Introduction: Prohibitin 1 (PHB1) is a versatile protein that is located in the inner mitochondri... more Introduction: Prohibitin 1 (PHB1) is a versatile protein that is located in the inner mitochondrial membrane, maintaining normal mitochondrial function and morphology. Prohibitin interacts with NADH dehydrogenase, a protein complex essential for the oxidoreductase activity within cells. However, its expression in lung epithelium, especially in patients with inflammatory lung diseases associated with increased oxidative stress, such as COPD, is unknown. Aim: To study PHB1 expression in lung tissue of non-smokers, non-COPD smokers and COPD patients. Methodology: Lung tissue specimens from 30 male subjects were studied: 15 COPD patients [age: 65.9±6.2 years, smoking: 88.9±35.2 pack-years, FEV1 (% pred): 58.4±16.4, FEV1/FVC (%): 66.2±8.6], 10 non-COPD smokers [age: 57.0±11.7 years, smoking: 67.1±39.9 pack-years, FEV1 (% pred): 84.0±15.9, FEV1/FVC (%): 80.0±3.8] and 5 non-smokers. Quantitative Real-Time PCR and Western Blot experiments were carried out for PHB1, using beta-actin as internal control. Results: Non-COPD smokers exhibited lower prohibitin levels when compared to non-smokers (0.55±0.06 vs. 0.90±0.06, p=0.011), while PHB1 mRNA levels were even further decreased in COPD patients (0.32±0.02), a finding statistically significant vs. both non-COPD smokers (p=0.012) and non-smokers (p=0.009). Western blot analysis verified the above results (non-smokers: 1.77±0.10; non-COPD smokers: 0.97±0.08; COPD patients: 0.62±0.09, p=0.028). Conclusion: The significantly downregulated prohibitin levels in non-COPD and COPD smokers in comparison with non-smokers possibly reflects a distorted mitochondrial function, resulting in decreased anti-oxidant activity, especially in the mitochondria of COPD patients.
European Journal of Clinical Investigation, Oct 22, 2013
Growth factors mediate various cellular responses to environmental stimuli. Specifically, exposur... more Growth factors mediate various cellular responses to environmental stimuli. Specifically, exposure of lung epithelium to oxidative stress induced by cigarette smoke stimulates aberrant epidermal growth factor receptor (ERBB) family activation. This study's objective was to evaluate the expression of ERBB1-4 receptors in the lung tissue of smokers with or without chronic obstructive pulmonary disease (COPD). ERBBs expression was measured by microarray analysis in lung tissue samples from five patients with COPD and five non-COPD smokers, and by quantitative real-time PCR in additional 20 patients with COPD (GOLD stage II), 15 non-COPD smokers and 10 nonsmoker controls. Microarray data analysis revealed that ERBB receptors expression was elevated in patients with COPD compared to non-COPD smokers, ranging from 1·62- to 2·45-fold, (P < 0·01). Real-time qPCR verified that patients with COPD had higher ERBB1-3 expression levels compared with non-COPD smokers (PERBB1 < 0·001; PERBB2 = 0·003; PERBB3 = 0·003) and nonsmokers (PERBB1 = 0·019; PERBB2 = 0·005; PERBB3 = 0·011). On the other hand, ERBB4 mRNA levels gradually increased from nonsmokers (0·74 ± 0·19) to non-COPD smokers (1·11 ± 0·05) to patients with COPD (1·57 ± 0·28) and were correlated with the degree of airflow obstruction (PFEV1 < 0·001). These data suggest that ERBB1-3 overexpression is not related only to smoking exposure but probably to epithelial remodelling and mucociliary system distortion, characterizing COPD. Additionally, the inverse correlation of ERBB4 with FEV1 exhibits a possible link between ERBB4 and COPD severity.
High incidence of genetic alterations at the microsatellite (MS) DNA level has been reported in a... more High incidence of genetic alterations at the microsatellite (MS) DNA level has been reported in asthmatic adults. The aim of this study was to investigate whether microsatellite instability (MSI) and loss of heterozygosity (LOH) were detectable phenomena in children with asthma. DNA was extracted from sputum and blood cells of 27 children (10.8 +/- 2.5 years) with mild to moderate asthma, and from 8 healthy, never-smoked young adults. Fourteen polymorphic MS markers, namely D5S207, D5S820, D5S637, D6S344, D6S2223, D6S263, SGC35231, D11S1253, D11S1337, D11S97, USAT24G1, D13S273, D14S258, and D14S292, located on chromosomes (chr) 5q, 6p, 11q, 13q, and 14q were used to assess MSI and LOH. None of the healthy subjects exhibited any genetic alteration. Five out of 27 children (18.5%) exhibited MSI or LOH in sputum cells versus blood samples from which 3 in the marker USAT24G1 (chr 13q14.1), 1 in the marker D14S258 (chr 14q23-q24.3), and 1 in the marker D5S637 (chr 5q12-q13). Compared to a previous study, with asthmatic adults, whereas MSI and/or LOH was exhibited in approximately 60% of the cases, the current study reported <20% of genetic alterations, at the MS DNA, in asthmatic children. Our results showed that genetic instability in the MS DNA, is present in asthmatic children, but to less extent than in adult asthmatics from previous studies. These findings may support the hypothesis that somatic mutations may be early acquired in the natural course of asthma and could represent another contributor to the molecular pathogenesis of the disease. However, further studies are needed to clarify this hypothesis.
