Bcl-2 family gene expression during severe hyperoxia induced lung injury.

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-X(L). The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-X(L) with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-X(L), no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-X(L) mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-X(L), but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.

Discordant pulmonary proinflammatory cytokine expression during acute hyperoxia in the newborn rabbit.

Newborn animals are resistant to oxygen toxicity. To investigate this phenomenon, the proinflammatory cytokines interleukin (IL)-1 beta, IL-8, and monocyte chemoattractant protein-1 (MCP-1) were measured during newborn rabbit hyperoxic lung injury. Pups were exposed to > 95% O2 for 8-9 days, followed by 60% O2 until 36 days of age. Lung lavage fluid, RNA, and tissue sections were collected at 0, 2, 4, 6, 8, 10, 12, 14, 22, and 36 days. Acute inflammation occurred by 6-10 days of hyperoxia, and fibrosis by 22 days. Northern hybridization of lung homogenates from hyperoxia-exposed pups showed elevated MCP-1 and IL-8 mRNA expression at 6 and 10 days, respectively, compared to age-matched, air-exposed controls. Lavage fluid IL-8 protein also peaked at 10 days, and was strongly correlated to neutrophil numbers in lavage. In situ hybridization revealed elevated IL-1 beta mRNA in macrophages, alveolar epithelial and interstitial cells at 2-10 days, elevated MCP-1 mRNA in similar cell types at 4-8 days, and elevated IL-8 mRNA in these cells and neutrophils at 4-10 days. IL-1 beta and IL-8 expression peaked during peak inflammation, whereas peak MCP-1 expression preceded macrophage influx. Comparing newborn and adult animals' chemokine response may help explain their differences in hyperoxia susceptibility.

Hyperoxia increases keratinocyte growth factor mRNA expression in neonatal rabbit lung.

Acute hyperoxic lung injury remains a major factor in the development of chronic lung disease in neonates. A critical step in the repair of acute lung injury is the proliferation of type II alveolar epithelial cells. Type II cell proliferation is stimulated by keratinocyte growth factor (KGF), an epithelial cell-specific mitogen. We sought to investigate KGF mRNA expression in relation to type II cell proliferation during hyperoxic lung injury. We studied a previously described newborn (NB) rabbit model of acute and chronic hyperoxic injury [C. T. D'Angio, J. N. Finkelstein, M. B. LoMonaco, A. Paxhia, S. A. Wright, R. B. Baggs, R. H. Notter, and R. M. Ryan. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L720-L730, 1997]. NB rabbits were placed in 100% O2 for 9 days and then recovered in 60% O2. RT-PCR was used to synthesize and amplify a 267-bp fragment of rabbit KGF cDNA from whole lung RNA. KGF mRNA expression was analyzed by ribonuclease protection assay, and mRNA abundance was quantified by phosphorimaging. Proliferating cell nuclear antigen immunohistochemistry was used on lung sections to identify proliferating cells. The rabbit partial cDNA sequenced was >95% homologous to human cDNA, and all amino acids were conserved. Whole lung KGF mRNA expression was increased 12-fold after 6 days of hyperoxia compared with control lungs, and remained increased throughout the 100% O2 exposure period. Proliferating cell nuclear antigen immunohistochemistry showed an increase in type II cell proliferation after 8-12 days of hyperoxia. NB rabbits exposed to hyperoxic injury exhibit increased whole lung KGF mRNA expression preceding type II cell proliferation. KGF may be an important mitogen in the regulation of alveolar epithelial repair after hyperoxic lung injury.

Fibronectin expression in bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic fibrotic lung disease of neonates. Fibronectin (FN), a component of the extracellular matrix, is increased in the tracheobronchial effluent of neonates destined to develop BPD. Pulmonary FN is derived from plasma and local cellular synthesis. In order to identify which pulmonary cells synthesize FN and to test the hypothesis that FN is more abundant in lungs with BPD, we examined the distribution of pulmonary FN by in situ hybridization (for mRNA) and immunohistochemistry (for protein) in neonatal autopsy lung specimens, comparing lungs with BPD to those without. We used a staging system in which BPD is characterized by disruption of alveolar architecture, severe vascular changes, airway epithelial necrosis, smooth muscle hypertrophy, and peribronchial fibrosis. FN mRNA and protein were found in vascular endothelium, macrophages, fibroblasts, vascular and airway smooth muscle, and chondrocytes as well as in the pulmonary parenchyma in neonates with and without BPD. Hyaline membranes, when present, immunostained intensely for FN protein. FN mRNA was not seen in airway epithelial cells of either group. FN mRNA and protein were first increased in early acute BPD with their levels appearing greatest during the chronic reparative stage of BPD. In long-standing "healed" BPD, lower levels of FN mRNA and protein were seen. These findings are consistent with the association of increased FN with adult fibrotic lung disease and the previously reported increase in FN tracheal effluent levels in infants with BPD. Our results suggest an important role for pulmonary cell-derived FN in the early inflammatory and later proliferative stages of BPD.

