Search tips
Search criteria

Results 1-25 (1654033)

Clipboard (0)

Related Articles

1.  Protecting the BBB Endothelium against Cigarette Smoke-Induced Oxidative Stress Using Popular Antioxidants: Are they really beneficial? 
Brain research  2015;1627:90-100.
Blood Brain Barrier (BBB) exposed to realistic concentrations (comparable to a chronic heavy smoker) of Cigarette Smoke Extract (CSE) triggers a strong endothelial inflammatory which can lead to the onset of neurological disorders. The involvement of Reactive Oxygen Species (ROS) in this inflammatory cascade is evident from the up-regulation of nuclear factor erythroid 2 related factor 2 (Nrf-2), a transcription factor involved in anti-oxidant response signaling in CSE exposed endothelial cells. We have shown that pre-treatment with α-tocopherol and/or ascorbic acid is highly protective for the BBB, thus suggesting that, prophylactic administration of antioxidants can reduce CSE and/or inflammatory-dependent BBB damage. We have assessed and ranked the protective effects of 5 popular OTC antioxidants (Coenzyme Q10, Melatonin, Glutathione, Lipoic acid and Resveratrol) against CSE-induced BBB endothelial damage using hCMEC/D3 cells. The analysis of pro-inflammatory cytokines release by ELISA revealed that, resveratrol, lipoic acid melatonin and Co-Q10 inhibited the BBB endothelial release of pro-inflammatory cytokines IL-6 & IL-8, reduced (not Co-Q10) CSE-induced up-regulation of Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1), Vascular Endothelial Cell Adhesion Molecule-1 (VCAM-1) & E-selectin and inhibited monocytes-endothelial cell adhesion. The anti-inflammatory effects correlated with the anti-oxidative protection endowed by these compounds as evidenced by upregulation of NADPH: Quinone Oxidoreductase 1 (NQO1) and reduced cellular oxidative stress. CSE-induced release of Vascular Endothelial Growth Factor (VEGF) was inhibited by all tested compounds although the effect was not strictly dose-dependent. Further in vivo studies are required to validate our results and expand our current study to include combinatorial treatments.
PMCID: PMC4636922  PMID: 26410779
Blood-Brain Barrier; Smoking; Oxidative stress; Inflammation; Countermeasure; Antioxidant; Alternative
2.  Cigarette Smoke–Induced CXCR3 Receptor Up-Regulation Mediates Endothelial Apoptosis 
Endothelial monocyte–activating polypeptide II (EMAP II) and interferon-inducible protein (IP)–10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke–exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke–induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II–induced and IP-10–induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II–induced and IP-10–induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II–induced and IP-10–induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II–induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema.
PMCID: PMC3547093  PMID: 22936405
CXCR3; endothelial; apoptosis; lung; emphysema
3.  Cigarette smoke but not electronic cigarette aerosol activates a stress response in human coronary artery endothelial cells in culture 
Drug and Alcohol Dependence  2016;163:256-260.
•Human coronary artery endothelial cells show a biological response to cigarette smoke.•This response was not seen following exposure to e-cigarette aerosol.•Using e-cigarettes instead of cigarettes may reduce immediate cardiovascular harms.
It is generally acknowledged that e-cigarettes are unlikely to be as harmful as conventional cigarettes, but there is little data that quantifies their relative harms. We investigated the biological response to e-cigarette aerosol exposure (versus conventional cigarette smoke exposure) at the cellular level, by exposing human coronary artery endothelial cells (HCAEC) to aqueous filtered extracts of e-cigarette aerosol or cigarette smoke and looking at gene expression changes consistent with a stress response. This included genes controlled by the oxidant-stress sensing transcription factor NFR2 (NFE2L2), and cytochrome P450 family members.
Cigarette smoke extract (CSE) was created using mainstream smoke from a single cigarette drawn through 10 ml of endothelial cell growth media MV2. Electronic cigarette aerosol extract (eCAE) was created using the same apparatus, using a constant power output of 10.8 w (4.2 V) and 18 mg/ml nicotine solution. eCAE was generated using 5 cycles of 5 s heat with at least 10 s in between each puff to allow the coil to cool, air being drawn through the device at 70 ml/minute.
HCAEC responded to the noxious components in CSE, resulting in activation of NRF2 and upregulation of cytochrome p450. However, eCAE did not induce NRF2 nuclear localisation, upregulation of NRF2-activated genes, or the upregulation of cytochrome p450.
The use of e-cigarettes as a substitute for conventional cigarettes is likely to reduce immediate tobacco-related harm, at least with respect to cardiovascular harms.
