Apoptosis In Rat Lungs Research Articles

Exogenous Apelin-13 Administration Ameliorates Cyclophosphamide- Induced Oxidative Stress, Inflammation, and Apoptosis in Rat Lungs.

Apelin-13 is an endogenous adipocytokine known for its antioxidant, antiinflammatory, and antiapoptotic properties. We aimed to investigate the possible protective effects of exogenous Apelin-13 administration on oxidative stress, inflammation, and apoptosis induced by the cytotoxic agent cyclophosphamide (CP) in the lungs. Twenty-four male Wistar albino rats were divided into four groups: Control (saline), CP (200 mg/kg), Apelin-13 (10 μg/kg/day), and CP+Apelin-13. CP was administered as a single dose on the fifth day, and apelin-13 was administered intraperitoneally for five days. Total oxidant status (TOS), total antioxidant status (TAS), and lipid peroxidation were determined with spectrophotometry, TNFα and IL1β were determined with ELISA, APJ, Sirt1, NF-κB, and p53 mRNA expressions were determined with qRT-PCR, cytochrome (Cyt) C and caspase-3 protein expressions were studied with western blotting in lung tissues. The oxidative stress index (OSI) was also calculated. Furthermore, serum surfactant protein-D (SP-D) and Krebs von den Lungen-6 (KL-6) levels were measured with ELISA. Compared to the control group, TOS, OSI, lipid peroxidation, TNFα, IL1β, cyt C, caspase-3, APJ, NF-κB, and p53 were higher, and Sirt1 was lower in the lung tissue of rats in the CP group. Serum KL-6 and SP-D levels were higher in the CP group. Co-administration of CP with Apelin-13 completely reversed the changes induced by CP administration. Exogenous Apelin-13 treatment protected lung tissue against injury by inhibiting cyclophosphamide-induced oxidative stress, inflammation, and apoptosis. This protective effect of apelin-13 was accompanied by upregulation of the Sirt1 and downregulation of NF-κB/p53 in the lungs.

Effect of refloated soil dust on oxidative stress and apoptosis in rat lung

Little study has illustrated the associations between inhalation of refloated soil dust and the morbidity of respiratory disease in animals. In this study, soil dust samples in arid and semiarid areas of China were collected for investigating the underlying biotoxicity and mechanisms of oxidative stress and apoptosis in rat lung induced by inhalation of refloated soil dust. The soil dust samples were collected from the arid and semiarid areas in Zhangjiakou city, located close to Inner Mongolia Plateau, China. After intratracheal instillation of soil dust, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities in rat lung were declined in a dose-dependent relationship, and pathology changes in lung tissue were also enhanced along with increased levels of soil dust. Meanwhile, apoptotic cells were scattered in the pulmonary epithelial and distributed in the mesenchyma, and the apoptotic index was significantly increased in rat lung instilled by soil dust. Besides, soil dust promoted Bax expression and inhibited Bcl2 expression, i.e., increased Bax/Bcl2 ratio in a concentration-dependent manner. In summary, short-term exposure to soil dust caused oxidative damage and induced apoptosis in rat lung through the regulation of Bax and Bcl2 expression.

Lead Nitrate Induces Inflammation and Apoptosis in Rat Lungs Through the Activation of NF-κB and AhR Signaling Pathways

Lead (Pb) is one of the most frequent hazardous air contaminants, where the lungs are particularly vulnerable to its toxicity. However, the Pb distribution and its impact on lung inflammation/apoptosis and particularly the involvement of nuclear factor kappa B (NF-κB) and aryl hydrocarbon receptor (AhR) signaling pathways in Pb-induced lung toxicity have not yet been fully investigated. Adult male Wistar albino rats were exposed to Pb nitrate 25, 50, and 100 mg/kg b.w. orally for 3 days. The histopathological changes of several rat organs were analyzed using hematoxylin and eosin staining. The concentrations of Pb ion in different organ tissues were quantified using inductive coupled plasma mass spectrometry, while gas chromatography-mass spectrometry was used to identify organic compounds. The changes in the mRNA and protein expression levels of inflammatory and apoptotic genes in response to Pb exposure were quantified by using RT-PCR and Western blot analyses, respectively. Treatment of rats with Pb for three consecutive days significantly increased the accumulation of Pb in lung tissues causing severe interstitial inflammation. Pb treatment also increased the percentage of lung apoptotic cells and modulated apoptotic genes (Bc2, p53, and TGF-α), inflammatory markers (IL-4, IL-10, TNF-α), and oxidative stress biomarkers (iNOS, CYP1A1, EphX) in rat lung tissues. These effects were associated with a significant increase in organic compounds, such as 3-nitrotyrosine and myeloperoxidase, and some inorganic elements, such as selenium. Importantly, the Pb-induced lung inflammation and apoptosis were associated with a proportional increase in the expression of NF-κB and AhR mRNAs and proteins. These findings clearly show that Pb induces severe inflammation and apoptosis in rat lungs and suggest that NF-κB and AhR may play a role in Pb-induced lung toxicity.

