Del-1 Plays a Protective Role against COPD Development by Inhibiting Inflammation and Apoptosis.

Neutrophilic inflammation is a prominent feature of chronic obstructive pulmonary disease (COPD). Developmental endothelial locus-1 (Del-1) has been reported to limit excessive neutrophilic inflammation by inhibiting neutrophil adhesion to the vascular endothelial cells. However, the effects of Del-1 in COPD are not known. We investigated the role of Del-1 in the pathogenesis of COPD. Del-1 protein expression was decreased in the lungs of COPD patients, especially in epithelial cells and alveolar macrophages. In contrast to human lung tissue, Del-1 expression was upregulated in lung tissue from mice treated with cigarette smoke extracts (CSE). Overexpression of Del-1 significantly suppressed IL-8 release and apoptosis in CSE-treated epithelial cells. In contrast, knockdown of Del-1 enhanced IL-8 release and apoptosis. In macrophages, overexpression of Del-1 significantly suppressed inflammatory cytokine release, and knockdown of Del-1 enhanced it. This anti-inflammatory effect was mediated by inhibiting the phosphorylation and acetylation of NF-κB p65. Nuclear factor erythroid 2-related factor 2 (Nrf2) activators, such as quercetin, resveratrol, and sulforaphane, increased Del-1 in both cell types. These results suggest that Del-1, mediated by Nrf2, plays a protective role against the pathogenesis of COPD, at least in part through anti-inflammatory and anti-apoptotic effects.

Machine Learning-Based Proteomics Reveals Ferroptosis in COPD Patient-Derived Airway Epithelial Cells Upon Smoking Exposure.

BACKGROUND: Proteomics and genomics studies have contributed to understanding the pathogenesis of chronic obstructive pulmonary disease (COPD), but previous studies have limitations. Here, using a machine learning (ML) algorithm, we attempted to identify pathways in cultured bronchial epithelial cells of COPD patients that were significantly affected when the cells were exposed to a cigarette smoke extract (CSE). METHODS: Small airway epithelial cells were collected from patients with COPD and those without COPD who underwent bronchoscopy. After expansion through primary cell culture, the cells were treated with or without CSEs, and the proteomics of the cells were analyzed by mass spectrometry. ML-based feature selection was used to determine the most distinctive patterns in the proteomes of COPD and non-COPD cells after exposure to smoke extract. Publicly available single-cell RNA sequencing data from patients with COPD (GSE136831) were used to analyze and validate our findings. RESULTS: Five patients with COPD and five without COPD were enrolled, and 7,953 proteins were detected. Ferroptosis was enriched in both COPD and non-COPD epithelial cells after their exposure to smoke extract. However, the ML-based analysis identified ferroptosis as the most dramatically different response between COPD and non-COPD epithelial cells, adjusted P value = 4.172 × 10-6, showing that epithelial cells from COPD patients are particularly vulnerable to the effects of smoke. Single-cell RNA sequencing data showed that in cells from COPD patients, ferroptosis is enriched in basal, goblet, and club cells in COPD but not in other cell types. CONCLUSION: Our ML-based feature selection from proteomic data reveals ferroptosis to be the most distinctive feature of cultured COPD epithelial cells compared to non-COPD epithelial cells upon exposure to smoke extract.

Downregulation of Lysosome-Associated Membrane Protein-2A Contributes to the Pathogenesis of COPD.

