Lung Cell Apoptosis Research Articles

Acrolein Induces Endoplasmic Reticulum Stress and Causes Airspace Enlargement

BackgroundGiven the relative abundance and toxic potential of acrolein in inhaled cigarette smoke, it is surprising how little is known about the pulmonary and systemic effects of acrolein. Here we test the hypothesis whether systemic administration of acrolein could cause endoplasmic reticulum (ER) stress, and lung cell apoptosis, leading to the enlargement of the alveolar air spaces in rats.MethodsAcute and chronic effects of intraperitoneally administered acrolein were tested. Mean alveolar airspace area was measured by using light microscopy and imaging system software. TUNEL staining and immunohistochemistry (IHC) for active caspase 3 and Western blot analysis for active caspase 3, and caspase 12 were performed to detect apoptosis. The ER-stress related gene expression in the lungs was determined by Quantitative real-time PCR analysis. Acrolein-protein adducts in the lung tissue were detected by IHC.ResultsAcute administration of acrolein caused a significant elevation of activated caspase 3, upregulation of VEGF expression and induced ER stress proteins in the lung tissue. The chronic administration of acrolein in rats led to emphysematous lung tissue remodeling. TUNEL staining and IHC for cleaved caspase 3 showed a large number of apoptotic septal cells in the acrolein-treated rat lungs. Chronic acrolein administration cause the endoplasmic reticulum stress response manifested by significant upregulation of ATF4, CHOP and GADd34 expression. In smokers with COPD there was a considerable accumulation of acrolein-protein adducts in the inflammatory, airway and vascular cells.ConclusionsSystemic administration of acrolein causes endoplasmic reticulum stress response, lung cell apoptosis, and chronic administration leads to the enlargement of the alveolar air spaces and emphysema in rats. The substantial accumulation of acrolein-protein adducts in the lungs of COPD patients suggest a role of acrolein in the pathogenesis of emphysema.

Protective effects of ischemic postconditioning on non-heart-beating donor in rat lung transplantation

To explore the protective effects of ischemic postconditioning on non-heart-beating donor (NHBD) in rat NHBD lung transplantation model. Forty Sprague-Dawley rats were randomized into the ischemic postconditioning group (IPO group) and the control group (C group), 10 pairs in each group in which left lung orthotopic transplantations from NHBDs were done with " two-cuff-one-stent technique". In the C group, perfusion was resumed by declamping pulmonary artery immediately after transplantation, whereas in the IPO group, 5 cycles of 1-min reperfusion and 1-min reocclusion of pulmonary artery were applied as postcontioning before full recovery of perfusion. Compared with the C group, water content of donor lungs was lower and pathological changes were milder in the IPO group, meanwhile compliance, structure and function of donor lungs were better preserved. Furthermore, the expression of cell apoptosis and MDA content in donor lungs were lower in the IPO group, while SOD content was higher. Ischemic postconditioning can reduce ischemic reperfusion injury of NHBD lung transplantation and preserve the structure and function of donor lungs. It can inhibit lipid peroxidation and cell apoptosis in NHBD lungs after transplantation.

Effect of sufentanil preconditioning on acute lung injury induced by intestinal ischemia-reperfusion in rats and the role of opioid receptors

