Cigarette Smoke Condensate Exposure Research Articles

Cigarette smoke condensate exposure delays follicular development and function in a stage-dependent manner

To determine the effects of cigarette smoke condensate (CSC) on follicular development and function from the early preantral stage through ovulation. Prospective laboratory study. Academic research environment. Female F1 hybrid (C57BL/6j×CBA/Ca) mice. Mouse early preantral follicles (100-130 μm) were exposed to increasing concentrations of CSC (0 μg/mL [control] to 130 μg/mL) during in vitro growth and ovulation. Follicular development, follicle survival, gonadal steroid output, expansion of the cumulus-oocyte complex, oocyte growth, and maturation. Cigarette smoke condensate exposure significantly inhibited follicular development in the preantral and antral stage and decreased follicle survival at 90 μg CSC/mL and higher. Estradiol output was significantly lower in CSC-exposed (90 and 130 μg/mL) follicles. Before ovulation, CSC significantly increased P output, which decreased thereafter. Cigarette smoke condensate exposure reduced cumulus-oocyte complex expansion and subsequently reduced the number of polar body oocytes. Cigarette smoke condensate exposure inhibits follicle development and leads to premature luteinization of the preovulatory follicle, with decreased oocyte maturation in a mouse isolated follicle culture system that mimics murine folliculogenesis in vivo.

Abstract 1332: Cigarette smoking attenuates TGF-β-mediated tumor suppression function through down-regulation of Smad3 in lung cancer

Abstract Cigarette smoking has been connected to an array of chronic lung diseases and one of the major causes of morbidity and mortality. Epidemiological studies have demonstrated the most cases of lung cancers (85-90%) are directly attributable to tobacco smoking. Although much information is gained about the effect of cigarette smoking on various signaling pathways causing lung cancer, no information is available about the effect cigarette smoking on the TGF-β-dependent initiation and progression of lung cancer. To address this issue, A549, a lung adenocarcinma cell line and HPL1A, an immortalized lung epithelial cell line were chronically treated with cigarette smoke condensate (CSC) to mimic the conditions of long-term cigarette smoking. Prolonged exposure of these cells to CSC resulted in a decrease in Smad3 and Smad4 complex formation and TGF-ß-mediated transcription due to reduced expression of Smad3. As a consequence, the cells were less apoptotic, were more viable, and lost TGF-β mediated growth inhibition. We found that the decrease in apoptosis is due to the up-regulation of Bcl2, which is a downstream target of Smad3. The repression of Smad3 expression is at the promoter level and is due to histone deacetylation. Withdrawal of CSC treatment resulted in the restoration of Smad3 expression, reduction in cell viability and increased TGF-β mediated growth inhibition. Rescue experiment with reintroduction of Smad3 in the 300 day CSC treated cells restored TGF-β signaling, increased apoptosis and decreased cell viability. Finally, three hundred days CSC exposure dramatically increased the tumorigenicity of cells in vivo, and Smad3 re-expression reverses this effect. In patient samples, there is a trend that expression of Smad3 goes down in smokers compared to non-smokers. Collectively, these data provide evidence that cigarette smoking promotes tumorigenicity partly by abrogating TGF-β mediated growth inhibition and apoptosis by reducing expression of Smad3. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1332. doi:10.1158/1538-7445.AM2011-1332

The effects of cigarette smoke condensate on vocal fold transepithelial resistance and inflammatory signaling in vocal fold fibroblasts

In response to chronic cigarette smoke exposure, a subset of patients present with edematous vocal folds, characteristically referred to as Reinke's edema. This phenotype differs from the tissue changes associated with prolonged smoke exposure in the lower airway, and the mechanism underlying Reinke's edema remains poorly described. We hypothesize that the effects of smoke are diffuse and involve both the epithelium and mucosa. In vitro, ex vivo experiment. Transepithelial resistance (R(T) ) was quantified in an ex vivo, viable, porcine vocal fold model. Excised tissue was exposed to cigarette smoke condensate (CSC) and R(T) was computed at baseline and 1 and 4 hours after exposure. In vitro, human vocal fold fibroblasts were exposed to CSC. Cyclooxygenase 2 (COX-2), microsomal prostaglandin E synthase-1, and 15-hydroxyprostaglandin dehydrogenase mRNA expression were assessed at 4 hours. Prostaglandin E2 (PGE2) synthesis was quantified via immunoassay following 24 hours of CSC exposure. CSC had no effect on R(T) . CSC did, however, induce COX-2 mRNA expression as well as its downstream lipid mediator PGE2. PGE2 metabolism appears to be regulated via both synthetic and degradative enzymes in response to cigarette smoke. In vitro, CSC initiates an inflammatory response in vocal fold fibroblasts. However, in isolation, the epithelial resistance is not altered by CSC, at least acutely. These data may suggest a role for the interaction between the inflammatory response in the mucosa and compromised epithelial barrier function, as has been shown in other tissues.