Background: Macroautophagy plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but the role of chaperone-mediated autophagy (CMA) has not been investigated. We investigated if and how CMA is involved in the pathogenesis of COPD. Methods: We measured the level of lysosome-associated membrane protein-2A (LAMP-2A), which is a critical component of CMA that functions as a receptor for cytosolic substrate proteins, in total lung tissues and primary human bronchial epithelial cells (HBECs) from healthy never smokers, smokers, and COPD patients. We assessed the effects of LAMP-2A knock-down on cigarette smoke extract (CSE)-induced aging, cell cycle arrest, and apoptosis in BEAS-2B cells and the expression levels of apoptosis hallmarks in primary HBECs and lung tissue sections. Results: We found that the protein levels of LAMP-2A in lung homogenates and primary HBECs from smokers and COPD patients were lower than those from never smokers. In addition, its level in primary HBECs was negatively correlated with years of smoking. CSE caused degradation of LAMP-2A protein via the lysosomal pathway by activating macroautophagy. Knock-down of LAMP-2A markedly enhanced CSE-induced expression of senescence markers such as p16, p21, p27, and p53. G2/M cell cycle arrest, up-regulation of cyclin B1, and apoptosis in BEAS-2B cells. Apoptosis was increased in CSE-treated primary HBECs and in lung tissues from smokers and COPD patients. Conclusion: Cigarette smoke-induced down-regulation of LAMP-2A is involved in acceleration of aging and apoptosis of lung epithelial cells, which might at least partially contribute to COPD pathogenesis.

YPL-001 Shows Various Beneficial Effects against Cigarette Smoke Extract-Induced Emphysema Formation: Anti-Inflammatory, Anti-Oxidative, and Anti-Apoptotic Effects.

Inflammation, oxidative stress, and apoptosis are thought to be important causes of chronic obstructive pulmonary disease (COPD). We investigated the effect of YPL-001 (under phase 2a study, ClinicalTrials.gov identifier NCT02272634), a drug derived from Pseudolysimachion rotundum var. subintegrum, on cigarette smoke extract (CSE)-induced inflammation, the anti-oxidative pathway, and apoptosis in human lung epithelial cells and on CSE-induced emphysema in mice. YPL-001 suppressed CSE-induced expression of IL8 mRNA and protein. This was due to the reduction in NF-κB transcriptional activity by YPL-001, which resulted from the blockade of acetylation of the NF-κB subunit p65 (Lys310). Histone deacetylases (HDACs) prevent gene transcription by condensing the DNA structure and affecting NF-κB nuclear binding. YPL-001 alone increased HDAC2 activity and enhanced CSE-induced activation of HDAC2. YPL-001-induced suppression of NF-κB transcriptional activity might be caused by increased HDAC2 activity. YPL-001 increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression via both degradation of its inhibitory protein, Kelch-like ECH-associated protein 1, and an increase in de novo protein synthesis. YPL-001 increased the DNA binding activity of Nrf2. Consequently, YPL-001 upregulated the expression of Nrf2-targeted anti-oxidant genes such as NAD(P)H quinone dehydrogenase 1 and heme oxygenase 1. Moreover, YPL-001 significantly suppressed CSE-induced apoptotic cell death. In vivo study showed that CSE-induced emphysematous changes, neutrophilic inflammation, protein leakage into bronchoalveolar space, and lung cell apoptosis in mice were suppressed by YPL-001 treatment. Taken together, these results suggest that YPL-001 is a good therapeutic candidate for the treatment of COPD by blocking inflammation and apoptosis and activating the anti-oxidative pathway.

Developmental endothelial locus-1 as a potential biomarker for the incidence of acute exacerbation in patients with chronic obstructive pulmonary disease.

BACKGROUND: Despite the high disease burden of chronic obstructive pulmonary disease (COPD) and risk of acute COPD exacerbation, few COPD biomarkers are available. As developmental endothelial locus-1 (DEL-1) has been proposed to possess beneficial effects, including anti-inflammatory effects, we hypothesized that DEL-1 could be a blood biomarker for COPD. OBJECTIVE: To elucidate the role of plasma DEL-1 as a biomarker of COPD in terms of pathogenesis and for predicting acute exacerbation. METHODS: Cigarette smoke extract (CSE) or saline was intratracheally administered to wild-type (WT) and DEL-1 knockout (KO) C57BL/6 mice. Subsequently, lung sections were obtained to quantify the degree of emphysema using the mean linear intercept (MLI). Additionally, plasma DEL-1 levels were compared between COPD and non-COPD participants recruited in ongoing prospective cohorts. Using negative binomial regression analysis, the association between the plasma DEL-1 level and subsequent acute exacerbation risk was evaluated in patients with COPD. RESULTS: In the in vivo study, DEL-1 KO induced emphysema (KO saline vs. WT saline; P = 0.003) and augmented CSE-induced emphysema (KO CSE vs. WT CSE; P < 0.001) in 29 mice. Among 537 participants, patients with COPD presented plasma log (DEL-1) levels lower than non-COPD participants (P = 0.04), especially non-COPD never smokers (P = 0.019). During 1.2 ± 0.3 years, patients with COPD in the lowest quartile of Log(DEL-1) demonstrated an increased risk of subsequent acute exacerbation, compared with those in the highest quartile of Log(DEL-1) (adjusted incidence rate ratio, 3.64; 95% confidence interval, 1.03-12.9). CONCLUSION: Low DEL-1 levels are associated with COPD development and increased risk of subsequent COPD acute exacerbation. DEL-1 can be a useful biomarker in patients with COPD.