Objective To evaluate the effect of sufentanil preconditioning on acute lung injury induced by intestinal ischemia-reperfusion (I/R) in rats and the role of opioid receptors.Methods Forty-eight male Wistar rats weighing 200-250 g were randomly divided into 6 groups ( n =8 each):sham operation group (group S) ; I/R group; sufentanil preconditioning group (group SPC) ; COTP (μ receptor antagonist) + SPC group; NTD (δ receptor antagonist) + SPC group and nor-BNI (κ receptor antagonist) + SPC group.Intestinal I/R was produced by occlusion of the superior mesenteric artery for 45 min followed by 2 h reperfusion in groups I/R,SPC,COTP +SPC,NTD + SPC and nor-BNI + SPC.Sufentanil 10 μg/kg was injected intravenously at 10 min before ischemia in groups SPC,COTP + SPC,NTD + SPC and nor-BNI + SPC.COTP 1 mg/kg and NTD 5 mg/kg were injected intravenously at 10 min before sufentanil injection in groups COTP + SPC and NTD + SPC respectively,while nor-BNI 5 mg/kg was injected intravenously at 15 min before sufentanil administration in group nor-BNI + SPC.The animals were sacrificed at 2 h of reperfusion,the intestinal tissue was removed for microscopic examination and intestinal damage was assessed and scored according to Chiu.Left lung tissue was also removed for microscopic examination (1 =normal,4 =severely injured).The apoptosis in lung cells was detected using TUNEL and apoptosis index (AI) was calculated.The expression of Bcl-2 and Bax proteins in lung tissues was detected using immuno-histochemistry and Bcl-2/Bax ratio was calculated.Results Compared with group S,Chiu's score,lung injury score and AI were significantly increased,expression of Bcl-2 and Bax proteins was up-regulated,Bcl-2/Bax ratio was significantly decreased in the other groups ( P ＜ 0.01 ).Compared with group I/R,Chiu' s score,lung injury score and AI were significantly decreased,Bcl-2 protein expression was up-regulated,Bax protein expression was downregulated,and Bcl-2/Bax ratio was significantly increased in group SPC (P ＜ 0.01 ).Compared with group SPC,Chiu's score,lung injury score and AI were significantly increased,Bcl-2 protein expression was down-regulated,Bax protein expression was up-regulated,and Bcl-2/Bax ratio was significantly decreased in groups COTP + SPC,NTD + SPC and nor-BNI + SPC ( P ＜ 0.01 ).Conclusion Sufentanil preconditioning can attenuate lung injury induced by intestinal I/R by activation of opioid receptors in rats. Key words: Sufentanil; Ischemic preconditioning; Reperfusion injury; Intestines; Respiratory distress syndrome, adult; Receptors, opioid

Shen-Fu injection attenuates postresuscitation lung injury in a porcine model of cardiac arrest

ObjectiveTo investigate the effects of Shen-Fu injection (SFI) on postresuscitation lung injury in a porcine model of cardiac arrest. MethodsTwenty-four anaesthetised male Landrace pigs were subjected to 4min of untreated ventricular fibrillation (VF), followed by standard cardiopulmonary resuscitation. Sixteen successfully resuscitated pigs were randomised into two groups (eight pigs per group); one group received an SFI infusion and the other group received a normal saline infusion, at an infusion rate of 0.24mg/min from 15min after the return of spontaneous circulation (ROSC) until 6h after ROSC. ResultsOxygenation index, respiratory index, oxygen delivery, oxygen consumption, oxygen extraction, dynamic lung compliance, airway resistance, external vascular lung water index, and pulmonary vascular permeability index at 15min, 30min, 1h, 2h, 4h, and 6h after ROSC were all worse than baseline in the saline group, and were all better in the SFI group than in the saline group. The pulmonary protective effects of SFI were further confirmed by histopathological and ultrastructural observations of lung tissue. SFI infusion resulted in lower apoptosis index, caspase-3 protein expression, and malondialdehyde content of lung tissue after ROSC, and increased Bcl-2 protein expression and superoxide dismutase, Na+-K+-ATPase, and Ca2+-ATPase activity compared with the saline group. ConclusionShen-Fu injection can attenuate postresuscitation lung injury through suppression of lung cell apoptosis and improvement of energy metabolism and antioxidant capacity.

Overexpression of Tumor Necrosis Factor-α in the Lungs Alters Immune Response, Matrix Remodeling, and Repair and Maintenance Pathways

Increased production of tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) is a feature of inflammatory lung diseases, including emphysema and fibrosis, but the divergent pathological characteristics that result indicate involvement of other processes in disease pathogenesis. Transgenic mice overexpressing TNF-α in type II alveolar epithelial cells under the control of the surfactant protein (SP)-C promoter develop pulmonary inflammation and emphysema but are resistant to induction of fibrosis by administration of bleomycin or transforming growth factor-β. To study the molecular mechanisms underlying the development of this phenotype, we used a microarray approach to characterize the pulmonary transcriptome of SP-C/TNF-α mice and wild-type littermates. Four-month-old SP-C/TNF-α mice displayed pronounced pulmonary inflammation, airspace enlargement, increased MMP-2 and MMP-9 levels, and altered expression of 2332 probes. The functional assessment of genes with increased expression revealed enrichment of inflammatory/immune responses and proteases, whereas genes involved in protease inhibition, angiogenesis, cross-linking of basement membrane proteins, and myofibroblast differentiation were predominantly decreased. Comparison with multiple lung disease models identified a set of genes unique to the SP-C/TNF-α model and revealed that lack of extracellular matrix production distinguished SP-C/TNF-α mice from fibrosis models. Activation of inflammatory and proteolytic pathways and disruption of maintenance and repair processes are central features of emphysema in this TNF-overexpression model. Impairment of myofibroblast differentiation and extracellular matrix production may underlie resistance to induction of fibrosis.