Background: COPD is a multifactorial degenerative disease, mainly caused by smoking. However, sev... more Background: COPD is a multifactorial degenerative disease, mainly caused by smoking. However, several other mechanisms contribute to the development and progression of COPD, including inflammatory response by infiltrating macrophages and lymphocytes, abnormal cellular growth, increased apoptosis, extracellular matrix destruction, oxidative DNA damage repair and transcription activation. Objective: To identify, with the help of genome-wide analysis tools, the intracellular pathways, and the individual genes within them, that are associated with COPD. Methods: Gene expression microarrays, conducted on lung tissue samples from 5 selected COPD patients (GOLD stage II) and 5 non-COPD smokers using the U133 Plus 2.0 Array chip by Affymetrix, were clustered according to gene biological functions. Results: Cluster analysis within biological pathways that are associated with COPD revealed numerous genes of interest: A) Apoptosis: IL-1B, Perforin 1, K-RAS, AKT1, FGF2, TP63; B) Aging: TIMP3, Decorin, JUN, PTEN, TGFB3; C) Oxidative stress: TLR4, peroxiredoxins, OGG1, APOE, DUOX1; D) Inflammation: CCL18, Thrombospondin 1, ANXA1, HDAC9, IL-1A; E) Extracellular matrix: fibronectin 1, CCDC80, VHL, NF1; F) Transcription regulation: MAPK1, HIF1A, STAT1, HDAC1, SMAD5, SP7. Additionally, several less known pathways were also flagged: I) respiratory gaseous exchange; II) chromatin organization; III) protein methylation; IV) oxidative demethylation; V) surfactant homeostasis. Conclusions: Our findings suggest that apart from well described genes that possibly play a significant role in COPD, there are biological pathways and gene clusters that are also worth investigating.
Introduction: Prohibitin 1 (PHB1) is a versatile protein that is located in the inner mitochondri... more Introduction: Prohibitin 1 (PHB1) is a versatile protein that is located in the inner mitochondrial membrane, maintaining normal mitochondrial function and morphology. Prohibitin interacts with NADH dehydrogenase, a protein complex essential for the oxidoreductase activity within cells. However, its expression in lung epithelium, especially in patients with inflammatory lung diseases associated with increased oxidative stress, such as COPD, is unknown. Aim: To study PHB1 expression in lung tissue of non-smokers, non-COPD smokers and COPD patients. Methodology: Lung tissue specimens from 30 male subjects were studied: 15 COPD patients [age: 65.9±6.2 years, smoking: 88.9±35.2 pack-years, FEV1 (% pred): 58.4±16.4, FEV1/FVC (%): 66.2±8.6], 10 non-COPD smokers [age: 57.0±11.7 years, smoking: 67.1±39.9 pack-years, FEV1 (% pred): 84.0±15.9, FEV1/FVC (%): 80.0±3.8] and 5 non-smokers. Quantitative Real-Time PCR and Western Blot experiments were carried out for PHB1, using beta-actin as internal control. Results: Non-COPD smokers exhibited lower prohibitin levels when compared to non-smokers (0.55±0.06 vs. 0.90±0.06, p=0.011), while PHB1 mRNA levels were even further decreased in COPD patients (0.32±0.02), a finding statistically significant vs. both non-COPD smokers (p=0.012) and non-smokers (p=0.009). Western blot analysis verified the above results (non-smokers: 1.77±0.10; non-COPD smokers: 0.97±0.08; COPD patients: 0.62±0.09, p=0.028). Conclusion: The significantly downregulated prohibitin levels in non-COPD and COPD smokers in comparison with non-smokers possibly reflects a distorted mitochondrial function, resulting in decreased anti-oxidant activity, especially in the mitochondria of COPD patients.
