New Findings
What is the central question of this study?
What is the effect of cigarette smoke on cell death, oxidative damage, expression of heat shock proteins (HSPs) and activation of ...mitogen‐activated protein kinases (MAPKs) in A549 alveolar epithelial cells?
What is the main finding and its importance?
Cigarette smoke induces cytotoxicity and oxidative damage to A549 cells, increases expression of different HSPs and activates MAPK signalling pathways. This could be related to inflammatory response and apoptosis observed in lungs of patients with smoking‐related diseases.
Cigarette smoking is one of the main risk factors for development of chronic obstructive pulmonary disease (COPD). We previously reported that cigarette smoke (CS) induces damage to proteins and their ineffective degradation. Here, we hypothesize that CS could induce oxidative stress and cytotoxicity in lung epithelial cells through alterations of heat shock protein (HSP) expression and mitogen‐activated protein kinase (MAPK) signalling pathways. We exposed A549 alveolar epithelial cells to various concentrations of cigarette smoke extract (CSE). Higher concentrations of CSE caused apoptosis of A549 cells after 4 h, while after 24 h cell viability was decreased, and lactate dehydrogenase in cell culture medium was increased as well as the number of necrotic cells. Concentrations of malondialdehyde (MDA) were elevated, while total thiol groups were decreased. Changes in the expression of HSPs (HSP70, HSP32 and HSP27) were time‐dependent. After 6 h, CSE caused an increase in the expression of HSP70 and HSP32, while after 8 h all examined HSPs were up‐regulated and remained increased up to 48 h. Treatment of A549 cells with CSE stimulated phosphorylation of extracellular signal‐regulated kinase and p38 in a dose‐dependent manner, while c‐Jun N‐terminal kinase activation was not detected. By using specific inhibitors, we demonstrated that MAPKs and HSPs interplay in CSE effects. In conclusion, our results show that MAPKs and HSPs are involved in the mechanism underlying CSE‐induced cytotoxicity and oxidative damage to A549 alveolar epithelial cells. These processes could be related to inflammatory response and apoptosis observed in lungs of patients with smoking‐related diseases, such as COPD.
New findings
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What is the central question of this study?
The endoplasmic reticulum stress response caused by cigarette smoke may lead to excessive apoptosis with disruption of the epithelial ...barrier, thus contributing to chronic obstructive pulmonary disease. One way of promoting cell survival is to facilitate degradation of cigarette smoke‐induced protein damage through the ubiquitin–proteasome pathway. Direct effects of gas‐phase cigarette smoke on proteasomal activities have not been demonstrated previously.
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What is the main finding and what is its importance?
We show that cigarette smoke induces protein damage and triggers the endoplasmic reticulum stress response in human alveolar epithelial cells. A significant reduction of all three proteasomal activities was found. Ineffective degradation of damaged proteins could lead to a sustained epithelial stress response and development of chronic obstructive pulmonary disease.
Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease. Cigarette smoke (CS) causes oxidative stress and severe damage to proteins in the lungs. One of the main systems to protect cells from the accumulation of damaged proteins is the ubiquitin–proteasome pathway. In the present study, we aimed to find out whether exposure of alveolar epithelial cells to CS induces an endoplasmic reticulum (ER) stress response by accumulation of damaged proteins that are inefficiently degraded by the proteasomes. The hypothesis was tested in a human alveolar epithelial cell line (A549) exposed to gas‐phase CS. Exposure to gas‐phase CS for 5 min caused an increase in the amount of ubiquitin–protein conjugates within 4 h. Cigarette smoke exposure also induced the ER stress response marker eIF2α, followed by a significant reduction of nascent protein synthesis and increase in the level of free intracellular amino acids. Moreover, CS exposure significantly reduced all three proteasomal activities (caspase‐, trypsin‐ and chymotrypsin‐like activity) within 4 h, which was still present after 24 h. It can be concluded that gas‐phase CS induces ER stress in A549 alveolar epithelial cells, leading to inadequate protein turnover caused by an accumulation of damaged proteins, reduction in nascent protein synthesis and inhibition of the proteasome. We suggest that prolonged ER stress may lead to excessive cell death with disruption of the epithelial barrier, contributing to development of chronic obstructive pulmonary disease.