Regular and moderate exercise before experimental sepsis reduces the risk of lung and distal organ injury

Physical activity modulates inflammation and immune response in both normal and pathologic conditions. We investigated whether regular and moderate exercise before the induction of experimental sepsis reduces the risk of lung and distal organ injury and survival. One hundred twenty-four BALB/c mice were randomly assigned to two groups: sedentary (S) and trained (T). Animals in T group ran on a motorized treadmill, at moderate intensity, 5% grade, 30 min/day, 3 times a week for 8 wk. Cardiac adaptation to exercise was evaluated using echocardiography. Systolic volume and left ventricular mass were increased in T compared with S group. Both T and S groups were further randomized either to sepsis induced by cecal ligation and puncture surgery (CLP) or sham operation (control). After 24 h, lung mechanics and histology, the degree of cell apoptosis in lung, heart, kidney, liver, and small intestine villi, and interleukin (IL)-6, KC (IL-8 murine functional homolog), IL-1β, IL-10, and number of cells in bronchoalveolar lavage (BALF) and peritoneal lavage (PLF) fluids as well as plasma were measured. In CLP, T compared with S groups showed: 1) improvement in survival; 2) reduced lung static elastance, alveolar collapse, collagen and elastic fiber content, number of neutrophils in BALF, PLF, and plasma, as well as lung and distal organ cell apoptosis; and 3) increased IL-10 in BALF and plasma, with reduced IL-6, KC, and IL-1β in PLF. In conclusion, regular and moderate exercise before the induction of sepsis reduced the risk of lung and distal organ damage, thus increasing survival.

Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model

We hypothesized that bone marrow-derived mononuclear cell (BMDMC) therapy protects the lung and consequently the heart in experimental elastase-induced emphysema. Twenty-four female C57BL/6 mice were intratracheally instilled with saline (C group) or porcine pancreatic elastase (E group) once a week during 4 weeks. C and E groups were randomized into subgroups receiving saline (SAL) or male BMDMCs (2×106, CELL) intravenously 3h after the first saline or elastase instillation. Compared to E-SAL group, E-CELL mice showed, at 5 weeks: lower mean linear intercept, neutrophil infiltration, elastolysis, collagen fiber deposition in alveolar septa and pulmonary vessel wall, lung cell apoptosis, right ventricle wall thickness and area, higher endothelial growth factor and insulin-like growth factor mRNA expressions in lung tissue, and reduced platelet-derived growth factor, transforming growth factor-β, and caspase-3 expressions. In conclusion, BMDMC therapy was effective at modulating the inflammatory and remodeling processes in the present model of elastase-induced emphysema.

Proteomics Investigation Reveals Apoptosis-Associated Proteins in Aryl Hydrocarbon Receptor-Deficient Human Lung Cells Treated with 2,3,7,8-Tetrachlorobenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the most potent dioxin, binds to aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, which subsequently alters gene expression. Epidemiological studies suggested that exposure to TCDD highly enhanced the risk of pulmonary diseases. In this study, we adopted a comparative proteomic approach using two-dimensional gel electrophoresis (2DE) to identify the differential expression of proteins between TCDD-treated control and AhR-deficient lung adenocarcinoma cells. Fifteen proteins from seventeen differential protein spots were identified that four of which were down-regulated and eleven were up-regulated. Western blotting analysis confirmed differential expression for five identified proteins, including GRB10, YWHAG, HSP27, LGALS1 and ACTB. Further protein-protein interaction network analysis of the identified proteins illustrated that twelve proteins were linked to a network containing caspase-3, p53, MDM2 and Akt/PKB, which is known to be associated with apoptosis pathway. Among the identified proteins, GRB10 may play a critical role to regulate the development of apoptosis in human lung cells by TCDD exposure. According to our study, it is expected to lead to new insights into the apoptosis pathway in lung adenocarcinoma by TCDD exposure.