Chronic exposure to cigarette smoke condensate in vitro induces epithelial to mesenchymal transition-like changes in human bronchial epithelial cells, BEAS-2B

Cigarette smoke causes lung tumorigenesis; however, the mechanisms underlying transformation are unknown. We investigated if tobacco compounds induce DNA promoter hypermethylation in BEAS-2B cells treated with low doses of cigarette smoke condensate (CSC) for one month. Transcriptional profiles and anchorage-independent growth were explored using Affymetrix microarray and soft agar assay, respectively. To investigate if tobacco compounds induce hypermethylation, CSC/dimethyl sulfoxide (DMSO)-treated cells were further treated with 5-Aza-2′-deoxycytidine (5AzaC) and trychostatin A (TSA). This treatment was followed by transcriptional profiling. CSC-exposed cells acquired a fibroblast-like shape with enhanced anchorage-independent growth. Silencing of epithelial cadherin, the hallmark of epithelial to mesenchymal transition (EMT), was observed upon exposure to CSC. Changes in the expression of genes involved in epidermal development, intercellular junction formation, and cytoskeleton formation were identified. Gene expression profiles from 5AzaC- and TSA-treated cells revealed 130 genes possibly methylated due to chronic CSC exposure. Our results suggest that E-cadherin may also be silenced by hypermethylation in an in vitro model of chronic exposure to low doses of CSC. This study demonstrates evidence for a tobacco compound induced EMT-like process in vitro and provides insight into possible mechanisms of gene silencing occurring during this treatment.

Cigarette Smoke Induces C/EBP-β-Mediated Activation of miR-31 in Normal Human Respiratory Epithelia and Lung Cancer Cells

BackgroundLimited information is available regarding mechanisms by which miRNAs contribute to pulmonary carcinogenesis. The present study was undertaken to examine expression and function of miRNAs induced by cigarette smoke condensate (CSC) in normal human respiratory epithelia and lung cancer cells.MethodologyMicro-array and quantitative RT-PCR (qRT-PCR) techniques were used to assess miRNA and host gene expression in cultured cells, and surgical specimens. Software-guided analysis, RNA cross-link immunoprecipitation (CLIP), 3′ UTR luciferase reporter assays, qRT-PCR, focused super-arrays and western blot techniques were used to identify and confirm targets of miR-31. Chromatin immunoprecipitation (ChIP) techniques were used to evaluate histone marks and transcription factors within the LOC554202 promoter. Cell count and xenograft experiments were used to assess effects of miR-31 on proliferation and tumorigenicity of lung cancer cells.ResultsCSC significantly increased miR-31 expression and activated LOC554202 in normal respiratory epithelia and lung cancer cells; miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure. miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. CLIP and reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over-expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC-mediated decreases in Dkk-1 and DACT-3 expression. Furthermore, over-expression of miR-31 diminished SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. CSC increased H3K4Me3, H3K9/14Ac and C/EBP-β levels within the LOC554202 promoter. Knock-down of C/EBP-β abrogated CSC-mediated activation of LOC554202. Over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells; knock-down of miR-31 inhibited growth of these cells.ConclusionsCigarette smoke induces expression of miR-31 targeting several antagonists of cancer stem cell signaling in normal respiratory epithelia and lung cancer cells. miR-31 functions as an oncomir during human pulmonary carcinogenesis.

