Search

Permissive hypercapnia, achieved using low tidal volume ventilation, has been an effective protective strategy in patients with acute respiratory distress syndrome. To date, no such protective effect has been demonstrated for the chronic neonatal lung injury, bronchopulmonary dysplasia. The objective of our study was to determine whether evolving chronic neonatal lung injury, using a rat model, is resistant to the beneficial effects of hypercapnia or simply requires a less conservative approach to hypercapnia than that applied clinically to date. Neonatal rats inhaled air or 60% [O.sub.2] for 14 days with or without 5.5% C[O.sub.2]. Lung parenchymal neutrophil and macrophage numbers were significantly increased by hyperoxia alone, which was associated with interstitial thickening and reduced secondary crest formation. The phagocyte influx, interstitial thickening, and impaired alveolar formation were significantly attenuated by concurrent hypercapnia. Hyperoxic pups that received 5.5% C[O.sub.2] had a significant increase in alveolar number relative to air-exposed pups. Increased tyrosine nitration, a footprint for peroxynitrite-mediated reactions, arteriolar medial wall thickening, and both reduced small peripheral pulmonary vessel number and VEGF and angiopoietin-1 (Ang-1) expression, which were observed with hyperoxia, was attenuated by concurrent hypercapnia. We conclude that evolving chronic neonatal lung injury in a rat model is responsive to the beneficial effects of hypercapnia. Inhaled 5.5% C[O.sub.2] provided a significant degree of protection against parenchymal and vascular injury in an animal model of chronic neonatal lung injury likely due, at least in part, to its inhibition of a phagocyte influx. carbon dioxide; lung growth; inflammation doi: 10.1152/ajplung.00139.2009.

Classical tissue recombination experiments have reported that at early gestation both tracheal and distal lung epithelium have the plasticity to respond to mesenchymal signals. Herein we examined the role of epithelial-mesenchymal interactions in maintaining epithelial differentiation at late (E19-E21, term = 22 days) fetal gestation in the rat. Isolated distal lung epithelial cells were recombined with mesenchymal cells from lung, skin, and intestine, and the homotypic or heterotypic recombinant cell aggregates were cultured for up to 5 days. Recombining lung epithelial cells with mesenchyme from various sources induced a morphological pattern that was specific to the type of inducing mesenchyme. In situ analysis of surfactant protein (SP)-C, SP-B, and Clara cell secretory protein (CCSP) expression, as well as SP-C and CCSP promoter transactivation experiments, revealed that distal lung epithelium requires lung mesenchyme to maintain the alveolar, but not bronchiolar, phenotype. Incubation of lung recombinants with an anti-FGF7 antibody resulted in a partial inhibition of mesenchyme-induced SP-C promoter transactivation. Immunoreactivity for Delta and Lunatic fringe, components of the Notch pathway that regulates cell differentiation, was downregulated in the heterotypic recombinants. In contrast, Hes1 mRNA expression was increased in these recombinants. Cumulatively, these results suggest that at late fetal gestation, distal lung epithelial cells are not fully committed to a specific phenotype and still have the plasticity to respond to various signals. Their alveolar phenotype is likely maintained by Notch/Notch ligand interactions and mesenchymal factors, including FGF7. epithelial-mesenchymal interactions; cell differentiation; Notch signaling; fibroblast growth factors

Newborn rats exposed to 60% [O.sub.2] for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% [O.sub.2] on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked [O.sub.2]-induced upregulation of PDGF-[alpha] and -[beta] receptors (PDGF-[alpha]R and -[beta]R) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% [O.sub.2]-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the [O.sub.2]-induced upregulation of PDGF-[beta]R, but not PDGF[alpha]R, on arterial smooth muscle. PDGF-BB, a major PDGF-[beta]R ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-[beta]R ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-[beta]R intervention, which attenuated SMC proliferation induced by exposure to 60% [O.sub.2] Collectively, these data are consistent with a major role for PDGF-[beta]R-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% [O.sub.2]-induced pulmonary hypertension. pulmonary oxygen toxicity; vascular smooth muscle; growth factors; soluble receptors