TLR4-dependent GM-CSF protects against lung injury in Gram-negative bacterial pneumonia

Toll-like receptors (TLRs) are required for protective host defense against bacterial pathogens. However, the role of TLRs in regulating lung injury during Gram-negative bacterial pneumonia has not been thoroughly investigated. In this study, experiments were performed to evaluate the role of TLR4 in pulmonary responses against Klebsiella pneumoniae (Kp). Compared with wild-type (WT) (Balb/c) mice, mice with defective TLR4 signaling (TLR4(lps-d) mice) had substantially higher lung bacterial colony-forming units after intratracheal challenge with Kp, which was associated with considerably greater lung permeability and lung cell death. Reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA and protein was noted in lungs and bronchoalveolar lavage fluid of TLR4 mutant mice postintratracheal Kp compared with WT mice, and primary alveolar epithelial cells (AEC) harvested from TLR4(lps-d) mice produced significantly less GM-CSF in vitro in response to heat-killed Kp compared with WT AEC. TLR4(lps-d) AEC underwent significantly more apoptosis in response to heat-killed Kp in vitro, and treatment with GM-CSF protected these cells from apoptosis in response to Kp. Finally, intratracheal administration of GM-CSF in TLR4(lps-d) mice significantly decreased albumin leak, lung cell apoptosis, and bacteremia in Kp-infected mice. Based on these observations, we conclude that TLR4 plays a protective role on lung epithelium during Gram-negative bacterial pneumonia, an effect that is partially mediated by GM-CSF.

Imbalance of Apoptosis and Cell Proliferation Contributes to the Development and Persistence of Emphysema

We postulate that in adults there is an established lung structure maintenance program and that lung alveolar septal cells are undergoing both continuous apoptosis and proliferation. Whereas lung cell apoptosis has been recognized in human emphysema, little is known about cell proliferation. Using a novel rat model of emphysema, induced by intratracheal instillation of cigarette smoke extract (CSE), we investigated the dynamics of emphysematous lung destruction. Emphysematous lung destruction was determined by measuring mean linear intercept and destructive index. Lung injury and repair were assessed by immunohistochemistry and Western blot analysis for active caspase-3 and proliferating cell nuclear antigen (PCNA) after 4, 8, and 12 weeks of CSE instillations. The emphysematous lung tissue destruction was present at 4 weeks of CSE treatment and progressed to 8 weeks. Spontaneous repair began at 12 weeks. Treatment with a peroxisome proliferator activated receptor (PPAR)α+γ agonist or granulocyte and macrophage-colony stimulating factor (GM-CSF) for 4 weeks prevented the progression of emphysematous lung destruction and decreased the number of caspase-3-positive cells. Apoptosis and cell proliferation occur in this new model of emphysema. Treatment with a PPARα+γ agonist or GM-CSF can inhibit the progression of emphysematous alveolar septal destruction by decreasing alveolar cell apoptosis.

Hydrogen inhalation ameliorates lipopolysaccharide-induced acute lung injury in mice

Acute lung injury (ALI) is a serious illness, the incidence and mortality of which are very high. Free radicals, such as hydroxyl radicals (OH) and peroxynitrite (ONOO−), are considered to be the final causative molecules in the pathogenesis of ALI. Hydrogen, a new antioxidant, can selectively reduce OH and ONOO−. In the present study, we investigated the hypothesis that hydrogen inhalation could ameliorate ALI induced by intra-tracheal lipopolysaccharide (LPS, 5mg/kg body weight). Mice were randomized into three groups: sham group (physiological saline+2% hydrogen mixed gas), control group (LPS+normal air) and experiment group (LPS+2% hydrogen mixed gas). Bronchoalveolar lavage fluid (BALF) was performed to determine the total protein concentrations and pro-inflammatory cytokines. Lung tissues were assayed for oxidative stress variables, wet/dry (W/D) ratio, histological, immunohistochemistry and Western blotting examinations. Our experiments exhibited that hydrogen improved the survival rate of mice and induced a decrease in lung W/D ratio. In addition, hydrogen decreased malonaldehyde and nitrotyrosine content, inhibited myeloperoxidase and maintained superoxide dismutase activity in lung tissues and associated with a decrease in the expression of TNF-α, IL-1β, IL-6 and total protein concentrations in the BALF. Hydrogen further attenuated histopathological alterations and mitigated lung cell apoptosis. Importantly, hydrogen inhibited the activation of P-JNK, and also reversed changes in Bax, Bcl-xl and caspase-3. In conclusion, our data demonstrated that hydrogen inhalation ameliorated LPS-induced ALI and it may be exerting its protective role by preventing the activation of ROS–JNK–caspase-3 pathway.

Hypo-CpG Methylation Controls PTEN Expression and Cell Apoptosis in Irradiated Lung

Purpose: The current study was designed to address our hypothesis that oxidative stress secondary to the ionizing event upregulates phosphatase and tensin homolog (PTEN) mRNA and protein in the lun...