Summary
Extracellular Hsp70 (eHsp70) exerts its biological actions via Toll‐like receptors 2 and 4, and is increased in sera of chronic obstructive pulmonary disease (COPD) patients. The aim of this ...study was to explore the pro‐inflammatory effects and cytotoxicity of eHsp70 alone and in combination with bacterial components lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on NCI‐H292 airway epithelial cells. NCI‐H292 cells were treated with recombinant human Hsp70 protein (rhHsp70), LPS, LTA and their combinations for 4, 12, 24 and 48 hours. IL‐6, IL‐8 and TNF‐α levels were measured by an ELISA method. Cell viability was determined by the MTS method, and caspase‐3/7, caspase‐8 and caspase‐9 assays. rhHsp70 induced secretion of IL‐6 and IL‐8 in a concentration‐ and time‐dependent manner, with the highest secretion at 24 hours. rhHsp70 combined with LTA had antagonistic and with LPS synergistic effect on IL‐6 secretion, while the interactions between rhHsp70 and LPS or LTA on IL‐8 were synergistic. TNF‐α was not detected in the applied conditions. rhHsp70, LPS or LTA did not affect cell viability, and rhHsp70 even suppressed caspase‐3/7 activities. We suggest that pro‐inflammatory effects of eHsp70, together with other damaging molecules and/or COPD risk factors, might contribute to the aggravation of chronic inflammation in human bronchial epithelium.
Chronic obstructive pulmonary disease (COPD) is a complex inflammatory condition that can affect haemostasis. This study aimed to determine differences in platelet-related parameters between controls ...and COPD subjects. The hypothesis was that platelet indices are disturbed in COPD patients, and this would be accompanied by increased C-reactive protein (CRP), fibrinogen (Fbg) and white blood cells (WBC). Therefore, platelet count (Plt), platelet-related parameters - mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (Pct), their ratios (MPV/Plt, MPV/Pct, PDW/Plt, PDW/Pct), platelet to lymphocyte ratio (PLR), Plt index as well as CRP, Fbg and WBC were assessed.
Study included 109 patients with stable COPD and 95 control subjects, recruited at Clinical Department for Lung Diseases Jordanovac, University Hospital Centre Zagreb (Zagreb, Croatia). Complete blood count was performed on Sysmex XN-1000, CRP on Cobas c501, and Fbg on BCS XP analyser. Data were analysed with MedCalc statistical software.
Platelet (P = 0.007) and PLR (P = 0.006) were increased, while other platelet indices were decreased in COPD patients compared to controls. Combined model that included PLR, PDW and WBC showed great diagnostic performances, and correctly classified 75% of cases with an AUC of 0.845 (0.788 - 0.892), P < 0.001. Comorbidities (cardiovascular or metabolic diseases) had no effect on investigated parameters, while inhaled corticosteroids/long-acting β
-agonists (ICS/LABA) therapy increased MPV and PDW values in COPD patients.
Platelet indices were altered in COPD patients and they could be valuable as diagnostic markers of COPD development, especially if combined with already known inflammatory markers.
Chronic obstructive pulmonary disease (COPD), an increasing global health problem, may be complicated by acute atherothrombotic events. Although systemic inflammation plays the leading role in ...atherothrombotic processes, platelet activation and increased coagulation together with oxidative stress can significantly exacerbate atherosclerosis in COPD patients. In this study we determined platelet count, mean platelet volume (MPV) and classical markers of systemic inflammation - serum C-reactive protein (CRP), white blood cell (WBC) count and the relative proportion of segmented neutrophils in COPD patients, and compared them to those from the healthy controls. The most important and novel finding of this study was that patients with COPD had a significantly increased platelet count, along with a reduced MPV when compared to healthy controls (286 vs. 260 × 109/l; 9.6 vs. 8.7 fL, respectively). Cigarette smoking had no influence on these results. The presence of systemic inflammation was clearly proved by the increase in classical inflammatory markers (CRP, WBC and segmented neutrophil count).