Abstract 4250: Cigarette smoke induces expression of the putative stem cell marker ABCG2 in cultured esophageal adenocarcinoma cells

Abstract Limited information is available regarding epigenetic mechanisms by which cigarette smoke mediates initiation and progression of esophageal cancer. In order to examine this issue, esophageal adenocarcinoma cell (EsC) lines (NCI-EsC1 and NCI-EsC2) were cultured in normal media (NM) with or without cigarette smoke condensate (CSC) under clinically relevant exposure conditions. Microarray analysis revealed that CSC exposure significantly upregulated expression of ABCG2, which encodes a xenobiotic pump protein believed to be highly expressed in cancer stem cells, and recently shown to correlate with aggressive phenotype of primary esophageal squamous cell carcinomas. Analysis revealed an average of 11 fold upregulation of ABCG2 as well as a 4 fold upregulation of another ABC transporter, ABCC3. Quantitative reverse transcription-PCR (RT-PCR), western blot, and immunohistochemistry experiments confirmed upregulation of ABCG2 in esophageal cancer cells exposed to CSC. Interestingly, basal as well as CSC-induced levels of ABCG2 were considerably higher in NCI-EsC2 derived from a smoker, relative to NCI-EsC1, which was established from a never-smoker. Time course experiments revealed that induction of ABCG2 commenced approximately 8 hours and peaked 48 hours following initiation of CSC exposure; thereafter, ABCG2 expression was sustained at these levels with continuous CSC exposure. HDAC inhibitors such as TSA and depsipeptide repressed basal levels of ABCG2 expression, and reduced CSC-mediated induction of ABCG2 in EsC cells. HDAC inhibitors in combination with verapamil (a ABCC3 inhibitor) and Depsipeptide virtually abolished basal, as well as CSC-induced ABCG2 expression in these cells. Preliminary pyrosequencing and chromatin immunoprecipitation (ChIP) experiments revealed that CSC-mediated ABCG2 induction was independent of promoter DNA demethylation, and coincided with hyperacetylation of H3K9 within the ABCG2 promoter. Flow cytometry analysis utilizing hoescht dye with and without fumitremorgin C, an ABCG2 inhibitor, revealed an increased side population in CSC-treated ESC cells relative to untreated controls. Experiments are in progress to further delineate the mechanisms by which CSC induces ABCG2 expression, and to ascertain if ABCG2 induction enhances the malignant phenotype of esophageal adenocarcinoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4250.

Abstract 1693: Assessment of Nrf2 luciferase reporter assay and heme oxygenase expression as models of oxidative stress in in vitro cultures of lung cells exposed to cigarette smoke condensate

Abstract The Nrf2 signaling pathway is an important regulator associated with oxidative stress, xenobiotic metabolism, DNA damage and anti-inflammatory response. A key responsive gene in the Nrf2 regulatory pathway is heme oxygense 1 (HO-1), a cytoprotective enzyme that serves in an antioxidant capacity. An imbalance in antioxidant capacity observed in animal models or individuals with specific Nrf2 and HO-1 polymorphisms has been associated with susceptibility to lung diseases. The objective of this study was to characterize the response of Nrf 2 promoter regulation and HO-1 expression following exposure to cigarette smoke in lung models for oxidative stress. Cigarette smoke condensate (CSC) was prepared from Kentucky 3R4F (K3R4F) reference cigarettes. Nrf2 promoter regulation and HO-1 expression were evaluated in lung cells exposed to K3R4F CSC (0 − 90μg total particulate matter/ml media). Nrf2 promoter regulation was evaluated in transiently-transfected Nrf2 luciferase reporter cells exposed to K3R4F CSC for 0.5, 1, 2, 6 or 24 hours followed by quantification of luciferase activity. HO-1 expression was assessed following exposure to the K3R4F CSC in normal human bronchial epithelial (NHBE) cells for 3, 6 or 24 hours using real-time quantitative RT/PCR and enzyme linked immunoassay (ELISA). Nrf2 reporter cells exhibited dose-dependent increases in promoter activation following CSC exposure. The maximum response was observed between 6 and 24 hours of exposure with 15-, 25-, and 40-fold increases across the exposure range. The specificity of the promoter response was confirmed with co- exposure of the inhibitor all-trans retinoic acid with CSC. The CSC-induced Nrf2 activation was reduced towards control levels in the presence of the inhibitor. CSC also induced dose- and time-dependent increases on HO-1 mRNA and protein levels in NHBE cells. The mRNA changes ranged from 10- to 70-fold with maximal responses observed between 3 and 6 hours. The dynamic range of HO-1 protein response was observed and reached a level of 11-fold increase by 60 μg/ml. Collectively, these responses are consistent with our previously reported study which identified oxidative stress responsive genes in NHBE cells exposed to repetitive applications of CSC. The data suggest that changes in the Nrf2 regulatory pathway may serve as an effective indicator of oxidative stress and the impact of tobacco products in in vitro lung cell culture models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1693.

Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate Paxillin

Paternal smoking is associated with infertility, birth defects and childhood cancers. Our earlier studies using cigarette smoke condensate (CSC) demonstrated several deleterious changes in male germ cells. Here, we hypothesize that chronic paternal exposure to CSC causes molecular and phenotypic changes in the sire and the offspring, respectively. In this mouse study, CSC caused DNA damage and cytotoxicity in testes via accumulation of benzo(a)pyrene (B[a]P) and cotinine. Decreased expression of growth arrest and DNA damage inducible alpha (Gadd45a), aryl hydrocarbon receptor (Ahr), and cyclin-dependent kinase inhibitor 1A (P21) was seen in CSC exposed testes. Apoptotic germ cell death was detected by induction of Fas, FasL, and activated caspase-3. The CSC-exposed males displayed reduction in sperm motility and fertilizing ability and sired pups with reduced body weight and crown-rump length, and smaller litter size with higher numbers of resorption. This model of CSC exposure demonstrates testicular toxicity and developmental defects in the offspring.

Identification of in vitro differential cell secretions due to cigarette smoke condensate exposure using nanoflow capillary liquid chromatography and high-resolution mass spectrometry

Secreted proteins, the secretome, can be isolated from biological fluids (e.g., blood) and are often responsible for the regulation of biological processes such as cell signaling, growth, and apoptosis. The identification of secreted proteins can lead to an understanding of disease mechanisms and they can serve as early candidate biomarkers of disease and exposure. However, it is time-consuming and costly to conduct in vivo interrogations of the human secretome. The purpose of this article is to provide a detailed description of a rapid in vitro technique for the analysis of differential protein secretion due to exposure to smoking-machine-generated cigarette smoke (CS) condensate (total particulate matter, TPM). Endothelial cells were exposed to CS-TPM, the supernatant was collected, and the secretome was elucidated by nano liquid chromatography coupled with high-resolution mass spectrometry. A total of 1,677 unique peptides were identified in the cell culture supernatants. Several proteins were differentially expressed following CS-TPM exposure that relate to several biological processes, such as metabolism, development, communication, response to stimulus, and response to stress.

Cigarette Smoke Condensate affects Dendritic Cell (DC) Maturation by Modulating DC-Epithelial Cell Cross-talk