Fas/FasL pathway-mediated alveolar macrophage apoptosis involved in human silicosis

In vitro and in vivo studies have demonstrated that lung cell apoptosis is associated with lung fibrosis; however the relationship between apoptosis of alveolar macrophages (AMs) and human silicosis has not been addressed. In the present study, AM apoptosis was determined in whole-lung lavage fluid from 48 male silicosis patients, 13 male observers, and 13 male healthy volunteers. The relationships between apoptosis index (AI) and silica exposure history, soluble Fas (sFas)/membrane-bound Fas (mFas), and caspase-3/caspase-8 were analyzed. AI, mFas, and caspase-3 were significantly higher in lung lavage fluids from silicosis patients than those of observers or healthy volunteers, but the level of sFas demonstrated a decreasing trend. AI was related to silica exposure, upregulation of mFas, and activation of caspase-3 and -8, as well as influenced by smoking status after adjusting for confounding factors. These results indicate that AM apoptosis could be used as a potential biomarker for human silicosis, and the Fas/FasL pathway may regulate this process. The present data from human lung lavage samples may help to understand the mechanism of silicosis and in turn lead to strategies for preventing or treating this disease.

Early and late effects of bone marrow-derived mononuclear cell therapy on lung and distal organs in experimental sepsis

We tested the hypothesis that bone marrow-derived mononuclear cells (BMDMCs) at an early phase of cecal ligation and puncture (CLP)-induced sepsis may have lasting effects on: (1) lung mechanics and histology, (2) the structural remodelling of lung parenchyma, (3) lung, kidney, and liver cell apoptosis, and (4) pro- and anti-inflammatory cytokines and growth factors. At day 1, BMDMC significantly reduced mortality, as well as caspase-3, interleukin (IL)-6 and IL-1β, vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, and transforming growth factor-β, but increased IL-10 mRNA expression in lung tissue in septic mice contributing to endothelium and epithelium alveolar repair and improvement of lung mechanics. BMDMC also prevented the increase of apoptotic cells in lung, liver, and kidney. At day 7, these early functional and morphological effects were preserved or further improved. In conclusion, in the present model of sepsis, the beneficial effects of early administration of BMDMCs on lung and distal organs were preserved, possibly by paracrine mechanisms.

Prenatal PFOS exposure induces oxidative stress and apoptosis in the lung of rat off-spring

Perfluorooctane sulfonate (PFOS) could induce neonatal pulmonary injuries in rodents. The aim of this study was to investigate the underlying mode of action. Pregnant rats were dosed orally with PFOS (0, 0.1 and 2.0mg/kgd) from gestation days (GD) 1 to 21. Lung samples from postnatal day (PND) 0 and 21 pups were analyzed for the toxic effects of PFOS. The results showed that maternal exposure to 2.0mg/kgd PFOS caused severe histopathological changes along with marked oxidative injuries and cell apoptosis in offspring lungs; at the same time, the ratio of Bax to Bcl-2, release of cytochrome c (Cyt c) from mitochondria to cytoplasm, expressions of Fas and Fas-L, and activities of caspase-3, -8 and -9 were up-regulated correspondingly. The results indicate that oxidative stress and both intrinsic and extrinsic cell death pathways were involved in prenatal PFOS exposure-induced injuries in postnatal lungs.

Toxicity of Graphene in Normal Human Lung Cells (BEAS-2B)

Graphite nanomaterials such as thermally exfoliated graphite oxide (GO) are versatile in many applications. However, little is known about its effects on biological systems. In this study we characrerized the GO using dynamic light scattering (DLS) along with the toxicological aspects related to cytotoxicity and apoptosis in normal human lung cells (BEAS-2B). A significant concentration and time dependent decrease in cell viability was observed at different concentrations (10-100 microg/ml) by the MTT assay after 24 and 48 h of exposure and significant increase of early and late apoptotic cells was observed as compared to control cells. Our study demonstrates that GO induces cytotoxicity and apoptosis in human lung cells.