The impact of organic extracts of seasonal PM2.5 on primary human lung epithelial cells and their chemical characterization.

Lung epithelial cells serve as the first line of defense against various inhaled pollutant particles. To investigate the adverse health effects of organic components of fine particulate matter (PM2.5) collected in Seoul, South Korea, we selected 12 PM2.5 samples from May 2016 to January 2017 and evaluated the effects of organic compounds of PM2.5 on inflammation, cellular aging, and macroautophagy in human lung epithelial cells isolated directly from healthy donors. Organic extracts of PM2.5 specifically induced neutrophilic chemokine and interleukin-8 expression via extracellular signal-regulated kinase activation. Moreover, PM2.5 significantly increased the expression of aging markers (p16, p21, and p27) and activated macroautophagy. Average mass concentrations of organic and elemental carbon had no significant correlations with PM2.5 effects. However, polycyclic aromatic hydrocarbons and n-alkanes were the most relevant components of PM2.5 that correlated with neutrophilic inflammation. Vegetative detritus and residential bituminous coal combustion sources strongly correlated with neutrophilic inflammation, aging, and macroautophagy activation. These data suggest that the chemical composition of PM2.5 is important for determining the adverse health effects of PM2.5. Our study provides encouraging evidence to regulate the harmful components of PM2.5 in Seoul.

Synergistic cycles of protease activity and inflammation via PPARγ degradation in chronic obstructive pulmonary disease.

Inflammation, oxidative stress, and protease-antiprotease imbalance have been suggested to be a pathogenic triad in chronic obstructive pulmonary disease (COPD). However, it is not clear how proteases interact with components of inflammatory pathways. Therefore, this study aimed to evaluate the effect of neutrophil elastase (NE) on lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) production and determine the molecular mechanism in human bronchial epithelial cells (HBECs). Immortalized bronchial epithelial cells and primary HBECs were used to investigate the impact of NE on LPS-induced IL-8 production. The molecular mechanism by which NE modulated LPS-induced IL-8 production was confirmed in elastase-treated C57BL/6 mice and primary HBECs obtained from COPD patients and healthy controls. The results showed that NE treatment synergistically augmented LPS-induced IL-8 production in both immortalized bronchial epithelial cells and primary HBECs. NE partially degraded peroxisome proliferator-activated receptor gamma (PPARγ), which is known to regulate IL-8 production in the nucleus. Treatment with a PPARγ agonist and overexpression of PPARγ reversed the NE-induced synergistic increase in LPS-induced IL-8 production. Moreover, PPARγ levels were lower in lung homogenates and lung epithelial cells from elastase-treated mice than in those from saline-treated mice. In accordance with the findings in mice, PPARγ levels were lower in primary HBECs from COPD patients than in those from healthy never-smokers or healthy smokers. In conclusion, a vicious cycle of mutual augmentation of protease activity and inflammation resulting from PPARγ degradation plays a role in the pathogenesis of COPD.

Cereblon contributes to the development of pulmonary fibrosis via inactivation of adenosine monophosphate-activated protein kinase α1.