Thal protects against paraquat-induced lung injury through a microRNA-141/HDAC6/IκBα-NF-κB axis in rat and cell models.

The protective functions of thalidomide in paraquat (PQ)‐induced injury have been reported. But the mechanisms remain largely unknown. In this research, a PQ‐treated rat model was established and further treated with thalidomide. Oedema and pathological changes, oxidative stress, inflammation, fibrosis and cell apoptosis in rat lungs were detected. A PQ‐treated RLE‐6TN cell model was constructed, and the viability and apoptosis rate of cells were measured. Differentially expressed microRNAs (miRNAs) after thalidomide administration were screened out. Binding relationship between miR‐141 and histone deacetylase 6 (HDAC6) was validated. Altered expression of miR‐141 and HDAC6 was introduced to identify their involvements in thalidomide‐mediated events. Consequently, thalidomide administration alone exerted no damage to rat lungs; in addition it reduced PQ‐induced oedema. The oxidative stress, inflammation and cell apoptosis in rat lungs were reduced by thalidomide. In RLE‐6TN cells, thalidomide increased cell viability and decreased apoptosis. miR‐141 was responsible for thalidomide‐mediated protective events by targeting HDAC6. Overexpression of HDAC6 blocked the protection of thalidomide against PQ‐induced injury via activating the IkBα‐NF‐κB signalling pathway. Collectively, this study evidenced that thalidomide protects lung tissues from PQ‐induced injury through a miR‐141/HDAC6/IkBα‐NF‐κB axis.

Mitochondrial separation protein inhibitor inhibits cell apoptosis in rat lungs during intermittent hypoxia.

Obstructive sleep apnoea (OSA) is a very common sleep and breathing disorder that occurs in worldwide. It is important to develop a more effective treatment for OSA to overcome lung cell apoptosis during intermittent hypoxia (IH). A mitochondrial separation protein inhibitor (Mdivi-1) has been demonstrated to be a powerful tool for inhibiting apoptosis. In the present study, the protective effect and possible mechanism of apoptosis in lung cells during IH was investigated using in vivo and in vitro experiments. Following IH exposure for 4 weeks, the lung tissues of Sprague Dawley rats exhibited interstitial lesions, while Mdivi-1 reduced these pulmonary interstitial lesions. B-cell lymphoma (Bcl)-2 mRNA and protein expression levels were decreased however caspase-3, caspase-9 and dynamin-related protein 1 (Drp-1) mRNA and protein expression levels were increased. Following Mdivi-1 intervention, Bcl-2 mRNA and protein expression levels were increased while caspase-3, caspase-9 and Drp-1 mRNA and protein expression levels were decreased (P<0.05). After exposure to IH for 12 h, the apoptosis rate of WTRL1 cells in rats increased gradually with the IH time (P<0.05). Bcl-2 mRNA and protein expression levels were decreased, whereas caspase-3, caspase-9, cytochrome C (Cyt-C) and Drp-1 mRNA levels were increased, and caspase-3, caspase-9 and Drp-1 protein expression levels were increased. After Mdivi-1 intervention, Bcl-2 mRNA and protein expression levels were increased but caspase-3, caspase-9, Cyt-C and Drp-1 mRNA levels were decreased along with caspase-9, Cyt-C and Drp-1 protein expression levels which were decreased (P<0.05). The results of the present study suggest that Mdivi-1 may be a potential agent for treating OSA because it inhibits the mitochondrial pathway and reduces apoptosis.