RationaleCigarette smoke suppresses dendritic cell (DC) function and also affects cytokine expression in epithelial cell (EC) cultures. Since smoke affects these cells simultaneously in vivo, it was reasoned that the smoke effects on DC may be modulated by its effects on ECs. Therefore, the role of cigarette smoke condensate (CSC) induced phenotypic and functional changes in DC and DC-EC cultures were compared.MethodsConfluent, monolayer EC were cultured in the presence or absence of 10 μg/ml (low) or 50 μg/ml (high) CSC for 12 hours and poly I:C stimulated. Monocyte-derived immature dendritic cells (iDC) were then added to the EC culture or cultured alone in the presence or absence of CSC and Poly I:C for 24 hours. CSC-exposed iDC were stained for viability, surface maturation markers and analyzed by flow cytometry. Cytokine levels were examined by a 14-plex bead array and endocytosis was evaluated by FITC-dextran uptake.ResultsCSC exposure in both DC and EC-DC cultures have minimal effects on viability. Both doses of CSC reduce CD80, CD83, CD86 maturation markers, including CD54 (ICAM-1). DC cultures exhibit an increase in endocytosis while the EC-DC cultures show a significant decrease in endocytosis. Analysis of the cytokine profiles shows that the expression of IL1-beta, IL-6, IL-10, IL-12p40 and IL-13 increase in EC-DC cultures as compared to DC alone. When pre-stimulated with poly I:C, CSC enhances expression of IL-10, IL-12p40 and decreases expression of IL-13 in EC-DC co-cultures.ConclusionCSC modulates EC-DC cross-talk by inducing production of cytokines that do not affect DC maturation but affect their function. RationaleCigarette smoke suppresses dendritic cell (DC) function and also affects cytokine expression in epithelial cell (EC) cultures. Since smoke affects these cells simultaneously in vivo, it was reasoned that the smoke effects on DC may be modulated by its effects on ECs. Therefore, the role of cigarette smoke condensate (CSC) induced phenotypic and functional changes in DC and DC-EC cultures were compared. Cigarette smoke suppresses dendritic cell (DC) function and also affects cytokine expression in epithelial cell (EC) cultures. Since smoke affects these cells simultaneously in vivo, it was reasoned that the smoke effects on DC may be modulated by its effects on ECs. Therefore, the role of cigarette smoke condensate (CSC) induced phenotypic and functional changes in DC and DC-EC cultures were compared. MethodsConfluent, monolayer EC were cultured in the presence or absence of 10 μg/ml (low) or 50 μg/ml (high) CSC for 12 hours and poly I:C stimulated. Monocyte-derived immature dendritic cells (iDC) were then added to the EC culture or cultured alone in the presence or absence of CSC and Poly I:C for 24 hours. CSC-exposed iDC were stained for viability, surface maturation markers and analyzed by flow cytometry. Cytokine levels were examined by a 14-plex bead array and endocytosis was evaluated by FITC-dextran uptake. Confluent, monolayer EC were cultured in the presence or absence of 10 μg/ml (low) or 50 μg/ml (high) CSC for 12 hours and poly I:C stimulated. Monocyte-derived immature dendritic cells (iDC) were then added to the EC culture or cultured alone in the presence or absence of CSC and Poly I:C for 24 hours. CSC-exposed iDC were stained for viability, surface maturation markers and analyzed by flow cytometry. Cytokine levels were examined by a 14-plex bead array and endocytosis was evaluated by FITC-dextran uptake. ResultsCSC exposure in both DC and EC-DC cultures have minimal effects on viability. Both doses of CSC reduce CD80, CD83, CD86 maturation markers, including CD54 (ICAM-1). DC cultures exhibit an increase in endocytosis while the EC-DC cultures show a significant decrease in endocytosis. Analysis of the cytokine profiles shows that the expression of IL1-beta, IL-6, IL-10, IL-12p40 and IL-13 increase in EC-DC cultures as compared to DC alone. When pre-stimulated with poly I:C, CSC enhances expression of IL-10, IL-12p40 and decreases expression of IL-13 in EC-DC co-cultures. CSC exposure in both DC and EC-DC cultures have minimal effects on viability. Both doses of CSC reduce CD80, CD83, CD86 maturation markers, including CD54 (ICAM-1). DC cultures exhibit an increase in endocytosis while the EC-DC cultures show a significant decrease in endocytosis. Analysis of the cytokine profiles shows that the expression of IL1-beta, IL-6, IL-10, IL-12p40 and IL-13 increase in EC-DC cultures as compared to DC alone. When pre-stimulated with poly I:C, CSC enhances expression of IL-10, IL-12p40 and decreases expression of IL-13 in EC-DC co-cultures. ConclusionCSC modulates EC-DC cross-talk by inducing production of cytokines that do not affect DC maturation but affect their function. CSC modulates EC-DC cross-talk by inducing production of cytokines that do not affect DC maturation but affect their function.

Green tea polyphenol EGCG suppresses cigarette smoke condensate-induced NF-κB activation in normal human bronchial epithelial cells

Cigarette smoke is a powerful inducer of inflammatory responses resulting in disruption of major cellular pathways with transcriptional and genomic alterations driving the cells towards carcinogenesis. Cell culture and animal model studies indicate that (-)-epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent anti-inflammatory and antiproliferative activity capable of selectively inhibiting cell growth and inducing apoptosis in cancer cells without adversely affecting normal cells. Here, we demonstrate that EGCG pretreatment (20-80 microM) of normal human bronchial epithelial cells (NHBE) resulted in significant inhibition of cigarette smoke condensate (CSC)-induced cell proliferation. Nuclear factor-kappaB (NF-kappaB) controls the transcription of genes involved in immune and inflammatory responses. In most cells, NF-kappaB prevents apoptosis by mediating cell survival signals. Pretreatment of NHBE cells with EGCG suppressed CSC-induced phosphorylation of IkappaBalpha, and activation and nuclear translocation of NF-kappaB/p65. NHBE cells transfected with a luciferase reporter plasmid containing an NF-kappaB-inducible promoter sequence showed an increased reporter activity after CSC exposure that was specifically inhibited by EGCG pretreatment. Immunoblot analysis showed that pretreatment of NHBE cells with EGCG resulted in a significant downregulation of NF-kappaB-regulated proteins cyclin D1, MMP-9, IL-8 and iNOS. EGCG pretreatment further inhibited CSC-induced phosphorylation of ERK1/2, JNK and p38 MAPKs and resulted in a decreased expression of PI3K, AKT and mTOR signaling molecules. Taken together, our data indicate that EGCG can suppress NF-kappaB activation as well as other pro-survival pathways such as PI3K/AKT/mTOR and MAPKs in NHBE cells, which may contribute to its ability to suppress inflammation, proliferation and angiogenesis induced by cigarette smoke.