Changes in surfactant protein gene expression in a neonatal rabbit model of hyperoxia-induced fibrosis.

Lung injuries, including bronchopulmonary dysplasia, alter the surfactant system. We developed a newborn rabbit model of acute, followed by chronic, hyperoxic injury to study surfactant protein (SP) gene expression. Initial litters were exposed to >95% O2 until 50% died (LD50; 7-11 days old). Subsequent litters were exposed to >95% O2 for 8 days, followed by 60% O2 until 22-36 days. Controls were exposed to room air. LD50 animals displayed acute pulmonary inflammation, edema, protein leak, and surfactant dysfunction. These changes resolved, and fibrosis developed by 22 days. Whole lung SP-A mRNA expression (measured by membrane hybridization) was twice control levels at 4 days of >95% O2, with specific elevations in terminal bronchioles and type II cells at 4 days and the LD50 by in situ hybridization. Whole lung SP-B and SP-C mRNA were unchanged from control throughout exposure. However, in situ hybridization showed elevations in SP-B and SP-C mRNA in type II cells in inflamed areas at the LD50. SP mRNA alterations resolved by 22-36 days. The surfactant system recovers from acute hyperoxic injury, despite continued 60% O2 exposure.

Differential cytokine mRNA expression by neonatal pulmonary cells.

The purpose of this study was to describe cytokine profiles of human neonatal pulmonary cells isolated by tracheal aspiration (TA) and by deep pulmonary lavage (DPL). We hypothesized that mRNA phenotyping, using the technique of reverse transcriptase polymerase chain reaction (RT-PCR), would reveal differences in cytokine expression patterns between cells from proximal and distal airway compartments. We reasoned that cells derived by DPL may reflect pathogenic pathways indicative for the development of bronchopulmonary dysplasia in the premature infant. Here we have described the detection of mRNA for IL-1 alpha, IL-1 beta, IL-6, IL-8, and tumor necrosis factor-alpha. Fourteen paired TA and DPL samples from six premature infants were collected at 1, 7, or 28 d of age. Two of 14 samples were negative for beta-actin (a ubiquitous mRNA) by RT-PCR and were excluded from further analysis. Each of the remaining 12 samples expressed IL-8. Furthermore, each cytokine could be expressed by TA or DPL cells. Cytokine mRNA phenotype profiles were found to differ between TA and DPL cells in four of five paired samples. Our results show that cells retrieved from these two pulmonary compartments are sources for these cytokines and suggest that RT-PCR of TA/DPL cells can be used to test hypothetical predictive markers for the development of bronchopulmonary dysplasia.

Retinal vascular endothelial growth factor (VEGF) mRNA expression is altered in relation to neovascularization in oxygen induced retinopathy.

The temporal and spatial expression of vascular endothelial cell growth factor (VEGF) mRNA was studied in normal developing cat retina, and in oxygen induced retinopathy. Unexposed control and oxygen-exposed animals (80 h of 80% oxygen from day 3, n = 16) were studied at 1, 2, 4, and 6 weeks after birth. India ink injected retinal flat mounts were used to study vessel progression, and in situ hybridizations using retinal cross sections were used to assess VEGF mRNA accumulation. In controls, as the retina matured, VEGF mRNA hybridization was evident in the ganglion cell layer in a scattered line of distinct cells prior to the ingrowth of vessels, involved the most cells in regions just peripheral to invading vessels and persisted in a fewer positive cells, widely spaced in the vascularized retinas of control, six week animals. In the inner nuclear layer, hybridization initially appeared diffusely and later became localized to a narrow portion of that layer and persisted there. In animals with oxygen induced retinopathy, a substantial increase in hybridization was observed in both the ganglion cell and inner nuclear layers of the avascular retina anterior to the advancing neovascularization. VEGF hybridization decreased abruptly to background levels in both layers at the point were neovascularization met avascular retina. By six weeks, when the neovascularization reached the ora, there was a return of VEGF mRNA in the inner nuclear layer which was similar to normal control expression. A low level of unchanging expression was also observed in the retinal pigment epithelium in both groups at all ages. These results indicate that VEGF mRNA abundance is regulated during retinal vascularization and is increased in relation to oxygen induced neovascularization, suggesting that VEGF may play an important role in both normal retinal vessel development and in the pathophysiology of retinopathy of prematurity.