Pulmonary fibrosis is a progressive and lethal lung disease characterized by the proliferation and differentiation of lung fibroblasts and the accumulation of extracellular matrices. Since pulmonary fibrosis was reported to be associated with adenosine monophosphate-activated protein kinase (AMPK) activation, which is negatively regulated by cereblon (CRBN), we aimed to determine whether CRBN is involved in the development of pulmonary fibrosis. Therefore, we evaluated the role of CRBN in bleomycin (BLM)-induced pulmonary fibrosis in mice and in transforming growth factor-beta 1 (TGF-ß1)-induced differentiation of human lung fibroblasts. BLM-induced fibrosis and the mRNA expression of collagen and fibronectin were increased in the lung tissues of wild-type (WT) mice; however, they were significantly suppressed in Crbn knockout (KO) mice. While the concentrations of TGF-ß1/2 in bronchoalveolar lavage fluid were increased via BLM treatment, they were similar between BLM-treated WT and Crbn KO mice. Knockdown of CRBN suppressed TGF-ß1-induced activation of small mothers against decapentaplegic 3 (SMAD3), and overexpression of CRBN increased it. TGF-ß1-induced activation of SMAD3 increased α-smooth muscle actin (α-SMA) and collagen levels. CRBN was found to be colocalized with AMPKα1 in lung fibroblasts. CRBN overexpression inactivated AMPKα1. When cells were treated with metformin (an AMPK activator), the CRBN-induced activation of SMAD3 and upregulation of α-SMA and collagen expression were significantly suppressed, suggesting that increased TGF-ß1-induced activation of SMAD3 via CRBN overexpression is associated with AMPKα1 inactivation. Taken together, these data suggest that CRBN is a profibrotic regulator and maybe a potential target for treating lung fibrosis.

Cigarette smoke extract-induced downregulation of p300 is responsible for the impaired inflammatory cytokine response of macrophages.

Patients with chronic obstructive pulmonary disease (COPD) are susceptible to infection owing to the impaired immune function of alveolar macrophages. This is presumed to be caused, at least partially, by cigarette smoke (CS), which is a major risk factor for COPD. Although CS has been reported to inhibit Toll-like receptor (TLR) function and phagocytosis in macrophages, the molecular mechanism of CS-mediated impairment of macrophage immune function has not been completely elucidated. We investigated the effects of CS extracts (CSE) on macrophage immune function and its molecular mechanism. We assessed lipopolysaccharide (LPS, TLR4 ligand)-, Pam3CSK4 (TLR2 ligand)-, or CpG-oligodeoxynucleotide (TLR9 ligand)-induced IL-6, TNF-α, and IL-1ß production in macrophages. Upregulation of IL-6, TNF-α, and IL-1ß mRNA and protein by TLR ligands was suppressed on treatment with CSE. However, LPS-induced MAP kinase activation, IκBα degradation, and nuclear translocation of NF-κB were not impeded by CSE. In contrast, CSE significantly suppressed NF-κB transcriptional activity in the nucleus. We found that p300, which acetylates RelA/p65 at lysine 310, and acetyl-p65 (K310) were downregulated upon CSE treatment. Knock-down of p300 suppressed LPS-induced acetylation of NF-κB p65 and production of inflammatory cytokine. To summarize, these results suggest that CSE impair cytokine response by decreasing the expression levels of p300.

Cigarette smoke extract decreased basal and lipopolysaccharide-induced expression of MARCO via degradation of p300.