Effect of Cigarette Smoke on the Activation of Transcription Factors in Mouse Lung

The effect of cigarette smoke on in vitro and in vivo transcription factor activation in the lung was examined. We first examined if NF-κB is activated by cigarette smoke condensate (CSC) in vitro. Using two different lung cell lines (H1299 and H-838), we did not observe activation of NF-κB following CSC exposure. We next examined if cigarette smoke could activate NF-κB in vivo. Mice were exposed to cigarette smoke for 1, 2, 3, or 6 hr, and 2, 5, 10, 15, 20, 42, or 56 days (6 hr/day, 5 days/wk). For all times except 20 and 42 days, the DNA binding activity of NF-κB in the lung and liver was either not changed or decreased by exposure to cigarette smoke. For the 20- and 42-day time points, the DNA binding activity in the lung was slightly increased. We also examined two other transcription factors: AP-1 and hypoxia inducible factor (HIF). Similar to NF-κB, the DNA binding activity of AP-1 and HIF was slightly elevated at 42 days but decreased at 56 days. These studies show that cigarette smoke does not activate NF-κB, AP-1 or HIF in the lung. Cigarette smoke activation of other transcription factors in the lung is currently being studied. (Supported by Kentucky Lung Cancer Research Program)

Kinetic analysis of cytokine response to cigarette smoke condensate by human endothelial and monocytic cells

Atherosclerosis is generally considered an inflammatory disease characterized by the accumulation of lipid in large and medium elastic arteries. Individuals who smoke are at increased risk for developing atherosclerosis and the clinical events associated with this disease. Underlying the mechanisms involved in atherosclerotic lesion development exists a complex pattern of signaling, involving molecules (cytokines and chemokines) that mediate the progression of arterial lesions. The unique nature of exposure to tobacco-related toxicants during the process of smoking prompted our investigation of the time-dependent responses of two critical cell types to cigarette smoke condensate exposure. In this study, we examined the kinetic responses, using suspension array technology and RT-PCR of 17 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17 GM-CSF, G-CSF, INF-γ, TNF-α, MCP-1 and MIP-1β) in human aortic endothelial cells (HAECs) and THP-1 monocyte macrophages following exposure to cigarette smoke condensate (CSC) for 24 h. In HAECs, IL-8 and IL-4 were rapidly stimulated by CSC exposure while, surprisingly, MCP-1 expression was downregulated. In THP-1 macrophages, IL-6, MIP-1β, MCP-1 and IL-1β protein expression were suppressed upon CSC exposure. All other measurable cytokines in THP-1 cells exposed to CSC had levels of protein and mRNA similar to controls. Depending on cell type, CSC uniquely influences the expression of cytokines. The complex interplay of these signaling molecules within the framework of atherosclerosis points to the ability of cigarette smoke components to alter such signaling following acute exposure, and by this mechanism may alter the course of both atherogenesis initiation and progression.

Exposure to tobacco-derived materials induces overproduction of secreted proteinases in mast cells

Mast cells reside at interfaces with the environment, including the mucosa of the respiratory and gastrointestinal tracts. This localization exposes mast cells to inhaled, or ingested, environmental challenges. In the airways of smokers, resident immune cells will be in contact with the condensed components of cigarette smoke. Mast cells are of particular interest due to their ability to promote airway remodeling and mucus hypersecretion. Clinical data show increased levels of mast cell-secreted tryptase and increased numbers of degranulated mast cells in the lavage and bronchial tissue of smokers. Since mast cell-secreted proteinases (MCPTs), including tryptases, contribute to pathological airway remodeling, we investigated the relationship between mast cell proteinases and smoke exposure. We exposed a mast cell line to cigarette smoke condensate (CSC). We show that CSC exposure increases MCPT levels in mast cells using an assay for tryptase-type MCPT activity. We hypothesized that this increase in MCPT activity reflects a CSC-induced increase in the cytosolic pool of proteinase molecules, via stimulation of MCPT transcription. Transcript array data suggested that mRNA changes in response to CSC were limited in number and peaked after 3 h of CSC exposure. However, we noted marked transcriptional regulation of several MCPT genes. CSC-induced changes in the mRNA levels for MCPTs were confirmed using quantitative RT-PCR. Taken together, our data suggest that chronic exposure to cigarette smoke up-regulates MCPT levels in mast cells at both the protein and the mRNA level. We suggest that the pathological airway remodeling that has been described in clinical studies of smoke inhalation may be attributable to MCPT overproduction in vivo.