PMCID: PMC4907307  PMID: 27137404
Cigarettes; E-cigarettes; Human coronary artery cells; Stress response
4.  Inflammatory Transcriptome Profiling of Human Monocytes Exposed Acutely to Cigarette Smoke 
PLoS ONE  2012;7(2):e30120.
Cigarette smoking is responsible for 5 million deaths worldwide each year, and is a major risk factor for cardiovascular and lung diseases. Cigarette smoke contains a complex mixture of over 4000 chemicals containing 1015 free radicals. Studies show smoke is perceived by cells as an inflammatory and xenobiotic stimulus, which activates an immune response. The specific cellular mechanisms driving cigarette smoke-induced inflammation and disease are not fully understood, although the innate immune system is involved in the pathology of smoking related diseases.
Methodology/Principle findings
To address the impact of smoke as an inflammagen on the innate immune system, THP-1 cells and Human PBMCs were stimulated with 3 and 10% (v/v) cigarette smoke extract (CSE) for 8 and 24 hours. Total RNA was extracted and the transcriptome analysed using Illumina BeadChip arrays. In THP-1 cells, 10% CSE resulted in 80 genes being upregulated and 37 downregulated by ≥1.5 fold after 8 hours. In PBMCs stimulated with 10% CSE for 8 hours, 199 genes were upregulated and 206 genes downregulated by ≥1.5 fold. After 24 hours, the number of genes activated and repressed by ≥1.5 fold had risen to 311 and 306 respectively. The major pathways that were altered are associated with cell survival, such as inducible antioxidants, protein chaperone and folding proteins, and the ubiquitin/proteosome pathway.
Our results suggest that cigarette smoke causes inflammation and has detrimental effects on the metabolism and function of innate immune cells. In addition, THP-1 cells provide a genetically stable alternative to primary cells for the study of the effects of cigarette smoke on human monocytes.
PMCID: PMC3281820  PMID: 22363418
5.  Cellular and molecular mechanisms of cigarette smoke-induced lung damage and prevention by vitamin C 
Cigarette smoke-induced cellular and molecular mechanisms of lung injury are not clear. Cigarette smoke is a complex mixture containing long-lived radicals, including p-benzosemiquinone that causes oxidative damage. Earlier we had reported that oxidative protein damage is an initial event in smoke-induced lung injury. Considering that p-benzosemiquinone may be a causative factor of lung injury, we have isolated p-benzosemiquinone and compared its pathophysiological effects with cigarette smoke. Since vitamin C is a strong antioxidant, we have also determined the modulatory effect of vitamin C for preventing the pathophysiological events.
Vitamin C-restricted guinea pigs were exposed to cigarette smoke (5 cigarettes/day; 2 puffs/cigarette) for 21 days with and without supplementation of 15 mg vitamin C/guinea pig/day. Oxidative damage, apoptosis and lung injury were assessed in vitro, ex vivo in A549 cells as well as in vivo in guinea pigs. Inflammation was measured by neutrophilia in BALF. p-Benzosemiquinone was isolated from freshly prepared aqueous extract of cigarette smoke and characterized by various physico-chemical methods, including mass, NMR and ESR spectroscopy. p-Benzosemiquinone-induced lung damage was examined by intratracheal instillation in guinea pigs. Lung damage was measured by increased air spaces, as evidenced by histology and morphometric analysis. Oxidative protein damage, MMPs, VEGF and VEGFR2 were measured by western blot analysis, and formation of Michael adducts using MALDI-TOF-MS. Apoptosis was evidenced by TUNEL assay, activation of caspase 3, degradation of PARP and increased Bax/Bcl-2 ratio using immunoblot analysis and confocal microscopy.
Exposure of guinea pigs to cigarette smoke resulted in progressive protein damage, inflammation, apoptosis and lung injury up to 21 days of the experimental period. Administration of 15 mg of vitamin C/guinea pig/day prevented all these pathophysiological effects. p-Benzosemiquinone mimicked cigarette smoke in causing protein modification and apoptosis in vitro and in A549 cells ex vivo as well as apoptosis and lung damage in vivo. All these pathophysiological events were also prevented by vitamin C.
p-Benzosemiquinone appears to be a major causative factor of cigarette smoke-induced oxidative protein damage that leads to apoptosis and lung injury. The pathophysiological events are prevented by a moderately large dose of vitamin C.
PMCID: PMC2615750  PMID: 19014449
6.  Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells 
Pleiotrophin (PTN) is a cytokine that is expressed by monocytes/macrophages in ischemic tissues and that promotes neovascularization, presumably by stimulating proliferation of local endothelial cells. However, the effect of PTN on monocytes/macrophages remains unknown. We investigated the role of PTN in regulating the phenotype of monocytes/macrophages.