BACKGROUND AND OBJECTIVE: Alveolar macrophages of patients with COPD display impaired cytokine release and diminished phagocytosis. COPD exacerbations exhibit immune dysfunction towards the respiratory pathogens. CS and CSE were reported to aggravate bacterial infections in COPD patients. METHODS: MARCO is highly expressed in lungs and is involved in pathogen clearance. We investigated the effect of CSE on MARCO expression and its regulatory mechanisms. After relevant siRNA transfection and treatment with CSE and/or LPS, we measured the levels of MARCO by q-RT PCR, immunoblotting and flow cytometry. Immunofluorescence staining and immunoprecipitation were used to evaluate the mechanism. RESULTS: CSE decreased LPS-induced expression of MARCO mRNA and protein. Upregulation of MARCO by LPS was Nrf2-dependent. Nrf2 knockdown significantly suppressed LPS-induced increase in MARCO transcripts. CSE did not block nuclear translocation of Nrf2 in LPS-treated cells, but rather CSE itself strongly accumulated Nrf2 in the nucleus through the degradation of its cytoplasmic inhibitor, KEAP1. However, CSE markedly suppressed LPS-induced Nrf2 acetylation. Histone acetyltransferase p300/CBP directly acetylates Nrf2, which augments promoter-specific DNA binding of Nrf2. Our results reveal CSE-induced polyubiquitinylation and subsequent degradation of p300 via the proteasome. Pretreatment with proteasome inhibitors completely blocked CSE-induced degradation of p300 and suppression of MARCO expression. CONCLUSION: These findings suggest that CSE decreases MARCO expression via the proteasomal degradation of p300 in macrophages, which may be in part responsible for impaired bacterial phagocytosis.

Proteasome Inhibitor-Induced IκB/NF-κB Activation is Mediated by Nrf2-Dependent Light Chain 3B Induction in Lung Cancer Cells.

IκB, a cytoplasmic inhibitor of nuclear factor-κB (NF-κB), is reportedly degraded via the proteasome. However, we recently found that long-term incubation with proteasome inhibitors (PIs) such as PS-341 or MG132 induces IκBα degradation via an alternative pathway, lysosome, which results in NF-κB activation and confers resistance to PI-induced lung cancer cell death. To enhance the anti-cancer efficacy of PIs, elucidation of the regulatory mechanism of PI-induced IκBα degradation is necessary. Here, we demonstrated that PI upregulates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) via both de novo protein synthesis and Kelch-like ECH-associated protein 1 (KEAP1) degradation, which is responsible for IκBα degradation via macroautophagy activation. PIs increased the protein level of light chain 3B (LC3B, macroautophagy marker), but not lysosome-associated membrane protein 2a (Lamp2a, the receptor for chaperone-mediated autophagy) in NCI-H157 and A549 lung cancer cells. Pretreatment with macroautophagy inhibitor or knock-down of LC3B blocked PI-induced IκBα degradation. PIs up-regulated Nrf2 by increasing its transcription and mediating degradation of KEAP1 (cytoplasmic inhibitor of Nrf2). Overexpression of dominant-negative Nrf2, which lacks an N-terminal transactivating domain, or knock-down of Nrf2 suppressed PI-induced LC3B protein expression and subsequent IκBα degradation. Thus, blocking of the Nrf2 pathway enhanced PI-induced cell death. These findings suggest that Nrf2-driven induction of LC3B plays an essential role in PI-induced activation of the IκB/NF-κB pathway, which attenuates the anti-tumor efficacy of PIs.

Globular Adiponectin Exerts a Pro-Inflammatory Effect via IκB/NF-κB Pathway Activation and Anti-Inflammatory Effect by IRAK-1 Downregulation.

Adiponectin, a hormone produced by adipose tissue, is very abundant in plasma, and its anti- and pro-inflammatory effects are reported. However, the mechanisms of these pro- and anti-inflammatory effects are not fully defined. Herein, we evaluated the dual inflammatory response mechanism of adiponectin in macrophages. Short-term globular adiponectin (gAd) treatment induced IκBα degradation, NF-κB nuclear translocation, and TNF-α production in RAW 264.7 cells. Polymyxin B pretreatment did not block gAd-induced IκBα degradation, and heated gAd was unable to degrade IκBα, suggesting that the effects of gAd were not due to endotoxin contamination. gAd activated IKK and Akt, and inhibition of either IKK or Akt by dominant-negative IKKß (DN-IKKß) or DN-Akt overexpression blocked gAd-induced IκBα degradation, suggesting that short-term incubation with gAd mediates inflammatory responses by activating the IκB/NF-κB and PI3K/Akt pathways. Contrastingly, long-term stimulation with gAd induced, upon subsequent stimulation, tolerance to gAd, lipopolysaccharide, and CpG-oligodeoxynucleotide, which is associated with gAd-induced downregulation of IL-receptor-associated kinase-1 (IRAK-1) due to IRAK-1 transcriptional repression. Conclusively, our findings demonstrate that the pro- and anti-inflammatory responses to gAd in innate immune cells are time-dependent, and mediated by the activation of the IκB/NF-κB pathway, and IRAK-1 downregulation, respectively.

