/NF-kappaB pathway, leading to increased type II cell maturation. These data implicate an important signaling mechanism in chorioamnionitis and suggest the TLR4/NF-kappaB pathway can influence lung development.     

Lipopolysaccharide disrupts the directional persistence of alveolar myofibroblast migration through EGF receptor

Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification with decreased alveolar number and increased airspace size. Formation of alveoli involves a process known as secondary septation triggered by myofibroblasts. This study investigated the underlying mechanisms of altered lung morphogenesis in a rat model of BPD induced by intra-amniotic injection of lipopolysaccharide (LPS). Results showed that LPS disrupted alveolar morphology and led to abnormal localization of myofibroblasts in the lung of newborn rats, mostly in primary septa with few in secondary septa. To identify potential mechanisms, in vitro experiments were carried out to observe the migration behavior of myofibroblasts. The migration speed of lung myofibroblasts increased with LPS treatment, whereas the directional persistence decreased. We found that LPS induced activation of EGFR and overexpression of its ligand, TGF-α in myofibroblasts. AG1478, an EGFR inhibitor, abrogated the enhanced locomotivity of myofibroblasts by LPS and also increased the directional persistence of myofibroblast migration. Myofibroblasts showed a high asymmetry of phospho-EGFR localization, which was absent after LPS treatment. Application of rhTGF-α to myofibroblasts decreased the directional persistence. Our findings indicated that asymmetry of phospho-EGFR localization in myofibroblasts was important for cell migration and its directional persistence. We speculate that LPS exposure disrupts the asymmetric localization of phospho-EGFR, leading to decreased stability of cell polarity and final abnormal location of myofibroblasts in vivo, which is critical to secondary septation and may contribute to the arrested alveolar development in BPD.     

Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: role of PKCζ-mediated p47phox phosphorylation

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKCζ pseudosubstrate peptide (a specific inhibitor of PKCζ) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKCζ also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKCζ in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKCζ-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKCζ or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD.     

Lipopolysaccharide changes the subcellular distribution of aquaporin 5 and increases plasma membrane water permeability in mouse lung epithelial cells

Aquaporin-5 (AQP5), a major water channel in lung epithelial cells, plays an important role in maintaining water homeostasis in the lungs. Cell surface expression of AQP5 is regulated by not only mRNA and protein synthesis but also changes in subcellular distribution. We investigated the effect of lipopolysaccharide (LPS) on the subcellular distribution of AQP5 in a mouse lung epithelial cell line (MLE-12). LPS caused significant increases in AQP5 in the plasma membrane at 0.5-2 h. Immunofluorescence and Western blotting strongly suggested that LPS altered AQP5 subcellular distribution from an intracellular vesicular compartment to the plasma membrane. The specific p38 MAP kinase inhibitor SB 203580 apparently prevented LPS-induced changes in AQP5 distribution. Furthermore, LPS increased the osmotic water permeability of MLE-12 cells. These findings demonstrate that LPS increases cell surface AQP5 expression by changing its subcellular distribution and increases membrane osmotic water permeability through activation of p38 MAP kinase.     

Antenatal inflammation induced TGF-beta1 but suppressed CTGF in preterm lungs

Chorioamnionitis is frequently associated with preterm birth and increases the risk of adverse outcomes such as bronchopulmonary dysplasia (BPD). Transforming growth factor (TGF)-beta1 is a key regulator of lung development, airway remodeling, lung fibrosis, and regulation of inflammation, and all these processes contribute to the development of BPD. Connective tissue growth factor (CTGF) is a downstream mediator of some of the profibrotic effects of TGF-beta1, vascular remodeling, and angiogenesis. TGF-beta1-induced CTGF expression can be blocked by TNF-alpha. We asked whether chorioamnionitis-associated antenatal inflammation would regulate TGF-beta1, the TGF-beta1 signaling pathway, and CTGF in preterm lamb lungs. Fetal sheep were exposed to 4 mg of intra-amniotic endotoxin or saline for 5 h, 24 h, 72 h, or 7 days before preterm delivery at 125 days gestation (full term = 150 days). Intra-amniotic endotoxin increased lung TGF-beta1 mRNA and protein expression. Elevated TGF-beta1 levels were associated with TGF-beta1-induced phosphorylation of Smad2. CTGF was selectively expressed in lung endothelial cells in control lungs, and intra-amniotic endotoxin caused CTGF expression to decrease to 30% of control values and TNF-alpha protein to increase. The antenatal inflammation-induced TGF-beta1 expression and Smad signaling in the fetal lamb lung may contribute to impaired lung alveolarization and reduced lung inflammation. Decreased CTGF expression may inhibit vascular development or remodeling and limit lung fibrosis during remodeling. These effects may contribute to the impaired alveolar and pulmonary vascular development that is the hallmark of the new form of BPD.     