Inositol monophosphatase 1 as a novel interacting partner of RAGE in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation end products (RAGE) plays an important role in PAH by promoting proliferation of pulmonary vascular cells. RAGE is also known to mediate activation of Akt signaling, although the particular molecular mechanism remains unknown. This study aimed to identify the interacting partner of RAGE that could facilitate RAGE-mediated Akt activation and vascular remodeling in PAH. The progressive angioproliferative PAH was induced in 24 female Sprague-Dawley rats ( n = 8/group) that were randomly assigned to develop PAH for 1, 2, or 5 wk [right ventricle systolic pressure (RVSP) 56.5 ± 3.2, 63.6 ± 1.6, and 111.1 ± 4.5 mmHg, respectively, vs. 22.9 ± 1.1 mmHg in controls]. PAH triggered early and late episodes of apoptosis in rat lungs accompanied by RAGE activation. Mass spectrometry analysis has identified IMPA1 as a novel PAH-specific interacting partner of RAGE. The proximity ligation assay (PLA) confirmed the formation of RAGE/IMPA1 complex in the pulmonary artery wall. Activation of IMPA1 in response to increased glucose 6-phosphate (G6P) is known to play a critical role in inositol synthesis and recycling. Indeed, we confirmed a threefold increase in G6P ( P = 0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), membrane translocation of PI3K, and a threefold increase in membrane Akt levels ( P = 0.02) and Akt phosphorylation. We conclude that the formation of the newly discovered RAGE-IMPA1 complex could be responsible for the stimulation of inositol pathways and activation of Akt signaling in PAH.

Exposure to Cerium Oxide Nanoparticles Is Associated With Activation of Mitogen-activated Protein Kinases Signaling and Apoptosis in Rat Lungs.

Objectives:With recent advances in nanoparticle manufacturing and applications, potential exposure to nanoparticles in various settings is becoming increasing likely. No investigation has yet been performed to assess whether respiratory tract exposure to cerium oxide (CeO2) nanoparticles is associated with alterations in protein signaling, inflammation, and apoptosis in rat lungs.Methods:Specific-pathogen-free male Sprague-Dawley rats were instilled with either vehicle (saline) or CeO2 nanoparticles at a dosage of 7.0 mg/kg and euthanized 1, 3, 14, 28, 56, or 90 days after exposure. Lung tissues were collected and evaluated for the expression of proteins associated with inflammation and cellular apoptosis.Results:No change in lung weight was detected over the course of the study; however, cerium accumulation in the lungs, gross histological changes, an increased Bax to Bcl-2 ratio, elevated cleaved caspase-3 protein levels, increased phosphorylation of p38 MAPK, and diminished phosphorylation of ERK-1/2-MAPK were detected after CeO2 instillation (p<0.05).Conclusions:Taken together, these data suggest that high-dose respiratory exposure to CeO2 nanoparticles is associated with lung inflammation, the activation of signaling protein kinases, and cellular apoptosis, which may be indicative of a long-term localized inflammatory response.

The endoplasmic reticulum stress pathway involving protein kinase R-like ER kinase-activating transcription factor 4-CCAAT/enhancer binding protein homologous protein implicated in apoptosis in lungs of rats with bronchopulmonary dysplasis