Methods and Results
RT-PCR, real-time PCR, and fluorescence-activated cell sorter analysis revealed that the expression of PTN by monocytic cells led to a downregulation of CD68, c-fms, and CD14 monocytic cell markers and an upregulation of FLK-1, Tie-2, vascular endothelial-cadherin, platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor, CD34, GATA-2, and GATA-3 endothelial cell markers. Fibrin gel assays showed that the treatment of mouse and human monocytic cells with PTN led to the formation of tube-like structures. In vivo studies showed that PTN-expressing monocytic cells incorporated into the blood vessels of the quail chorioallantoic membrane. The intracardial injection of PTN-expressing monocytic cells into chicken embryos showed that cells integrated only into the developing vasculature. Finally, the injection of PTN-expressing monocytes into a murine ischemic hindlimb model significantly improved perfusion of the ischemic tissue.
PTN expression by monocytes/macrophages led to a downregulation of their monocytic cell markers and an upregulation of endothelial cell characteristics, thus inducing the transdifferentiation of monocytes into functional endothelial cells.
PMCID: PMC3579570  PMID: 16614316
transdifferentiation; pleiotrophin; macrophage; endothelial cell
7.  Extracts from presumed “reduced harm” cigarettes induce equivalent or greater toxicity in antigen-presenting cells 
Toxicology  2015;335:46-54.
The tobacco industry has promoted certain cigarette products with claims that their use may be less harmful to the smoker as they purportedly deliver lower amounts of toxic chemicals compared to conventional cigarettes. This study was designed to compare the relative antigen presenting cellular toxicity of Eclipse, a presumed reduced exposure product (PREP) cigarette, when compared with the reference research 3R4F cigarettes (Kentucky University). Utilizing a murine macrophage cell line, murine bone marrow derived dendritic cells (DCs) and human monocyte-derived DCs incubated with extracts generated from Eclipse and Kentucky reference 3R4F cigarettes, we determined the relative toxic effects of the different cigarette smoke extracts on cellular viability, oxidative stress, T-helper-1 (Th-1) polarizing cytokine production and general gene expression. Eclipse and 3R4F cigarette smoke extracts induced equivalent oxidatively-mediated cellular heme oxygenase-1 (HO-1) protein levels in macrophages and DCs. Cellular viability determination demonstrated greater induction of cell death by apoptosis and necrosis by Eclipse extracts in DCs. The production of the key Th-1 polarizing cytokine interleukin-12 (IL-12) by activated DCs or macrophages was suppressed to an equivalent or greater extent by Eclipse extracts. Microarray studies performed on bone marrow derived murine DCs incubated with Eclispe or 3R4F cigarette extracts showed identical genotoxic profiles. These studies imply that presumed reduced harm Eclipse cigarettes induce equivalent or greater antigen presenting cell dysfunction relative to 3R4F cigarettes and illustrate the importance of independent validation and testing of similar products claimed to be associated with reduced toxicity relative to other cigarettes.
PMCID: PMC4532619  PMID: 26169828
cigarette smoke extract; antigen presenting cell; gene array; Eclipse; dendritic cell
8.  Nicotine-mediated induction of E-selectin in aortic endothelial cells requires Src kinase and E2F1 transcriptional activity 
Smoking is highly correlated with enhanced likelihood of atherosclerosis by inducing endothelial dysfunction. In endothelial cells, various cell-adhesion molecules including E-selectin, are shown to be upregulated upon exposure to nicotine, the addictive component of tobacco smoke; however, the molecular mechanisms underlying this induction are poorly understood. Here we demonstrate that nicotine induced E-selectin transcription in human aortic endothelial cells (HAECs) could be significantly blocked by α7-nAChR subunit inhibitor, α-BT, Src-kinase inhibitor, PP2, or siRNAs against Src or β-Arrestin-1 (β-Arr1). Further, chromatin immunoprecipitations show that E-selectin is an E2F1 responsive gene and nicotine stimulation results in increased recruitment of E2F1 on E-selectin promoter. Inhibiting E2F1 activity using RRD-251, a disruptor of the Rb-Raf-1 kinase interaction, could significantly inhibit the nicotine induced recruitment of E2F1 to the E-selectin promoter as well as E-selectin expression. Interestingly, stimulation of HAECs with nicotine results in increased adhesion of U937 monocytic cells to HAECs and could be inhibited by pre-treatment with RRD-251. Similarly, depletion of E2F1 or Src using RNAi blocked the increased adhesion of monocytes to nicotine stimulated HAECs. These results suggest that nicotine stimulated adhesion of monocytes to endothelial cells is dependent on the activation of α7-nAChRs, β-Arr1 and cSrc regulated increase in E2F1-mediated transcription of E-selectin gene. Therefore, agents such as RRD-251 that can target activity of E2F1 may have potential therapeutic benefit against cigarette-smoke induced atherosclerosis.