MMP-1 polymorphic expression in aortic endothelial cells: possible role in lesion development in smokers and nonsmokers.

Matrix metalloproteinase-1 (MMP-1) is involved in the degradation of extra-cellular matrix components and is thought to play a key role in atherogenesis. The objective of the present study was to determine: (1) whether MMP-1 differential gene expression observed in primary cultures of human aortic endothelial cells (HAEC) exposed to cigarette smoke condensate (CSC) was associated with a single nucleotide polymorphism (SNP) in its promoter region and (2) if the SNP altered atherosclerotic lesion development in smokers and nonsmokers. Genotype analysis of six HAEC lines revealed that those homozygous for the 1G-variant exhibited low levels of gene expression. Cells homozygous for the 2G-variant or heterozygous (1G/2G) had elevated levels of both mRNA and protein. These relative levels were also seen after CSC expo-sure. MMP-1 genotypes of 104 Caucasian males acquired from the Pathobiological Determinants of Atherosclerosis in Youth study were compared with the extent of atherosclerotic lesion development in the aorta. The results indicate that, although the MMP-1 SNP influences MMP-1 gene expression in cultured HAEC exposed to CSC, differences in lesion development were not observed based on this polymorphism in young Caucasian males.

Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells.

BackgroundTo demonstrate the involvement of tobacco smoking in the pathophysiology of lung disease, the responses of pulmonary epithelial cells to cigarette smoke condensate (CSC) — the particulate fraction of tobacco smoke — were examined.MethodsThe human alveolar epithelial cell line A549 and normal human bronchial epithelial cells (NHBEs) were exposed to 0.4 μg/ml CSC, a concentration that resulted in >90% cell survival and <5% apoptosis. Changes in gene expression and signaling responses were determined by RT-PCR, western blotting and immunocytofluorescence.ResultsNHBEs exposed to CSC showed increased expression of the inflammatory mediators sICAM-1, IL-1β, IL-8 and GM-CSF, as determined by RT-PCR. CSC-induced IL-1β expression was reduced by PD98059, a blocker of mitogen-actived protein kinase (MAPK) kinase (MEK), and by PDTC, a NFκB inhibitor. Analysis of intracellular signaling pathways, using antibodies specific for phosphorylated MAPKs (extracellular signal-regulated kinase [ERK]-1/2), demonstrated an increased level of phosphorylated ERK1/2 with increasing CSC concentration. Nuclear localization of phosphorylated ERK1/2 was seen within 30 min of CSC exposure and was inhibited by PD98059. Increased phosphorylation and nuclear translocation of IκB was also seen after CSC exposure. A549 cells transfected with a luciferase reporter plasmid containing a NFκB-inducible promoter sequence and exposed to CSC (0.4 μg/ml) or TNF-α (50 ng/ml) had an increased reporter activity of approximately 2-fold for CSC and 3.5-fold for TNF-α relative to untreated controls.ConclusionThe acute phase response of NHBEs to cigarette smoke involves activation of both MAPK and NFκB.

Glutathione homeostasis in alveolar epithelial cells in vitro and lung in vivo under oxidative stress

We studied the acute effects of cigarette smoke condensate (CSC), H2O2, and tumor necrosis factor (TNF)-alpha on the glutathione (GSH) redox system in a human type II epithelial cell line (A549) in vitro. CSC, in vitro and in vivo after intratracheal instillation of CSC in the rat, produced a depletion of intracellular soluble GSH, concomitant with GSH-conjugate formation, without significant elevation of oxidized GSH (GSSG), protein-GSH mixed disulfides (PrSSG), nor any GSH efflux from the cells. By contrast, H2O2 (500 microM) after 5-min exposure to A549 cells caused significant depletion of intracellular GSH associated with an efflux of GSSG and a significant increase in the formation of PrSSG. TNF-alpha, in concentrations of 100 U/ml and 1,000 U/ml, produced a significant depletion of GSH in A549 cells after 4- and 24-h exposure, with an associated elevation of GSSG. The activities of glutathione peroxidase, gamma-glutamylcysteine synthetase, and glucose-6-phosphate dehydrogenase were significantly decreased in epithelial cells and in rat lungs after CSC exposure, without change in glutathione S-transferase and glutathione reductase activities. By contrast, H2O2 and TNF-alpha did not alter these enzyme activities in epithelial cells. Thus GSH depletion and alteration in enzyme activities in alveolar epithelial cells by CSC, H2O2, and TNF-alpha occur by different mechanisms.