Objective To investigate the role of endoplasmic reticulum stress (ERS) pathway involving protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) in apoptosis in lungs of rats with bronchopulmonary dysplasis (BPD). Methods Forty-eight premature SD rats were divided into BPD group and control group according to random number table.Rats in BPD group were continually exposed to O2 with volumetric concentration factor of 850 mL/L, while rats in control group were exposed to air.Lung tissues in each group were obtained in 7, 14 and 21 days respectively.The apoptosis in lung cells was evaluated by terminal dexynucleotifyl transferase-mediated dUTP nick end labeling (TUNEL) assay.The mRNA levels of glucose regulated protein 78 (GRP78), PERK, ATF4 and CHOP were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of GRP78, phosphorylated PERK (pho-PERK), ATF4 and CHOP were detected by using Western blot. Results Compared with control group, the lung cells of the rats in BPD group developed more serious apoptosis.Furthermore, the apoptosis index (AI) in lung cells of the rats increased rapidly with the hyperoxia exposure time.This had been statistically verified by comparison with the control group at different timing(7 d: 15.50±0.58 vs 1.25±0.50, 14 d: 27.75±1.71 vs 3.25±0.96, 21 d: 50.50±3.70 vs 4.00±1.15; t=57.00, 20.58, 25.16, all P<0.01). The mRNA levels of GRP78, PERK, ATF4 and CHOP in BPD group increased significantly compared to the control group [GRP78: 7 d (33.88±3.73 )vs(11.65±1.00), 14 d (54.50±2.18)vs(12.84±1.41), 21 d (95.34±7.61)vs(12.43±0.59); PERK: 7 d (5.23±0.92)vs(1.45±0.46), 14 d (7.60±1.56)vs(2.18±0.97), 21 d (16.55±0.50)vs(2.90±1.18); ATF4: 7 d (23.04±2.45)vs(12.56±2.81), 14 d (28.66±2.66)vs(15.18±2.92), 21 d (36.63±2.99)vs(15.14±2.09); CHOP: 7 d (2.21±0.19)vs(0.81±0.02), 14 d (4.19±0.17)vs(0.90±0.08), 21 d (6.08±0.38)vs(0.88±0.10); all P<0.05]. The protein levels of GRP78, pho-PERK, ATF4 and CHOP in BPD group increased significantly as well [GRP78: 7 d (1.33±0.03 )vs(0.85±0.04), 14 d (1.31±0.02)vs(0.92±0.01), 21 d (1.82±0.28)vs(0.87±0.01); pho-PERK: 7 d (0.68±0.02)vs(0.54±0.01), 14 d (1.04±0.01)vs(0.65±0.01), 21 d (1.29±0.02)vs(0.73±0.01); ATF4: 7 d (1.26±0.01)vs(0.83±0.01), 14 d (1.39±0.02)vs(0.87±0.02), 21 d (1.67±0.02)vs(0.94±0.02); CHOP: 7 d (1.37±0.01)vs(0.47±0.06), 14 d (1.50±0.04)vs(0.74±0.05), 21 d (1.61±0.03)vs(0.55±0.02); all P<0.05]. Positive correlation was demonstrated between the expression levels of CHOP protein and AI, PERK, ATF4 in the BPD group (r=0.87, 0, 92, 0.93 respectively, all P<0.05). Conclusion PERK-ATF4-CHOP mediated ERS may participate in and contribute to the apoptosis mechanism in lungs of rats with BPD. Key words: Endoplasmic reticulum stress; Protein kinase R-like ER kinase; CCAAT/enhancer binding protein homologous protein; Bronchopulmonary dysplasis; Apoptosis

Endostatin, an angiogenesis inhibitor, ameliorates bleomycin-induced pulmonary fibrosis in rats

BackgroundRecent evidence has demonstrated the role of angiogenesis in the pathogenesis of pulmonary fibrosis. Endostatin, a proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis. The aim of our study was to assess whether endostatin has beneficial effects on bleomycin (BLM)-induced pulmonary fibrosis in rats.MethodsThe rats were randomly divided into five experimental groups: (A) saline only, (B) BLM only, (C) BLM plus early endostatin treatment, (D) BLM plus late endostatin treatment, and (F) BLM plus whole-course endostatin treatment. We investigated the microvascular density (MVD), inflammatory response and alveolar epithelial cell apoptosis in rat lungs in each group at different phases of disease development.ResultsEarly endostatin administration attenuated fibrotic changes in BLM-induced pulmonary fibrosis in rats. Endostatin treatment decreased MVD by inhibiting the expression of VEGF/VEGFR-2 (Flk-1) and the activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). Endostatin treatment also decreased the number of inflammatory cells infiltrating the bronchoalveolar lavage fluid during the early inflammatory phase of BLM-induced pulmonary fibrosis. In addition, the levels of tumour necrosis factor-α (TNF-α) and transforming growth factor β1 (TGF-β1) were reduced by endostatin treatment. Furthermore, endostatin decreased alveolar type II cell apoptosis and had an epithelium-protective effect. These might be the mechanism underlying the preventive effect of endostatin on pulmonary fibrosis.ConclusionsOur findings suggest that endostatin treatment inhibits the increased MVD, inflammation and alveolar epithelial cell apoptosis, consequently ameliorating BLM-induced pulmonary fibrosis in rats.

Regulatory T Cells Limit Vascular Endothelial Injury and Prevent Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood. To determine whether immune dysregulation due to absent T-cell populations directly contributes to the development of PAH. Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell-deficient rats but not in immune-reconstituted (IR) rats. T cell-lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH, whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4(+)CD25(hi) or CD4(+)CD25(-) T cell subsets prior to vascular injury attenuated the development of PAH. IR limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3(+), IL-10- and TGF-β-expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)-expressing cells, a receptor that activates endothelial cell survival pathways. PAH may arise when regulatory T-cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.