PMCID: PMC3273677  PMID: 22240023
E-selectin; atherosclerosis; monocyte adhesion; RRD-251; β-arrestin-1; Src; Rb
9.  Vapours of US and EU Market Leader Electronic Cigarette Brands and Liquids Are Cytotoxic for Human Vascular Endothelial Cells 
PLoS ONE  2016;11(6):e0157337.
The present study was conducted to provide toxicological data on e-cigarette vapours of different e-cigarette brands and liquids from systems viewed as leaders in the e-cigarette market and to compare e-cigarette vapour toxicity to the toxicity of conventional strong high-nicotine cigarette smoke. Using an adapted version of a previously constructed cigarette smoke constituent sampling device, we collected the hydrophilic fraction of e-cigarette vapour and exposed human umbilical vein endothelial cells (HUVECs) to the mixture of compounds present in the vapour of 4 different single-use e-cigarettes, 6 different liquid vapours produced by the same refillable e-cigarette, and one e-cigarette with an exchangeable liquid cartridge. After incubation of cells with various concentrations and for various periods of time we analysed cell death induction, proliferation rates, the occurrence of intra-cellular reactive oxygen species, cell morphology, and we also measured e-cigarette heating coil temperatures. Overall, conventional cigarette smoke extract showed the most severe impact on endothelial cells. However, some e-cigarette vapour extracts showed high cytotoxicity, inhibition of cell proliferation, and alterations in cell morphology, which were comparable to conventional high-nicotine cigarettes. The vapours generated from different liquids using the same e-cigarette show substantial differences, pointing to the liquids as an important source for toxicity. E-cigarette vapour-mediated induction of oxidative stress was significant in one out of the 11 analysed vapours. There is a high variability in the acute cytotoxicity of e-cigarette vapours depending on the liquid and on the e-cigarettes used. Some products showed toxic effects close to a conventional high-nicotine cigarette. Liquid nicotine, menthol content, and the formation of acute intracellular reactive oxygen species do not seem to be the central elements in e-cigarette vapour toxicity.
PMCID: PMC4924852  PMID: 27351725
10.  Dynamics of heat shock protein 60 in endothelial cells exposed to cigarette smoke extract 
Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the “HSP60 pathway.” It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the “auto-immune hypothesis of atherosclerosis.”
► Cigarette smoke alters the structure and function of mitochondria. ► Cigarette smoke potently induces HSP60 expression and translocation. ► Secondhand smokers are particularly prone to cigarette smoke-induced atherosclerosis.
PMCID: PMC3190135  PMID: 21798264
Cigarette smoking; Heat shock protein 60; Atherosclerosis; Autoimmunity; Live cell imaging
11.  Paracrine Potential of Fibroblasts Exposed to Cigarette Smoke Extract With Vascular Growth Factor Induction 
The Laryngoscope  2013;123(9):2228-2236.
Nicotine, a major constituent of cigarette smoke, can activate the cholinergic anti-inflammatory pathway by binding to α7-nicotinic acetylcholine receptor (α7nAChR) expressed on the surface of certain cells. Here, we ask whether cigarette smoke extract induced different paracrine factors compared to the in vivo regulator of inflammation, tumor necrosis factor-α, in human vocal fold fibroblasts (hVFFs) shown to express low levels of α7nAChR.
Study Design
In vitro.
α7nAChR was detected by nested polymerase chain reaction and immunohistochemistry. γH2AX, a marker for DNA double-stand breaks, was measured by immunofluorescence. Cigarette smoke extract was prepared in accordance with investigators studying effects of cigarette smoke. hVFFs treated for 3 hours had media replaced for an additional 24 hours. Cytokine, chemokine, and growth factor levels in media were assessed by multiplex analysis.
α7nAChR expression levels decreased with the passage number of fibroblasts. Tumor necrosis factor-α induced a significantly different profile of cytokines, chemokines, and growth factor compared to cigarette smoke extract exposure. Cigarette smoke extract at a concentration not associated with induction of γH2AX nuclear foci significantly increased vascular endothelial growth factor.