Effect of cigarette smoke condensate and vitamin A depletion on keratin expression patterns in cultured hamster tracheal epithelium

Keratin expression in hamster tracheal epithelium was investigated during organ culture in serum-free, hormone-supplemented medium using monospecific monoclonal antibodies. Generally, tracheal basal cells expressed keratins detected by antibodies RCK102 and RCK103, while columnar epithelial cells were stained positively by RGE53, RCK103, RCK105 and HCK19. Metaplastic squamous cell foci reacted with antibodies RKSE60, RCK103 and HCK19. Early metaplastic alterations were more clearly RKSE60-positive than the mature lesions. In the vitamin A-depleted tracheas basal cells were clearly RCK102-positive. Superficial cells in the central part of areas of squamous metaplasia induced by cigarette smoke condensate expressed the basal cell keratins, and were negative for the columnar cell keratin 18 detected by the RGE53 antibody. This finding suggests that in cigarette smoke condensate-induced squamous metaplasia basal cells play an important role. The mucus-producing cells at the edges of metaplastic squamous cell foci expressed the keratins specific to columnar cells. Cigarette smoke condensate exposure accelerated epithelial keratinization compared to the vitamin A-depleted epithelium. It was concluded that not only small mucous granule cells, but also basal cells are involved in the development and maintenance of induced squamous metaplasia in tracheal epithelium. Furthermore, in vitro vitamin A-depleted epithelium did not coexpress vimentin in addition to the different keratins.

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. II. A histomorphological study.

The effects of all-trans retinol and cigarette smoke condensate (CSC) on tissue morphology and cellular differentiation were investigated in vitamin A-deprived tracheal epithelium cultured in vitamin A-and serum-free hormone-supplemented medium. Physiological retinol concentrations prevented the development of hyperplasia and squamous metaplasia with or without keratinization, and induced differentiation to mucous cells. Squamous metaplastic foci with keratinization were observed during 12 days of culture with low retinol concentrations and with dimethylsulfoxide (DMSO) which was accompanied by an increased number of basal and indeterminate cells. CSC induced a dose-related hyperplasia and irregularly shaped foci of squamous metaplasia with atypical epithelial proliferation. In non-metaplastic epithelium, CSC exposure increased the number of ciliated cells. Hyperplasia and squamous metaplasia were inhibited if the tracheal rings were first treated with retinol followed by CSC exposure, or if the tracheas were simultaneously treated with retinol and CSC. CSC-exposure prior to retinol treatment induced similar histomorphological alterations as CSC alone.

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. I. A cell proliferation study.

The effects of cigarette smoke condensate (CSC) and all-trans retinol on the cell proliferative activity of vitamin A-deprived hamster tracheal epithelium have been studied in vitamin A-deficient, serum-free, hormone-supplemented medium in organ culture. In the absence of retinol, CSC induced a dose-dependent increase in labeling index (LI) during 12 days of culture. The basal cells were more sensitive to CSC exposure than non-basal cells during the first 6 to 8 culture days. However, in squamous metaplastic foci developing after culture day 6, both basal and non-basal cells in the mid-part of the epithelium were labeled. Physiological concentrations of all-trans retinol stimulated the non-basal LI and inhibited the basal cell LI. Compared with dimethylsulfoxide (DMSO), all retinol concentrations used in the present study inhibited the basal cell LI at each time point examined (4-12 days culture). Exposure of tracheal rings to retinol, either before or after exposure to CSC, or simultaneous exposure to retinol and CSC, clearly decreased the CSC-induced basal cell proliferative activity depending on the retinol concentration used. It is concluded from the present study that squamous metaplasia induced by vitamin A-deficiency or by CSC originates mainly from basal cells and that for the maintenance of these lesions, both basal and non-basal cells play a role. Furthermore, all-trans retinol inhibited CSC-induced basal cell proliferation.