Angiotensin Receptor Type I Blockade Inhibits Apoptosis in Meconium-Instilled Rabbit Lungs

Background and objectives: In prior studies we demonstrated that meconium-induced lung cell death by apoptosis was associated with activation of the local pulmonary renin-angiotensin system (RASL). Alveolar epithelial apoptosis requires the authocrine synthesis and proteolytic processing of angiotensinogen (AGT) to Angiotensin II (ANG II). Inhibitors of angiotensin converting enzyme (ACE) block meconium-induced apoptosis. ANG II plays an essential role in vascular homeostasis and lung injury. The objectives of this study were to evaluate expression of AGT, ANG II and Caspase 3 in meconium and saline treated newborn lungs and to study mechanisms of its inhibition by a selective antagonist of the AT1 receptor. Methods: Two week old rabbits were studied. Three treatment groups were studied (six rabbit pups in each group): Group 1: rabbits instilled with saline; Group 2: rabbits instilled with 10% meconium; Group 3: Losartan pretreated followed by meconium-instillation. Three groups of A549 human lung epithelial cells were studies as well. Group 4: AGT pretreated and then meconium-exposed cells; Group 5: ANG II pretreated and then meconium exposed cells and Group 6: Caspase 3 inhibitor ZVAD-fmk pretreated and then meconium exposed cells. AGT, ANG II and Caspase 3 were evaluated and compared with and without inhibitors in meconium and control groups. Results: In Situ End Labeling (ISEL) and Caspase 3 assays showed that purified ANG II induced dose dependent apoptosis in rabbit lung lavage cells and the human A549 lung epithelial cell line. Apoptosis also was induced by purified AGT. The increase in apoptotic cells was accompanied by increases in ANG II and Caspase 3 activities. In both airway epithelium and alveolar wall cells, measures of apoptosis were attenuated by Losartan or by the Caspase 3 inhibitor ZVAD-fmk. Conclusions: These data demonstrate the presence of a functional ANG II and Caspase 3 dependent apoptotic pathways in newborn meconium-instilled lungs. They also imply that meconium-induced apoptosis is modulated by the pulmonary RASL system in which ANG II plays a critical role. Both losartan and the Caspase 3 inhibitor ZVAD-fmk pretreatment significantly decreased meconium-induced Caspase-3 activation and lung cell apoptosis.

Inhibition of histone deacetylase causes emphysema

In patients with chronic obstructive pulmonary disease (COPD), histone deacetylase (HDAC) expression and activity are reduced in the lung tissue. However, whether HDAC activity controls the maintenance of the lung alveolar septal structures has not been investigated. To explore the consequences of HDAC inhibition and address the question of whether HDAC inhibition causes lung cell apoptosis and emphysema, male Sprague-Dawley rats and human pulmonary microvascular endothelial cells (HPMVEC) were treated with trichostatin A (TSA), a specific inhibitor of HDACs. Chronic TSA treatment increased the alveolar air space area, mean linear intercept, and the number of caspase-3-positive cells in rat lungs. TSA suppressed hypoxia-inducible factor-1α (HIF-1α), VEGF, and lysyl oxidase (LOX) and increased microtubule-associated protein-1 light chain 3 (LC3), p53, and miR34a microRNA expression in both rat lungs and cultured HPMVEC. Gene silencing of HDAC2 using small interfering RNA (siRNA) in cultured HPMVEC resulted in the suppression of HIF-1α, VEGF, and LOX and an increase of p53 expression. These data indicate that HDAC inhibition causes emphysema and that HDAC-dependent mechanisms contribute to the maintenance of the adult lung structure. Our results also suggest that the increase in apoptosis, as a consequence of HDAC inhibition, is associated with decreased VEGF and HIF-1α expression.

Inhibition of apoptosis by 60% oxygen: a novel pathway contributing to lung injury in neonatal rats

During early postnatal alveolar formation, the lung tissue of rat pups undergoes a physiological remodeling involving apoptosis of distal lung cells. Exposure of neonatal rats to severe hyperoxia (≥95% O(2)) both arrests lung growth and results in increased lung cell apoptosis. In contrast, exposure to moderate hyperoxia (60% O(2)) for 14 days does not completely arrest lung cell proliferation and is associated with parenchymal thickening. On the basis of similarities in lung architecture observed following either exposure to 60% O(2), or pharmacological inhibition of physiological apoptosis, we hypothesized that exposure to 60% O(2) would result in an inhibition of physiological lung cell apoptosis. Consistent with this hypothesis, we observed that the parenchymal thickening induced by exposure to 60% O(2) was associated with decreased numbers of apoptotic cells, increased expressions of the antiapoptotic regulator Bcl-xL, and the putative antiapoptotic protein survivin, and decreased expressions of the proapoptotic cleaved caspases-3 and -7. In summary, exposure of the neonatal rat lung to moderate hyperoxia results in an inhibition of physiological apoptosis, which contributes to the parenchymal thickening observed in the resultant lung injury.

Sphingolipid-mediated Inhibition of Apoptotic Cell Clearance by Alveolar Macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.