Cigarette smoke extract elicited a response important for regulation of angiogenesis and vascular permeability during inflammation, without evidence of DNA double-stand breaks associated with carcinogenesis. hVFFs are capable of participating in paracrine regulation of pathological blood vessel formation associated with cigarette smoking–related diseases (ie, Reinke edema). These cells express α7nAChR, an essential component of the cholinergic anti-inflammatory pathway regulated by the vagus nerve in certain tissues and a target of therapeutic agents.
PMCID: PMC4113205  PMID: 23494588
Vocal fold fibroblasts; cigarette smoke extract; γH2AX foci; vascular endothelial growth factor
12.  Potential proinflammatory effects of hydroxyapatite nanoparticles on endothelial cells in a monocyte–endothelial cell coculture model 
Currently, synthetic hydroxyapatite nanoparticles (HANPs) are used in nanomedicine fields. The delivery of nanomedicine to the bloodstream exposes the cardiovascular system to a potential threat. However, the possible adverse cardiovascular effects of HANPs remain unclear. Current observations using coculture models of endothelial cells and monocytes with HANPs to mimic the complex physiological functionality of the vascular system demonstrate that monocytes could play an important role in the mechanisms of endothelium dysfunction induced by the exposure to HANPs. Our transmission electron microscopy analysis revealed that both monocytes and endothelial cells could take up HANPs. Moreover, our findings demonstrated that at a subcytotoxic dose, HANPs alone did not cause direct endothelial cell injury, but they did induce an indirect activation of endothelial cells, resulting in increased interleukin-6 production and elevated adhesion molecule expression after coculture with monocytes. The potential proinflammatory effect of HANPs is largely mediated by the release of soluble factors from the activated monocytes, leading to an inflammatory response of the endothelium, which is possibly dependent on p38/c-Jun N-terminal kinase, and nuclear factor-kappa B signaling activation. The use of in vitro monocyte–endothelial cell coculture models for the biocompatibility assessment of HANPs could reveal their potential proinflammatory effects on endothelial cells, suggesting that exposure to HANPs possibly increases the risk of cardiovascular disease.
PMCID: PMC3956627  PMID: 24648726
coculture; monocytes; endothelial cells; inflammation; hydroxyapatite nanoparticles
13.  Cigarette smoke induces β2-integrin-dependent neutrophil migration across human endothelium 
Respiratory Research  2011;12(1):75.
Cigarette smoking induces peripheral inflammatory responses in all smokers and is the major risk factor for neutrophilic lung disease such as chronic obstructive pulmonary disease. The aim of this study was to investigate the effect of cigarette smoke on neutrophil migration and on β2-integrin activation and function in neutrophilic transmigration through endothelium.
Methods and results
Utilizing freshly isolated human PMNs, the effect of cigarette smoke on migration and β2-integrin activation and function in neutrophilic transmigration was studied. In this report, we demonstrated that cigarette smoke extract (CSE) dose dependently induced migration of neutrophils in vitro. Moreover, CSE promoted neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that Mac-1 (CD11b/CD18) is responsible for the cigarette smoke-induced firm adhesion of neutrophils to fibrinogen. Furthermore, neutrophils transmigrated through endothelium by cigarette smoke due to the activation of β2-integrins, since pre-incubation of neutrophils with functional blocking antibodies against CD11b and CD18 attenuated this transmigration.
This is the first study to describe that cigarette smoke extract induces a direct migratory effect on neutrophils and that CSE is an activator of β2-integrins on the cell surface. Blocking this activation of β2-integrins might be an important target in cigarette smoke induced neutrophilic diseases.
PMCID: PMC3128861  PMID: 21651795
14.  A Novel Anti-Inflammatory and Pro-Resolving Role for Resolvin D1 in Acute Cigarette Smoke-Induced Lung Inflammation 
PLoS ONE  2013;8(3):e58258.
Cigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation.
Primary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1β or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed.
RvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure.
RvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants.
PMCID: PMC3590122  PMID: 23484005
15.  The Economic Impact of Smoking and of Reducing Smoking Prevalence: Review of Evidence 
Tobacco Use Insights  2015;8:1-35.
Tobacco smoking is the cause of many preventable diseases and premature deaths in the UK and around the world. It poses enormous health- and non-health-related costs to the affected individuals, employers, and the society at large. The World Health Organization (WHO) estimates that, globally, smoking causes over US$500 billion in economic damage each year.
This paper examines global and UK evidence on the economic impact of smoking prevalence and evaluates the effectiveness and cost effectiveness of smoking cessation measures.
Search methods
We used two major health care/economic research databases, namely PubMed and the National Institute for Health Research (NIHR) database that contains the British National Health Service (NHS) Economic Evaluation Database; Cochrane Library of systematic reviews in health care and health policy; and other health-care-related bibliographic sources. We also performed hand searching of relevant articles, health reports, and white papers issued by government bodies, international health organizations, and health intervention campaign agencies.