We hypothesized that constitutive formation of reactive oxygen species by respiratory cells is a barrier to gene transfer when liposome-DNA complexes are used, by contributing to rapid degradation of plasmid DNA. When plasmid DNA is complexed to liposomes it is protected against [H.sub.2][O.sub.2]-mediated degradation but not that mediated by the hydroxyl radical. Treatment of distal rat fetal lung epithelial cells (RF[L.sub.19]Ep) with the vitamin E analog Trolox (50 [micro]M) reduced intracellular plasmid degradation. Both Trolox (50 [micro]M) and an iron chelator, phenanthroline (0.1 [micro]M), significantly increased transgene expression in RF[L.sub.19]Ep approximately twofold, consistent with a hydroxyl radical-mediated inhibition of transgene expression. When basic fibroblast growth factor (bFGF; 20 ng/ml), a growth factor with antioxidant properties, was included within liposomes, we observed a significantly greater enhancement of RF[L.sub.19]Ep transgene expression (~2-fold) over that seen with Trolox or phenanthroline. Inclusion of bFGF within liposomes also significantly enhanced (~4-fold) transgene expression in mice following intratracheal instillation. gene transfer; reactive oxygen species; growth factors; basic fibroblast growth factor

The effect of strain on fibronectin metabolism was investigated. This was due to the fact that normal lung growth requires proper coordination between cell proliferation and extracellular matrix remodeling. Organotypic cultures of fetal rat lung cells showed increased fibronectin content in the culture media. The results showed that strain differentially regulated fibronectin production of fetal lung cells at the transcriptional and posttranscriptional levels. Soluble fibronectin content was increased by mechanical strain by stimulating its synthesis and secretion.

A study was conducted to determine whether the induction of fetal distal lung epithelial cells (FDLE) sodium ion transport by postnatal oxygen was permanent or reversible, as well as to examine possible mechanisms for the increased gene expression and functional activity of epithelial Na+ channel (ENaC) in FDLEs. Results showed that the physiological increase in O2 concentration increased distal lung epithelial Na+ transport. This increase was a reversible phenomenon and was likely to be mediated by an increase in reactive oxygen species that cause increased gene expression of ENaC.

The individual responses of fibroblasts and epithelial cells to mechanical strain, as well as the effect of mesenchymal-epithelial interactions on strain-induced cell proliferation during fetal lung development were examined. Both cell types responded to strain with enhanced DNA synthesis throughout late fetal lung development. Strain-induced DNA synthesis was suppressed when cells from different gestations were recombined. The ontogenic response pattern of recombinants to mechanical strain was similar to that of fibroblasts but not of epithelial cells.

An in vitro model system for the transfection of primary epithelial cell cultures from the rat fetal lung was established. Epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity which could be attributed to a nonspecific delivery of exogenous DNA of other copurified factors. The DNA-dependent increase in superoxide-mediated cytotoxicity was inhibited by the addition of nicotinamide and chloroquine.

Hyperoxic exposure enhances the synthesis of insulin-like growth factor (IGF) and its type I receptor in the lungs of adult rats. The expression of growth-stimulating and growth-inhibiting insulin-like growth factor binding proteins (IGFBPs) was investigated to determine if their expression is consistent with the observed synthesis of IGF. Results reveal the most of the IGFBPs generally follow the modulation of IGF synthesis.

The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5[prime] genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternative splicing products, due to the activation of two cryptic splice sites in the CFTR coding region. Modification of the K18 intron and CFTR cDNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein expression as well as biological function. Transgenic expression analysis in mice showed that the modified expression cassette can direct efficient and epithelium-specific expression of the Escherichia coli LacZ gene in the airways of fetal lungs, with no detectable expression in lung fibroblasts or endothelial cells. This is the first expression cassette which selectively directs lung transgene expression for CFTR gene therapy to airway epithelia.