Cigarette smoke extract enhances neutrophil elastase-induced IL-8 production via proteinase-activated receptor-2 upregulation in human bronchial epithelial cells.

Although inflammation, oxidative stress, and protease-antiprotease imbalance have been referred to as a pathogenic triad in chronic obstructive pulmonary disease (COPD), little is known about how they interact. The objectives of this study were to elucidate the effect of cigarette smoke extract (CSE) on the neutrophil elastase (NE)-induced inflammatory response and its molecular mechanism in bronchial epithelial cells. We observed that NE activated extracellular signal-regulated kinase (ERK) and induced IL-8 production. Blocking ERK activation using a MEK inhibitor (U0126) suppressed NE-induced IL-8 secretion and knockdown of proteinase-activated receptor 2 (PAR2) using siRNAs inhibited both NE-induced ERK activation and subsequent IL-8 release, suggesting that NE-induced IL-8 production is dependent on PAR2-mediated ERK activation. Interestingly, pre-exposure to CSE markedly enhanced NE-induced IL-8 production. As PAR2 acts as a receptor for NE, we next investigated the effect of CSE on PAR2 expression as a molecular mechanism for the increased IL-8 production induced by NE in CSE exposed cells. CSE, but not NE, increased the expression of PAR2 mRNA and surface membrane protein. Inhibition of p38 MAPK reduced PAR2 expression induced by CSE while inhibition of the ERK and Akt pathway had no effect. Consequently, p38 inhibition significantly abrogated CSE-induced enhancement of IL-8 production in NE-treated cells. Of note, we observed increased PAR2 levels in lung homogenates and lung epithelial cells from CSE-treated mice and from both smokers and patients with COPD. Taken together, these results suggest that CSE upregulates PAR2 in normal human bronchial epithelial cells, thereby enhancing the inflammatory response to NE.

Cigarette Smoke Extract Enhances IL-17A-Induced IL-8 Production via Up-Regulation of IL-17R in Human Bronchial Epithelial Cells.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine mainly derived from T helper 17 cells and is known to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) has been considered as a primary risk factor of COPD. However, the interaction between CS and IL-17A and the underlying molecular mechanisms have not been clarified. In the current study, we investigated the effects of cigarette smoke extract (CSE) on IL-17A-induced IL-8 production in human bronchial epithelial cells, and sought to identify the underlying molecular mechanisms. IL-8 production was significantly enhanced following treatment with both IL-17A and CSE, while treatment with either IL-17A or CSE alone caused only a slight increase in IL-8 production. CSE increased the transcription of IL-17RA/RC and surface membrane expression of IL-17R, which was suppressed by an inhibitor of the phosphoinositide 3-kinase (PI3K)/Akt pathway (LY294002). CSE caused inactivation of glycogen synthase kinase-3ß (GSK-3ß) via the PI3K/Akt pathway. Blockade of GSK-3ß inactivation by overexpression of constitutively active GSK-3ß (S9A) completely suppressed the CSE-induced up-regulation of IL-17R expression and the CSE-induced enhancement of IL-8 secretion. In conclusion, inactivation of GSK-3ß via the PI3K/Akt pathway mediates CSE-induced up-regulation of IL-17R, which contributes to the enhancement of IL-17A-induced IL-8 production.

Exogenous neutrophil elastase enters bronchial epithelial cells and suppresses cigarette smoke extract-induced heme oxygenase-1 by cleaving sirtuin 1.