Parathyroid hormone-related protein reduces alveolar epithelial cell proliferation during lung injury in rats

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells and could be a regulatory factor for alveolar epithelial cell proliferation after lung injury. We investigated lung PTHrP expression in rats exposed to 85% oxygen. Lung levels of PTHrP were significantly decreased between 4 and 8 days of hyperoxia, concurrent with increased expression of proliferating cell nuclear antigen and increased incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA in lung corner cells. PTHrP receptor was present in both normal and hyperoxic lung. To test whether the fall in PTHrP was related to cell proliferation, we instilled PTHrP into lungs on the fourth day of hyperoxia. Eight hours later, BrdU labeling in alveolar corner cells was 3.2 +/- 0.4 cells/high-power field in hyperoxic PBS-instilled rats compared with 0.5 +/- 0.3 cells/high-power field in PTHrP-instilled rats (P < 0. 01). Thus PTHrP expression changes in response to lung injury due to 85% oxygen and may regulate cell proliferation.     

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.     

Bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) remains a major complication of prematurity resulting in significant morbidity and mortality. The pathology of BPD is multifactorial and leads to alveolar simplification and distal lung injury. Previous studies have shown a beneficial effect of systemic treatment with bone marrow-derived mesenchymal stromal cells (MSCs) and MSC-conditioned media (MSC-CM) leading to amelioration of the lung parenchymal and vascular injury in vivo in the hyperoxia murine model of BPD. It is possible that the beneficial response from the MSCs is at least in part due to activation of endogenous lung epithelial stem cells. Bronchioalveolar stem cells (BASCs) are an adult lung stem cell population capable of self-renewal and differentiation in culture, and BASCs proliferate in response to bronchiolar and alveolar lung injury in vivo. Systemic treatment of neonatal hyperoxia-exposed mice with MSCs or MSC-CM led to a significant increase in BASCs compared with untreated controls. Treatment of BASCs with MSC-CM in culture showed an increase in growth efficiency, indicating a direct effect of MSCs on BASCs. Lineage tracing data in bleomycin-treated adult mice showed that Clara cell secretory protein-expressing cells including BASCs are capable of contributing to alveolar repair after lung injury. MSCs and MSC-derived factors may stimulate BASCs to play a role in the repair of alveolar lung injury found in BPD and in the restoration of distal lung cell epithelia. This work highlights the potential important role of endogenous lung stem cells in the repair of chronic lung diseases.     

Chronic fetal exposure to Ureaplasma parvum suppresses innate immune responses in sheep

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different proinflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic (IA) injections of media (control) or Ureaplasma parvum serovar 3 either 7 or 70 d before preterm delivery. Another group received an IA injection of Escherichia coli LPS 2 d prior to delivery. To test for interactions, IA U. parvum-exposed animals were challenged with IA LPS and delivered 2 d later. All animals were delivered at 124 ± 1-d gestation (term = 150 d). Compared with the 2-d LPS exposure group, the U. parvum 70 d + LPS group had 1) decreased lung pro- and anti-inflammatory cytokine expression and 2) fewer CD3(+) T lymphocytes, CCL2(+), myeloperoxidase(+), and PU.1(+) cells in the lung. Interestingly, exposure to U. parvum for 7 d did not change responses to a subsequent IA LPS challenge, and exposure to IA U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGF-β1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70 d + LPS but not U. parvum 7 d + LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of downregulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis-exposed preterm infants may respond to lung injury and postnatal nosocomial infections.     

Serum cholestenoic acid as a potential marker of pulmonary cholesterol homeostasis: increased levels in patients with pulmonary alveolar proteinosis

The conversion of cholesterol into the more polar metabolites 27-hydroxycholesterol (27-OH) and cholestenoic acid by the cytochrome P450 sterol 27-hydroxylase is a cholesterol-removal mechanism used by almost all cells. Most of the cholestenoic acid present in the circulation originates from the lung, and it has been suggested that sterol 27-hydroxylase is of particular importance for cholesterol homeostasis in this organ. As an example of pulmonary cholesterol accumulation, a known disorder of surfactant homeostasis, pulmonary alveolar proteinosis (PAP), was studied. Analysis of bronchoalveolar lavage fluid from PAP patients revealed a significant accumulation of the cholesterol metabolites cholestenoic acid and 27-OH. This pattern was recapitulated in serum, with a significant increase in the levels of both cholestenoic acid (P=0.003) and 27-OH (P=0.017) in PAP patients compared with healthy controls. Analysis of PAP alveolar macrophages did not reveal a significant change in mRNA expression levels of either sterol 27-hydroxylase or the cholesterol-esterifying enzyme acyl-CoA:cholesterol acyltransferase-1. These results are consistent with the contention that substrate availability, rather than enzyme expression, is the key factor in regulating the production of cholestenoic acid by the lung and that serum cholestenoic acid may be a marker of pulmonary cholesterol homeostasis.     