Effect of different tidal volume ventilation on apoptosis and the expression of Bax and Bcl-2 in rat lungs

To determine the effect of different tidal volume(Vt) ventilation on apoptosis and the expression of Bax and Bcl-2 in rat lungs. Twenty-four healthy SD rats were randomly divided into 4 groups: a control group, a low Vt ventilation group (LV), a middle Vt ventilation group (MV), and a high Vt ventilation group (HV). Rats were subjected to different tidal volumes (10,20,and 40 mL/kg) for 2 h except the control group, which kept their own breath. We determined the lung histopathology score, W/D ratio and WBC in bronchoalceolar lavage fluid (BALF) to evaluate the lung injury and examine the apoptotic cell death, Bax and Bcl-2 protein expression by using TUNEL technique and immunohistochemistry 24 h after the operation. Compared with the control group, MV and HV increased lung histopathology score, W/D ratio, WBC in BALF, apoptosis index (AI )and Bax protein expression, but decreased Bcl-2 protein expression (P<0.05). These changes showed no significant difference between the control group and the low Vt ventilation group (P>0.05). Low Vt ventilation contributed little to apoptosis. Higher Vt ventilation can improve Bax while inhibit Bcl-2 expression to aggravate apoptosis in rat lungs.

Benzo( a)pyrene increases phosphorylation of p53 at serine 392 in relation to p53 induction and cell death in MCF-7 cells

Although benzo( a)pyrene (BP) induces apoptosis in vitro in murine Hepa1c1c7 cells and in vivo indications of apoptosis in rat lung exist, related cellular mechanisms in human cells are not known. p53 protein participates in several apoptotic processes. We found that BP induces cell death in human MCF-7 breast adenocarcinoma cells at 48 and 72 h but not in human A549 lung carcinoma cells. BP did not induce measurable caspase-3-like protease activity or internucleosomal DNA fragmentation in either cell types. However, procaspase-7 cleavage in MCF-7 cells by BP-treatment indicates activation of caspase-7 meaning that apoptosis is most likely involved in BP-induced MCF-7 cell death. BP-7,8-dihydrodiol-9,10-epoxide (BPDE)–DNA adducts and level of p53 protein increased dose-dependently, but more extensively in MCF-7 cells. Phosphorylation of p53 protein at serines 15, 20, 46 and 392 increased in MCF-7 cells. Increase in phosphorylation at serine 392 was clear already at 24 h by 1 μM concentration of BP. Increase of phosphorylation at other sites occurred only with higher concentrations or at later time points in relation to the increase of p53 protein. These results suggest that serine 392 phosphorylation is the first stabilizing event of p53 associated with BP exposure and subsequent cell death in MCF-7 cells.

Nitric oxide inhalation inhibits inducible nitric oxide synthase but not nitrotyrosine formation and cell apoptosis in rat lungs with meconium-induced injury1

To investigate the effects of inhaled nitric oxide (NO) on pulmonary inflammation, apoptosis, peroxidation and protein nitration in a rat model of acute lung injury (ALI) induced by meconium. Twenty-four healthy male Sprague-Dawley rats were randomly devided into 3 groups (n=8): meconium-induced ALI with intratracheal instillation of 1 mL/kg saline (Mec/saline group), continuous inhalation of NO at 20 muL/L. (Mec/iNO), and the control group (control). Electromicroscopic examination was used to determine the extent of epithelial apoptosis. TUNEL was used to detect DNA fragmentation in pulmonary apoptotic cells, expressed as the apoptosis index (AI). Western blotting was used to detect pulmonary inducible NO synthase (iNOS) expression. RT-PCR was used to detect interleukin (IL)-1beta mRNA expression. Cell count in bronchoalveolar lavage (BAL), myeloperoxidase (MPO) activity, as well as malondialdehyde (MDA) and nitrotyrosine formation, the markers of toxic NO-superoxide pathway in rat lung parenchyma specimens, were also examined. Expression of iNOS protein and IL-1beta mRNA were increased significantly in the Mec/saline group (both P<0.01) compared with the control group. BAL cell count, MPO activity, lung injury score, pulmonary AI, MDA level and nitrotyrosine formation were also increased significantly (all P<0.01). The meconium-induced iNOS protein and IL-1beta mRNA expression were inhibited significantly by NO inhalation when compared with the Mec/saline group (both P<0.01). BAL cell count, MPO activity and lung injury score were also decreased significantly (P<0.01 or P<0.05). However, there were no statistical differences in MDA level, nitrotyrosine formation or pulmonary AI between the Mec/saline and Mec/iNO groups. Electromicroscopic examination revealed a significant degree of epithelial apoptosis in both the Mec/saline and Mec/iNO groups. Early continuous inhalation of NO 20 muL/L may protect the lungs from inflammatory injury, but does not decrease epithelial apoptosis or lung nitrotyrosine formation. Inhalation of NO alone is not associated with a detectable increase in oxidant stress.