European Journal of Clinical Investigation, Oct 22, 2013
Growth factors mediate various cellular responses to environmental stimuli. Specifically, exposur... more Growth factors mediate various cellular responses to environmental stimuli. Specifically, exposure of lung epithelium to oxidative stress induced by cigarette smoke stimulates aberrant epidermal growth factor receptor (ERBB) family activation. This study's objective was to evaluate the expression of ERBB1-4 receptors in the lung tissue of smokers with or without chronic obstructive pulmonary disease (COPD). ERBBs expression was measured by microarray analysis in lung tissue samples from five patients with COPD and five non-COPD smokers, and by quantitative real-time PCR in additional 20 patients with COPD (GOLD stage II), 15 non-COPD smokers and 10 nonsmoker controls. Microarray data analysis revealed that ERBB receptors expression was elevated in patients with COPD compared to non-COPD smokers, ranging from 1·62- to 2·45-fold, (P < 0·01). Real-time qPCR verified that patients with COPD had higher ERBB1-3 expression levels compared with non-COPD smokers (PERBB1 < 0·001; PERBB2 = 0·003; PERBB3 = 0·003) and nonsmokers (PERBB1 = 0·019; PERBB2 = 0·005; PERBB3 = 0·011). On the other hand, ERBB4 mRNA levels gradually increased from nonsmokers (0·74 ± 0·19) to non-COPD smokers (1·11 ± 0·05) to patients with COPD (1·57 ± 0·28) and were correlated with the degree of airflow obstruction (PFEV1 < 0·001). These data suggest that ERBB1-3 overexpression is not related only to smoking exposure but probably to epithelial remodelling and mucociliary system distortion, characterizing COPD. Additionally, the inverse correlation of ERBB4 with FEV1 exhibits a possible link between ERBB4 and COPD severity.
High incidence of genetic alterations at the microsatellite (MS) DNA level has been reported in a... more High incidence of genetic alterations at the microsatellite (MS) DNA level has been reported in asthmatic adults. The aim of this study was to investigate whether microsatellite instability (MSI) and loss of heterozygosity (LOH) were detectable phenomena in children with asthma. DNA was extracted from sputum and blood cells of 27 children (10.8 +/- 2.5 years) with mild to moderate asthma, and from 8 healthy, never-smoked young adults. Fourteen polymorphic MS markers, namely D5S207, D5S820, D5S637, D6S344, D6S2223, D6S263, SGC35231, D11S1253, D11S1337, D11S97, USAT24G1, D13S273, D14S258, and D14S292, located on chromosomes (chr) 5q, 6p, 11q, 13q, and 14q were used to assess MSI and LOH. None of the healthy subjects exhibited any genetic alteration. Five out of 27 children (18.5%) exhibited MSI or LOH in sputum cells versus blood samples from which 3 in the marker USAT24G1 (chr 13q14.1), 1 in the marker D14S258 (chr 14q23-q24.3), and 1 in the marker D5S637 (chr 5q12-q13). Compared to a previous study, with asthmatic adults, whereas MSI and/or LOH was exhibited in approximately 60% of the cases, the current study reported <20% of genetic alterations, at the MS DNA, in asthmatic children. Our results showed that genetic instability in the MS DNA, is present in asthmatic children, but to less extent than in adult asthmatics from previous studies. These findings may support the hypothesis that somatic mutations may be early acquired in the natural course of asthma and could represent another contributor to the molecular pathogenesis of the disease. However, further studies are needed to clarify this hypothesis.
Background: COPD is a multifactorial degenerative disease, mainly caused by smoking. However, sev... more Background: COPD is a multifactorial degenerative disease, mainly caused by smoking. However, several other mechanisms contribute to the development and progression of COPD, including inflammatory response by infiltrating macrophages and lymphocytes, abnormal cellular growth, increased apoptosis, extracellular matrix destruction, oxidative DNA damage repair and transcription activation. Objective: To identify, with the help of genome-wide analysis tools, the intracellular pathways, and the individual genes within them, that are associated with COPD. Methods: Gene expression microarrays, conducted on lung tissue samples from 5 selected COPD patients (GOLD stage II) and 5 non-COPD smokers using the U133 Plus 2.0 Array chip by Affymetrix, were clustered according to gene biological functions. Results: Cluster analysis within biological pathways that are associated with COPD revealed numerous genes of interest: A) Apoptosis: IL-1B, Perforin 1, K-RAS, AKT1, FGF2, TP63; B) Aging: TIMP3, Decorin, JUN, PTEN, TGFB3; C) Oxidative stress: TLR4, peroxiredoxins, OGG1, APOE, DUOX1; D) Inflammation: CCL18, Thrombospondin 1, ANXA1, HDAC9, IL-1A; E) Extracellular matrix: fibronectin 1, CCDC80, VHL, NF1; F) Transcription regulation: MAPK1, HIF1A, STAT1, HDAC1, SMAD5, SP7. Additionally, several less known pathways were also flagged: I) respiratory gaseous exchange; II) chromatin organization; III) protein methylation; IV) oxidative demethylation; V) surfactant homeostasis. Conclusions: Our findings suggest that apart from well described genes that possibly play a significant role in COPD, there are biological pathways and gene clusters that are also worth investigating.
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