Abstract Mycotoxin fumonisin B1 (FB1 ) is a frequent contaminant of grain, particularly maize, but the mechanism of its toxicity in the kidney and liver is not fully understood. FB1 -stimulated ...oxidative stress might disturb cellular redox state and signal transduction pathways of the target cells. In this study we measured total intracellular glutathione (GSH), and assessed mitogen-activated protein kinases (MAPKs) activation and the expression of heat shock proteins (Hsps) Hsp25 and Hsp70 in the liver and kidney of male Wistar rats given 0.5 mg FB1 /kg b.w. intraperitoneally for 2 or 7 days. The effect of FB1 on GSH levels, MAPK activation and Hsp expression was found to be related to the type of tissue affected and the length of treatment. In rat liver, cellular GSH content increased, Hsp expression was up-regulated, and ERK and p38 were activated after the 7-day treatment, while even the 2-day treatment sufficed to produce phospho-JNK signal. In rat kidney, GSH levels decreased after the 2- and 7-day treatment with FB1 , while after the 7-day treatment all three MAPKs were activated, Hsp25 expression increased and Hsp70 expression decreased. In conclusion, FB1 alters cellular redox balance, which leads to tissue-specific activation and expression of redox-sensitive signalling molecules. It seems that kidney cells are more sensitive to adverse effects of FB1.
Mycotoxin fumonisin B
1 (FB
1) is a frequent contaminant of grain, particularly maize, but the mechanism of its toxicity in the kidney and liver is not fully understood. FB
1-stimulated oxidative ...stress might disturb cellular redox state and signal transduction pathways of the target cells. In this study we measured total intracellular glutathione (GSH), and assessed mitogen-activated protein kinases (MAPKs) activation and the expression of heat shock proteins (Hsps) Hsp25 and Hsp70 in the liver and kidney of male Wistar rats given 0.5
mg
FB
1/kg
b.w. intraperitoneally for 2 or 7 days. The effect of FB
1 on GSH levels, MAPK activation and Hsp expression was found to be related to the type of tissue affected and the length of treatment. In rat liver, cellular GSH content increased, Hsp expression was up-regulated, and ERK and p38 were activated after the 7-day treatment, while even the 2-day treatment sufficed to produce phospho-JNK signal. In rat kidney, GSH levels decreased after the 2- and 7-day treatment with FB
1, while after the 7-day treatment all three MAPKs were activated, Hsp25 expression increased and Hsp70 expression decreased. In conclusion, FB
1 alters cellular redox balance, which leads to tissue-specific activation and expression of redox-sensitive signalling molecules. It seems that kidney cells are more sensitive to adverse effects of FB
1.
Hemodialyzed patients have lower paraoxonase 1 (PON1) activity. Higher mortality risk from cardiovascular disease observed in these patients could be due to the low antiathetrogenic activity of PON1. ...Understanding the mechanism that causes lower PON1 activity could provide the possibility for modulation of enzyme activity in purpose of preventing and/or decreasing development of atherosclerosis.
87 healthy individuals and 71 hemodialyzed patients were enrolled in this study.
Hemodialyzed patients had reduced PON1 paraoxonase and arylesterase activity, concentrations of HDL, HDL
3 and HDL
2 and concentrations of free thiol groups. Distribution of HDL subfractions and distribution of PON1 phenotypes as well as concentrations of MDA were not different between two study groups. In the
in vitro experiment high concentrations of urea, creatinine, uric acid and addition of patient's sera ultrafiltrate did not significantly affect PON1 paraoxonase activity.
Decreased HDL concentration as well as lower PON1 concentration (shown indirectly by the enzyme arylesterase activity) might contribute, at least partly, to the reduced PON1 activity observed in hemodialyzed patients. Decreased concentration of free thiol groups in sera suggest that free thiol group (Cys284) in PON1 might also be oxidized, which can affect PON1 activity.
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