Interleukin-8 and monocyte chemoattractant protein-1 mRNAs in oxygen-injured rabbit lung.

The chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are neutrophil and monocyte attractants, respectively. We hypothesized that IL-8 and MCP-1 mRNA expression in alveolar macrophages (AM) lavaged from rabbit lung would be increased by oxygen exposure, which is known to induce inflammation. Adult rabbits were exposed to > 95% oxygen for up to 64 h and allowed to recover in room air for up to 72 h before killing and pulmonary lavage. Numbers of lavageable polymorphonuclear cells (PMN) and AM rose during the exposure protocol. Quantitative in situ hybridization with 3H-labeled cRNA probes showed both IL-8 and MCP-1 mRNA expression in AM during oxygen exposure, with peak levels of IL-8 mRNA at 56-h oxygen exposure and of MCP-1 mRNA at 64-h oxygen exposure with 24-h room air recovery. IL-8 mRNA was present in PMN between 48-h oxygen exposure and 64-h oxygen exposure with 24-h room air recovery. MCP-1 mRNA was not expressed in PMN. This pattern of chemokine mRNA expression emphasizes the importance of inflammatory cells as effectors in the pulmonary response to oxygen exposure.

Cell-specific expression of fibronectin in adult and developing rabbit lung.

Fibronectin (FN), a glycoprotein component of the extracellular matrix, plays a role in tissue morphogenesis and tissue-specific differentiation through its effects on cell adhesion, cell shape, and cytoskeletal organization. Immunohistochemistry has been used to show that during lung development FN deposition changes, yet the cell-specific sites of pulmonary FN synthesis have not been determined. Because cellular FN synthesis is reflected by FN mRNA abundance, we performed in situ hybridizations to identify pulmonary tissue with the capacity to synthesize FN. Both in situ mRNA hybridization and immunohistochemical staining were performed on tissue sections from lungs of adults and late gestation fetal and neonatal rabbits. In adults, FN transcripts and immunostaining were clearly seen in endothelial cells, smooth muscle cells, and chondrocytes. During lung development, FN transcripts were virtually ubiquitous except in airway epithelium. There was a gradual decrease in FN mRNA abundance with advancing fetal age, but low levels of FN mRNA persisted in neonatal and adult lungs. In contrast, parenchymal immunostaining increased throughout fetal development and remained elevated in the newborn. FN immunostaining was lower in adult lung. In all tissues examined, airway epithelial cells contained no FN transcripts above background. However, immunostaining was detected in airway basement membrane zones and on luminal surfaces of some epithelial cells. The lack of FN transcripts in airway epithelial cells suggests that FN synthesis does not normally occur in this cell type and that its associated FN immunostaining is from another source. The colocalization of FN mRNA and protein in pulmonary endothelial cells, smooth muscle cells, and chondrocytes in adults strongly suggests that these cells are sites of FN synthesis.

Increased fibronectin mRNA in alveolar macrophages following in vivo hyperoxia.

Oxygen-mediated lung injury can stimulate a fibroproliferative response resulting in the alteration of the pulmonary extracellular matrix and subsequent scarring of parenchymal tissue. Fibronectin (FN), a component of the extracellular matrix, appears in increased quantities in fibrotic lung disease. Alveolar macrophages (AMs) are a potential source of this molecule. Using quantitative in situ hybridization, we demonstrated that AMs from rabbits acutely exposed to 100% oxygen (hyperoxia) for up to 64 h have 20-fold greater levels of FN mRNA relative to cells from control animals. When animals were allowed to recover in room air for up to 72 h after maximal oxygen exposure, AM FN mRNA abundance approached baseline levels. Furthermore, in oxygen-exposed animals, the fraction of lavaged cells expressing FN mRNA was increased 10-fold relative to controls. Although there was marked cell-to-cell variation, we conclude that the AM is a potential source of FN in the events leading to hyperoxia-induced pulmonary fibrosis.

Molecular analysis of Drosophila melanogaster AdhnLA405 confirms reliability of DNA-sequencing methodology.

An alcohol dehydrogenase (ADH) null mutant of Drosophila melanogaster (AdhnLA405) originally recovered following X-ray irradiation of mature sperm (Aaron, 979) is analyzed by Southern blotting, Western blotting, and DNA sequencing. The genetic, immunologic, and nucleic acid sequence data are consistent with the hypothesis that a cross-over event, independent of X-irradiation, between parental chromosomes is responsible for the ADH null phenotype of AdhnLA405. By DNA-sequence analysis we show that molecular cloning of this locus (i.e., propagation in prokaryotic hosts) apparently does not introduce any spurious changes (substitutions, additions, deletions, or rearrangements) within the DNA.