Rat lungs exposed to 85% oxygen showed a significant increase in insulin-like growth factor I (IGF-I) mRNA and peptide after 6 and 14 days of exposure, with maximal expression in the airway and alveolar epithelium. IGF-receptor immunoreactivity (IGFR) also showed a significant increase 6 days after exposure andd localized to the perivascular, peribronchial and endothelial cells. At 14 days after exposure, IGFR localized to the alveolar epithelial cells.

Alveolar P(sub O2) increase at birth leads to a shift from Cl secretion to Na absorption by the distal lung of fetal rats. Epithelial permeability increases transiently when primary cultures of fetal distal lung epithelium are switched from a fetal to a postnatal oxygen environment. Transepithelial resistance changes correspond with changes in the histological appearance of the interepithelial tight junctions. However, the intracellular actin remains unaffected under these conditions. Amiloride-sensitive equivalent short-circuit current increases within 18 hrs of postnatal oxygen exposure.

The addition of antioxidants to surfactants can minimize oxidant injury in the lungs of newborns. A combination of the artificial surfactant Survanta, superoxide dismutase and catalase was readily taken into pulmonary Type II cells and were found to be more successful in preventing oxidant injury. The administered antioxidant and catalase also enhanced the activity of endogenous superoxide dismutase and their dispersal in tissue was greatly aided by the surface-tension-lowering character of Survanta.

The mechanisms by which fetal lung cell behavior are influenced by alterations in lung fibronectin and laminin were investigated by identifying the receptors binding fibronectin and laminin on fetal rat lung cells. Both alpha6-integrin and alpha3-integrin subunit mRNAs were detected in both cells. However, it was found that the alpha3-integrin subunit mRNA completely inhibited epithelial cell attachment to laminin, but only partially by the alpha6-integrin subunit. Binding of the epithelial cells to fibronectin was attributed to an alpha3beta1-integrin binding site peptide.

A mechanism for the transduction of fetal breathing movements in order to stimulate fetal lung growth is proposed from the upregulation of platelet-derived growth factor (PDGF) gene expression in fetal lung cells in response to strain. These physical forces regulates the fetal lung cell by controlling PDGF-B and PDGF-beta-R gene expression. This upregulation has a blocking effect by tyrphostin 9. This mechanism is discussed in detail.

An initial loss of type I pneumocytes is exhibited by lungs subjected to the exposure of increased oxygen exposure. These lungs also exhibit a loss of reparative type II pneumocyte hyperlasia. These losses are primarily caused by the elevation in the concentration of intrapulmonary basic fibroblast growth factor (bFGF). The bFGF is a powerful stimulator of type II pneumocyte DNA synthesis.

Lung damage is caused by the sudden exposure of oxygen starved lung to increased oxygen supply or pulmonary oxygen toxins. The investigation of the process of lung damage reveals that superoxide dismutase, which is delivered to the pulmonar alveolar epithelial cells, causes damage to the lungs.

Variation in the proteinase antiproteinase balance in the lung leads to an enhancement of type I collagen deposition, which is induced by oxygen exposure. The deposition of type I collagen is optimum after 14 days of exposure to 85% oxygen. There is an increase in the mRNA expression of interstitial collagenase, the tissue inhibitor of metalloproteinases and the transforming growth factor-beta1 prior to type I collagen increase. There are no variations in the mRNA expression of stromelysin.

Subunit-specific antibodies enable confirm the presence of alpha1beta1, alpha2beta1 and alpha3beta1 collagen integrins in the fibroblast surface, and the presence of only alpha3beta1 integrin in epithelial cells. The distal fetal lung epithelial cells with iodinated surfaces do not possess any adherence to collagen and do not express the mRNAs for the alpha1- and alpha2-integrin.