Selection criteria
The paper includes cost-effectiveness studies from medical journals, health reports, and white papers published between 1992 and July 2014, but included only eight relevant studies before 1992. Most of the papers reviewed reported outcomes on smoking prevalence, as well as the direct and indirect costs of smoking and the costs and benefits of smoking cessation interventions. We excluded papers that merely described the effectiveness of an intervention without including economic or cost considerations. We also excluded papers that combine smoking cessation with the reduction in the risk of other diseases.
Data collection and analysis
The included studies were assessed against criteria indicated in the Cochrane Reviewers Handbook version 5.0.0.
Outcomes assessed in the review
Primary outcomes of the selected studies are smoking prevalence, direct and indirect costs of smoking, and the costs and benefits of smoking cessation interventions (eg, “cost per quitter”, “cost per life year saved”, “cost per quality-adjusted life year gained,” “present value” or “net benefits” from smoking cessation, and “cost savings” from personal health care expenditure).
The main findings of this study are as follows: The costs of smoking can be classified into direct, indirect, and intangible costs. About 15% of the aggregate health care expenditure in high-income countries can be attributed to smoking. In the US, the proportion of health care expenditure attributable to smoking ranges between 6% and 18% across different states. In the UK, the direct costs of smoking to the NHS have been estimated at between £2.7 billion and £5.2 billion, which is equivalent to around 5% of the total NHS budget each year. The economic burden of smoking estimated in terms of GDP reveals that smoking accounts for approximately 0.7% of China’s GDP and approximately 1% of US GDP. As part of the indirect (non-health-related) costs of smoking, the total productivity losses caused by smoking each year in the US have been estimated at US$151 billion.The costs of smoking notwithstanding, it produces some potential economic benefits. The economic activities generated from the production and consumption of tobacco provides economic stimulus. It also produces huge tax revenues for most governments, especially in high-income countries, as well as employment in the tobacco industry. Income from the tobacco industry accounts for up to 7.4% of centrally collected government revenue in China. Smoking also yields cost savings in pension payments from the premature death of smokers.Smoking cessation measures could range from pharmacological treatment interventions to policy-based measures, community-based interventions, telecoms, media, and technology (TMT)-based interventions, school-based interventions, and workplace interventions.The cost per life year saved from the use of pharmacological treatment interventions ranged between US$128 and US$1,450 and up to US$4,400 per quality-adjusted life years (QALYs) saved. The use of pharmacotherapies such as varenicline, NRT, and Bupropion, when combined with GP counseling or other behavioral treatment interventions (such as proactive telephone counseling and Web-based delivery), is both clinically effective and cost effective to primary health care providers.Price-based policy measures such as increase in tobacco taxes are unarguably the most effective means of reducing the consumption of tobacco. A 10% tax-induced cigarette price increase anywhere in the world reduces smoking prevalence by between 4% and 8%. Net public benefits from tobacco tax, however, remain positive only when tax rates are between 42.9% and 91.1%. The cost effectiveness ratio of implementing non-price-based smoking cessation legislations (such as smoking restrictions in work places, public places, bans on tobacco advertisement, and raising the legal age of smokers) range from US$2 to US$112 per life year gained (LYG) while reducing smoking prevalence by up to 30%–82% in the long term (over a 50-year period).Smoking cessation classes are known to be most effective among community-based measures, as they could lead to a quit rate of up to 35%, but they usually incur higher costs than other measures such as self-help quit-smoking kits. On average, community pharmacist-based smoking cessation programs yield cost savings to the health system of between US$500 and US$614 per LYG.Advertising media, telecommunications, and other technology-based interventions (such as TV, radio, print, telephone, the Internet, PC, and other electronic media) usually have positive synergistic effects in reducing smoking prevalence especially when combined to deliver smoking cessation messages and counseling support. However, the outcomes on the cost effectiveness of TMT-based measures have been inconsistent, and this made it difficult to attribute results to specific media. The differences in reported cost effectiveness may be partly attributed to varying methodological approaches including varying parametric inputs, differences in national contexts, differences in advertising campaigns tested on different media, and disparate levels of resourcing between campaigns. Due to its universal reach and low implementation costs, online campaign appears to be substantially more cost effective than other media, though it may not be as effective in reducing smoking prevalence.School-based smoking prevalence programs tend to reduce short-term smoking prevalence by between 30% and 70%. Total intervention costs could range from US$16,400 to US$580,000 depending on the scale and scope of intervention. The cost effectiveness of school-based programs show that one could expect a saving of approximately between US$2,000 and US$20,000 per QALY saved due to averted smoking after 2–4 years of follow-up.Workplace-based interventions could represent a sound economic investment to both employers and the society at large, achieving a benefit–cost ratio of up to 8.75 and generating 12-month employer cost savings of between $150 and $540 per nonsmoking employee. Implementing smoke-free workplaces would also produce myriads of new quitters and reduce the amount of cigarette consumption, leading to cost savings in direct medical costs to primary health care providers. Workplace interventions are, however, likely to yield far greater economic benefits over the long term, as reduced prevalence will lead to a healthier and more productive workforce.