An imbalance between oxidative stress and antioxidant activity plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke, a major risk factor of COPD, induces cellular oxidative stress, but levels of antioxidants such as heme oxygenase-1 (HO-1) are reduced in individuals with severe COPD. In this study, we evaluated the molecular mechanism of reduced HO-1 expression in human bronchial epithelial cells. We found that cigarette smoke extract (CSE) increases HO-1 levels via activation of NFE2-related factor 2 (Nrf2). However, pretreating cells with the protease neutrophil elastase (NE) suppressed the CSE-induced expression of HO-1 mRNA and protein. NE also decreased the sirtuin 1 (SIRT1) level, but did not inhibit CSE-induced nuclear translocation and DNA-binding activity of Nrf2. Transfection of cells with a Myc/His-tagged SIRT1 expression vector completely blocked the NE-mediated suppression of HO-1 expression. We further noted that the NE-induced down-regulation of SIRT1 was not due to decreased transcription or proteasomal/lysosomal degradation or loss of solubility. Immunofluorescence staining revealed that NE enters the cell cytoplasm, and we observed that NE directly cleaved SIRT1 in vitro, indicating that SIRT1 levels are decreased via direct degradation by internalized NE. Of note, we observed decreased SIRT1 levels in NE-treated primary human bronchial epithelial cells and in lung homogenates from both smokers and patients with COPD. In conclusion, NE suppresses CSE-induced HO-1 expression by cleaving SIRT1. This finding indicates the importance of cross-talk between oxidative stress and protease responses in the pathogenesis of COPD.

The Impact of Autophagy on the Cigarette Smoke Extract-Induced Apoptosis of Bronchial Epithelial Cells.

BACKGROUND: Previous studies report that apoptosis and autophagy are involved in the pathogenesis of emphysema, and macroautophagy is one of the processes regulating the apoptosis pathway. However, few studies have evaluated whether chaperone-mediated autophagy (CMA) contributes to the regulation of apoptosis. In this study, we investigated the impact of autophagy, including both macroautophagy and CMA, on the apoptosis in bronchial epithelial cells. METHODS: Cigarette smoke extract (CSE) was injected intratracheally into C57BL/6 mice, and emphysema and apoptosis were evaluated in the lungs. After treatment with CSE, apoptosis, macroautophagy, and CMA were measured in BEAS2-B cells, and the impact of autophagy on the apoptosis was evaluated following knockdown of autophagy-related genes by short interfering RNAs (siRNAs). RESULTS: Intratracheal CSE injection resulted in the development of emphysema and an increase in apoptosis in mice. CSE increased the apoptosis in BEAS2-B cells, and also elevated the expression of proteins related to both macroautophagy and CMA in BEAS2-B cells. The knockdown experiment with siRNAs showed that macroautophagy increases apoptosis in BEAS2-B cells, while CMA suppresses apoptosis. CONCLUSION: The intratracheal injection of CSE induces pulmonary emphysema and an increase in apoptosis in mice. CSE also induces apoptosis, macroautophagy, and CMA of bronchial epithelial cells. Macroautophagy and CMA regulate apoptosis in opposite directions.

Effect of exercise-induced neurogenesis on cognitive function deficit in a rat model of vascular dementia.

Chronic cerebral hypoperfusion (CCH) is strongly correlated with progressive cognitive decline in neurological diseases, such as vascular dementia (VaD) and Alzheimer's disease. Exercise can enhance learning and memory, and delay age-related cognitive decline. However, exercise-induced hippocampal neurogenesis in experimental animals submitted to CCH has not been investigated. The present study aimed to investigate whether hippocampal neurogenesis induced by exercise can improve cognitive deficit in a rat model of VaD. Male Wistar rats (age, 8 weeks; weight, 292±3.05 g; n=12-13/group) were subjected to bilateral common carotid artery occlusion (2VO) or sham­surgery and each group was then subdivided randomly into no exercise and treadmill exercise groups. Exercise groups performed treadmill exercise daily at 15 m/min for 30 min for 4 weeks from the third to the seventh week after 2VO. It was demonstrated that the number of neural progenitor cells and mature neurons in the subgranular zone of 2VO rats was increased by exercise, and cognitive impairment in 2VO rats was attenuated by treadmill exercise. In addition, mature brain­derived neurotrophic factor (BDNF) levels in the hippocampus were increased in the exercise groups. Thus the present study suggests that exercise delays cognitive decline by the enhancing neurogenesis and increasing BDNF expression in the context of VaD.