Liu, Mingyao, A. Keith Tanswell, and Martin Post. Mechanical force-induced signal transduction in lung cells. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L667-L683, 1999.--The lung is a unique organ in that it is exposed to physical forces derived from breathing, blood flow, and surface tension throughout life. Over the past decade, significant progress has been made at the cellular and molecular levels regarding the mechanisms by which physical forces affect lung morphogenesis, function, and metabolism. With the use of newly developed devices, mechanical forces have been applied to a variety of lung cells including fetal lung cells, adult alveolar epithelial cells, fibroblasts, airway epithelial and smooth muscle cells, pulmonary endothelial and smooth muscle cells, and mesothelial cells. These studies have led to new insights into how cells sense mechanical stimulation, transmit signals intra- and intercellularly, and regulate gene expression at the transcriptional and posttranscriptional levels. These advances have significantly increased our understanding of the process of mechanotransduction in lung cells. Further investigation in this exciting research field will facilitate our understanding of pulmonary physiology and pathophysiology at the cellular and molecular levels. physical force; mechanical stimulation; cell proliferation; cell differentiation

Bronchopulmonary dysplasia (BPD) is a chronic lung injury characterized by impaired alveologenesis that may persist into adulthood. Rat models of BPD using varying degrees of hyperoxia to produce injury either cause early mortality or spontaneously recover following removal of the inciting stimulus, thus limiting clinical relevance. We sought to refine an established rat model induced by exposure to 60% O2 from birth by following hyperoxia with intermittent hypoxia (IH). Rats exposed from birth to air or 60% O2 until day 14 were recovered in air with or without IH (FIO2 = 0.10 for 10 min every 6 h) until day 28. Animals exposed to 60% O2 and recovered in air had no evidence of abnormal lung morphology on day 28 or at 10-12 wk. In contrast, 60% O2-exposed animals recovered in IH had persistently increased mean chord length, more dysmorphic septal crests, and fewer peripheral arteries. Recovery in IH also increased pulmonary vascular resistance, Fulton index, and arterial wall thickness. IH-mediated abnormalities in lung structure (but not pulmonary hypertension) persisted when reexamined at 10-12 wk, accompanied by increased pulmonary vascular reactivity and decreased exercise tolerance. Increased mean chord length secondary to IH was prevented by treatment with a peroxynitrite decomposition catalyst [5,10,15,20-Tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin iron (III) chloride, 30 mg/kg/day, days 14-28], an effect accompanied by fewer inflammatory cells. We conclude that IH during recovery from hyperoxia-induced injury prevents recovery of alveologenesis and leads to changes in lung and pulmonary vascular function lasting into adulthood, thus more closely mimicking contemporary BPD. [ABSTRACT FROM AUTHOR]

Neutrophil (PMNL) influx precedes lung macrophage (LM) influx into the lung following exposure of newborn pups to 60% O2. We hypothesized that PMNL were responsible for the signals leading to LM influx. This was confirmed when inhibition of PMNL influx with a CXC chemokine receptor-2 antagonist, SB-265610, also prevented the 60% O2-dependent LM influx, LM-derived nitrotyrosine formation, and pruning of small arterioles. Exposure to 60% O2 was associated with increased lung contents of neutrophil elastase and elastin, a marker of denatured elastin, and a decrease in elastin fiber density. This led us to speculate that neutrophil elastase-induced elastin fragments were the chemokines that led to a LM influx into the 60% O2-exposed lung. Inhibition of neutrophil elastase with sivelestat or elafin attenuated the LM influx. Sivelestat also attenuated the 60% O2-induced decrease in elastin fiber density. Daily injections of pups with an antibody to elastin prevented the 60% O2-dependent LM influx, impaired alveologenesis, and impaired small vessel formation. This suggests that neutrophil elastase inhibitors may protect against neonatal lung injury not only by preventing structural elastin degradation, but also by blocking elastin fragment-induced LM influx, thus preventing tissue injury from LM-derived peroxynitrite formation. [ABSTRACT FROM AUTHOR]