We conclude that the direct costs and externalities to society of smoking far outweigh any benefits that might be accruable at least when considered from the perspective of socially desirable outcomes (ie, in terms of a healthy population and a productive workforce). There are enormous differences in the application and economic measurement of smoking cessation measures across various types of interventions, methodologies, countries, economic settings, and health care systems, and these may have affected the comparability of the results of the studies reviewed. However, on the balance of probabilities, most of the cessation measures reviewed have not only proved effective but also cost effective in delivering the much desired cost savings and net gains to individuals and primary health care providers.
PMCID: PMC4502793  PMID: 26242225
smoking prevalence; economic impact; smoking cessation; effectiveness; cost effectiveness; cost–benefit analysis
16.  Modulation of gene expression in endothelial cells by hyperlipaemic postprandial serum from healthy volunteers 
Genes & Nutrition  2010;5(3):263-274.
A single high-fat challenge induces plasmatic pro-inflammatory and pro-oxidative responses in the postprandial state, even in healthy men. This period is also associated with vascular endothelial dysfunction, which is an early event in the development of cardiovascular diseases. However, knowledge about the mechanisms involved in postprandial hyperlipaemia-induced endothelial dysfunction is sparse. An objective of our study was to characterize the behaviour and gene expression of vascular endothelial cells exposed to postprandial hyperlipaemic sera. Human umbilical vein endothelial cells (HUVECs) were cultured in media containing 10% serum from healthy men withdrawn either before or 4 h after a high-fat challenge. Endothelial cell proliferation, adhesion and migration were then assessed. The transcriptomic profiles of endothelial cells exposed to pre and postprandial sera were also compared. Exposure to postprandial hyperlipaemic sera significantly decreased HUVEC proliferation when compared to preprandial serum (P < 0.0001), while no changes in migration or endothelial/monocyte interactions were observed. The transcriptomic analysis revealed changes in the expression of 675 genes, of which 431 have a known function. Among them, a set of differentially expressed genes was linked to cell cycle regulation and apoptosis and are regulated in favour of cell cycle arrest or death. This result was confirmed by measuring the induction of apoptosis after postprandial sera exposure (P = 0.011). Taken together, the transcriptomic results and pathway analysis showed that postprandial serum promotes apoptosis in HUVECs, potentially through the activation of the p53 network. We conclude that upon postprandial serum exposure, vascular endothelial cells transcriptionally regulate genes involved in the control of cell cycle and death to favour growth arrest and apoptosis. These findings support the hypothesis that postprandial hyperlipaemia is associated with vascular dysfunction and offer new insights into the mechanisms involved.
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-010-0166-x) contains supplementary material, which is available to authorized users.
PMCID: PMC2935529  PMID: 21052530
Postprandial hyperlipaemia; Vascular endothelial cells; Nutrigenomics; Apoptosis atherogenesis
17.  An Analysis of the Role of Tobacco-Specific Nitrosamines in the Carcinogenicity of Tobacco Smoke 
Cigarette smoke is a complex mixture consisting of more than 4500 chemicals, including several tobacco-specific nitrosamines (TSNA). TSNA typically form in tobacco during the post-harvest period, with some fraction being transferred into mainstream smoke when a cigarette is burned during use. The most studied of the TSNA is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK has been shown to be carcinogenic in laboratory animals. Studies examining the carcinogenicity of NNK frequently are conducted by injecting rodents with a single dose of 2.5 to 10 μmol of pure NNK; the amount of NNK contained in all of the mainstream smoke from about 3700 to 14,800 typical U.S. cigarettes. Extrapolated to a 70-kg smoker, the carcinogenic dose of pure NNK administered to rodents would be equivalent to the amount of NNK in all of the mainstream smoke of 22 to 87 million typical U.S. cigarettes. Furthermore, extrapolating results from rodent studies based on a single injection of pure NNK to establish a causative role for NNK in the carcinogenicity of chronic tobacco smoke exposure in humans is not consistent with basic pharmacological and toxicological principles. For example, such an approach fails to consider the effect of other smoke constituents upon the toxicity of NNK. In vitro studies demonstrate that nicotine, cotinine, and aqueous cigarette “tar” extract (ACTE) all inhibit the mutagenic activity of NNK. In vivo studies reveal that the formation of pulmonary DNA adducts in mice injected with NNK is inhibited by the administration of cotinine and mainstream cigarette smoke. Cigarette smoke has been shown to modulate the metabolism of NNK, providing a mechanism for the inhibitory effects of cigarette smoke and cigarette smoke constituents on NNK-induced tumorigenesis. NNK-related pulmonary DNA adducts have not been detected in rodents exposed to cigarette smoke, nor has the toxicity of tobacco smoke or tobacco smoke condensate containing marked reductions in TSNA concentrations been shown to be reduced in any biological assay. In summary, there is no experimental evidence to suggest that reduction of TSNA will reduce the mutagenic, cytotoxic, or carcinogenic potential of tobacco smoke.