Leptin mediates the parathyroid hormone-related protein paracrine stimulation of fetal lung maturation

Developing rat lung lipofibroblasts express leptin beginning on embryonic day (E) 17, increasing 7- to 10-fold by E20. Leptin and its receptor are expressed mutually exclusively by fetal lung fibroblasts and type II cells, suggesting a paracrine signaling "loop." This hypothesized mechanism is supported by the following experimental data: 1) leptin stimulates the de novo synthesis of surfactant phospholipid by both fetal rat type II cells (400% x 100 ng(-1) x ml(-1) x 24 h(-1)) and adult human airway epithelial cells (85% x 100 ng(-1) x 24 h(-1)); 2) leptin is secreted by lipofibroblasts in amounts that stimulate type II cell surfactant phospholipid synthesis in vitro; 3) epithelial cell secretions such as parathyroid hormone-related protein (PTHrP), PGE(2), and dexamethasone stimulate leptin expression by fetal rat lung fibroblasts; 4) PTHrP or leptin stimulate the de novo synthesis of surfactant phospholipid (2- to 2.5-fold/24 h) and the expression of surfactant protein B (SP-B; >25-fold/24 h) by fetal rat lung explants, an effect that is blocked by a leptin antibody; and 5) a PTHrP receptor antagonist inhibits the expression of leptin mRNA by explants but does not inhibit leptin stimulation of surfactant phospholipid or SP-B expression, indicating that PTHrP paracrine stimulation of type II cell maturation requires leptin expression by lipofibroblasts. This is the first demonstration of a paracrine loop that functionally cooperates to induce alveolar acinar lung development.     

Regulation of lipopolysaccharide-induced lung inflammation by plasminogen activator Inhibitor-1 through a JNK-mediated pathway

The neutrophil is of undoubted importance in lung inflammation after exposure to LPS. We have shown recently that systemic inhibition of JNK decreased neutrophil recruitment to the lung after exposure to LPS, although the mechanisms underlying this inhibition are incompletely understood. As plasminogen activator inhibitor-1 (PAI-1) accentuates cell migration, with JNK activation recently shown to up-regulate PAI-1 expression, this suggested that systemic JNK inhibition may down-regulate LPS-induced pulmonary neutrophil recruitment through a decrease in PAI-1 expression. We show in this study that exposure of mice to aerosolized LPS increased PAI-1 expression in the lung and alveolar compartment, which was decreased by pretreatment with the JNK inhibitor SP600125. Exogenous, intratracheally administered PAI-1 prevented the inhibition of pulmonary neutrophil recruitment in the setting of systemic JNK inhibition, thereby suggesting a role for PAI-1 in the JNK-mediated pathway regulating LPS-induced neutrophil recruitment. In addition, PAI-1(-/-) mice had a decrease in neutrophil recruitment to the alveolar compartment after exposure to LPS, compared with wild-type controls, further suggesting a role for PAI-1 in LPS-induced lung inflammation. An increase in the intravascular level of KC is a likely mechanism for the inhibition of pulmonary neutrophil recruitment after LPS exposure in the setting of decreased PAI-1 expression, as systemic KC levels after exposure to LPS were increased in PAI-1-deficient mice and in mice pretreated with SP600125, with augmentation of intravascular KC levels inhibiting neutrophil recruitment to the lung after exposure to LPS.     

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.     

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.     

Characterization of fibroblasts recruited from bone marrow-derived precursor in neonatal bronchopulmonary dysplasia mice

We sought to determine whether the extrapulmonary origin of fibroblasts derived from bone marrow (BM) progenitor cells is essential to lung fibrosis in bronchopulmonary dysplasia (BPD). Neonate mice were durably engrafted with BM isolated from transgenic reporter mice that expressed green fluorescent protein (GFP). Such chimera mice were subjected to 60% O(2) exposure for 14 days. A large number of fibroblast-specific protein-1 (FSP1) and GFP-positive fibroblasts were identified in active fibrotic lesions. More surprisingly, however, FSP1(+) fibroblasts also arose in considerable numbers from BM-derived alveolar type II cells (AT2) through epithelial-mesenchymal transition (EMT) during lung fibrogenesis. Cultured lung fibroblasts could express the CXC chemokine receptor (CXCR4) and responded chemotactically to their cognate ligand, chemokine (C-X-C motif) ligand 12 (CXCL12), which were elevated in the serum of BPD mice. These data suggest that lung fibroblasts in BPD fibrosis could variably arise from BM progenitor cells. This finding, which suggests the pathophysiological process of fibrosis, could contribute to a therapy for BPD that is characterized by extensive interstitial fibrosis.