Pulmonary reexpansion causes xanthine oxidase-induced apoptosis in rat lung

The pathogenesis of reexpansion pulmonary edema is not yet fully understood. We therefore studied its mechanism in a rat model in which the left lung was collapsed by bronchial occlusion for 1 h and then reexpanded and ventilated for an additional 3 h. We then evaluated the production of reactive oxygen species in the lungs using fluorescent imaging and cerium deposition electron microscopic techniques and the incidence of apoptosis using the TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. We found that pulmonary reexpansion induced production of reactive oxygen species and then apoptosis, mainly in endothelial and alveolar type II epithelial cells. Endothelial cells and alveolar type I and II epithelial cells in the reexpanded lung were positive for TUNEL and cleaved caspase-3. DNA fragmentation was also observed in the reexpanded lung. In addition, wet-dry ratios obtained with reexpanded lungs were significantly higher than those obtained with control lungs, indicating increased fluid content. All of these effects were attenuated by pretreating rats with a specific xanthine oxidase inhibitor, sodium (-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one. It thus appears that pulmonary reexpansion activates xanthine oxidase in both endothelial and alveolar type II epithelial cells and that the reactive oxygen species produced by the enzyme induce apoptosis among the endothelial and alveolar type I and II epithelial cells that make up the pulmonary water-air barrier, leading to reexpansion pulmonary edema.

Lung tissue eosinophils may be cleared through luminal entry rather than apoptosis: effects of steroid treatment.

Spontaneous or steroid-induced eosinophil apoptosis occurring in vitro has not been demonstrated in lung tissues in vivo. This study examines cell apoptosis in rat lungs using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) technique and transmission electron microscopy (TEM). After establishing sustained lung edema and eosinophilia by challenge with Sephadex beads intratracheally, budesonide treatment was started intratracheally. Sephadex alone increased the total number of apoptotic cells, which were not efficiently engulfed by macrophages or other cells, in vivo. Yet apoptotic tissue eosinophils were exceedingly rare (1 of 360 TEM-analyzed eosinophils). By contrast, approximately 20% of eosinophils in the airway lumen were apoptotic, and unengulfed. Budesonide promptly inhibited edema but 3 d of steroid treatment were required to reduce the established tissue eosinophilia. Not at any time point did budesonide induce eosinophil apoptosis (0 of 318 TEM-analyzed tissue eosinophils). We conclude that (1) eosinophil apoptosis can occur but is a rare event in vivo in respiratory tract tissues; (2) airway tissue eosinophils, rather than undergoing apoptosis, are eliminated by migration into airway lumen followed by apoptosis and mucociliary clearance; (3) anti-inflammatory steroid treatment may not increase eosinophil apoptosis in vivo nor may it affect the luminal entry of eosinophils; (4) steroids permit elimination of eosinophils into airway lumen and slowly resolve established lung eosinophilia.

Inhaled nitric oxide protects against hyperoxia-induced apoptosis in rat lungs.

Inhaled nitric oxide (NO), frequently administered in combination with hyperoxic gas mixtures, was recently shown to protect against the injurious consequences of prolonged hyperoxia. We investigated the possibility that this protective effect is attributable to the ability of NO to block pulmonary apoptosis. We show that rats exposed to 100% O2 for 60 h develop severe lung injury consisting of pronounced vascular leak and alveolar apoptosis as inferred from the presence of positive terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and DNA ladders in agarose gels and a decrease in constitutive procaspase-3 levels. However, the inclusion of NO (20 parts/million) in the hyperoxic gas mixture significantly attenuated both the vascular leak and apoptosis. NO reversed the hyperoxia-associated changes in the activity of the redox-sensitive transcription factors nuclear factor-kappaB, activator protein-1, and Sp1 after 24 h, lowered intercellular adhesion molecule-1 levels, and increased glutathione content. We therefore show, for the first time, that NO can protect against both hyperoxia-induced apoptosis and inflammation. The data suggest that this protection may occur at the transcriptional and caspase-activation levels.