Neutrophil Elastase Differentially Regulates Interleukin 8 (IL-8) and Vascular Endothelial Growth Factor (VEGF) Production by Cigarette Smoke Extract.

Inflammation by IL-8-induced neutrophil recruitment and apoptosis of epithelial cells by decreased expression of VEGF have been suggested as one of the complicated pathogenic mechanisms of chronic obstructive pulmonary disease (COPD). The role of neutrophil elastase (NE) in the development of COPD is also well known. However, little is known about how they interact. The objective of this study was to elucidate the effect of NE on cigarette smoke extract (CSE)-induced IL-8 and VEGF production and its molecular mechanism in bronchial epithelial cells. CSE increased both IL-8 and VEGF production in human bronchial epithelial cells (BEAS-2B). Although NE significantly enhanced CSE-induced IL-8 production, it suppressed VEGF production. This differential regulation was not CSE-specific. The effect of NE on IL-8 production, but not VEGF, was ERK-dependent. Interestingly, in contrast to decreased VEGF protein expression, NE accelerated VEGF transcription by CSE, suggesting post-translational modification. When cells were incubated with purified NE, it was detected in the cytoplasm, suggesting the intracellular translocation of NE. Furthermore, NE fragmented recombinant human VEGF in vitro but not recombinant human IL-8. These results indicate that VEGF down-regulation is due to direct degradation by NE, which is translocated into cells. Similar to in vitro cell experiments, elastase treatment increased CSE-induced IL-8; however, it suppressed VEGF production in bronchoalveolar lavage fluid of CSE-treated mice. Moreover, elastase treatment enhanced CSE-induced emphysema in mice. Considering the actions of IL-8 and VEGF, our results suggest that NE contributes to the pathogenesis of COPD by enhancing inflammation and apoptosis.

Role of neuronal NADPH oxidase 1 in the peri-infarct regions after stroke.

The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia-reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2'-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke.

17-Allylamino-17-Demethoxygeldanamycin and the Enhancement of PS-341-Induced Lung Cancer Cell Death by Blocking the NF-κB and PI3K/Akt Pathways.

PS-341 is a highly selective and potent proteasome inhibitor that is cytotoxic to various types of cancer. However, no objective response was seen in a clinical trial with PS-341 as a single agent in non-small cell lung cancer. Its antitumor activity is limited by the simultaneously activated antiapoptosis pathway. Recently, PS-341-induced NF-κB activation via IκBα degradation has been suggested to be one of its antiapoptotic effects. In this study, we investigated the effects of a combined application of the heat shock protein (Hsp) 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) with PS-341 in lung cancer cells. Hsp90 inhibition with 17-AAG was effective in enhancing PS-341-induced lung cancer cell death in vitro and in vivo. 17-AAG pretreatment induced the degradation of upstream regulators of IκB, IL-1R-associated kinase-1 (IRAK-1), and IκB kinases (IKKs), dose and time dependently, which resulted in blocking of PS-341-induced IκBα degradation, p65 nuclear translocation, transcriptional activity, and NF-κB-regulated antiapoptotic gene expressions such as COX-2. The concentrations of 17-AAG used for combinatorial treatment with PS-341 did not change cell viability or the activity of proteasome complex. Moreover, 17-AAG pretreatment decreased the level of phsophorylated Akt at serine 473 residue and suppressed active Akt-dependent inactivation of glycogen synthase kinase 3ß. 17-AAG mediated the dissociation of its client proteins (IRAK-1, IKKs, and Akt) from the Hsp90 complex. As a result, it induced degradation of target proteins. Our results suggest that the combination of 17-AAG and PS-341 could be an effective anticancer therapy that overcomes the limited effects of PS-341.