PMCID: PMC2651603  PMID: 19330121
mainstream cigarette smoke; tobacco-specific nitrosamines; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
18.  Modeling the Health Effects of Expanding e-Cigarette Sales in the United States and United Kingdom 
JAMA internal medicine  2015;175(10):1671-1680.
The prevalence of electronic cigarette (e-cigarette) use is increasing. Population health effects will depend on cigarette smoking behaviors, levels of dual use with conventional cigarettes, and e-cigarette toxicity.
To evaluate potential health effects of various scenarios of increasing promotion and use of e-cigarettes.
A base case model was developed using data on actual cigarette and e-cigarette use patterns that quantifies transitions from an initial state of no cigarette or e-cigarette use to 1 of 5 final states: never use of cigarettes or e-cigarettes, cigarette use, e-cigarette use, dual use of cigarettes and e-cigarettes, or quit. Seven scenarios were created that cover a range of use patterns, depending on how the e-cigarette market might develop, as well as a range of possible long-term health effects of e-cigarette use. Scenarios for changes from the base case were evaluated using Monte Carlo simulations. Separate sets of base case model parameters were evaluated for the US and UK populations.
We assigned unitless health “costs” for each final state on a scale of 0 to 100. Population health “costs” were compared with the base case (status quo) assuming e-cigarette use health “costs” from 1% to 50% as dangerous as conventional cigarette use health costs.
Compared with the base case, a harm reduction scenario in which e-cigarette use increases only among smokers who are interested in quitting with more quit attempts and no increased initiation of e-cigarette use among nonsmokers, and another scenario in which e-cigarettes are taken up only by youth who would have smoked conventional cigarettes, had population-level health benefits regardless of e-cigarette health costs in both the United States and United Kingdom. Conversely, scenarios in which e-cigarette promotion leads to renormalization of cigarette smoking or e-cigarettes are used primarily by youth who never would have smoked showed net health harms across all e-cigarette health costs. In other scenarios, the net health effect varied on the basis of the health cost of e-cigarettes.
According to this analysis, widespread promotion of e-cigarettes may have a wide range of population-level health effects, depending on both e-cigarette health risks and patterns of use. Absent the primary effect of e-cigarette promotion being only to divert current or future conventional cigarette smokers to e-cigarette use, the current uncertainty about the health risks of e-cigarettes, increasing e-cigarette use among youth, and the varying health effects at different e-cigarette health costs suggest a potential for harm.
PMCID: PMC4594196  PMID: 26322924
19.  Cigarette smoke and α,β-unsaturated aldehydes elicit VEGF release through the p38 MAPK pathway in human airway smooth muscle cells and lung fibroblasts 
British Journal of Pharmacology  2011;163(3):649-661.
Vascular endothelial growth factor (VEGF) is an angiogenic factor known to be elevated in the sputum of asymptomatic smokers as well as smokers with bronchitis type of chronic obstructive pulmonary disease. The aim of this study was to investigate whether acute exposure to cigarette smoke extract altered VEGF production in lung parenchymal cells.
We exposed human airway smooth muscle cells (ASMC), normal human lung fibroblasts (NHLF) and small airways epithelial cells (SAEC) to aqueous cigarette smoke extract (CSE) in order to investigate the effect of cigarette smoke on VEGF expression and release.
Vascular endothelial growth factor release was elevated by sub-toxic concentrations of CSE in both ASMC and NHLF, but not in SAEC. CSE-evoked VEGF release was mimicked by its component acrolein at concentrations (10–100 µM) found in CSE, and prevented by the antioxidant and α,β-unsaturated aldehyde scavenger, N-acetylcysteine (NAC). Both CSE and acrolein (30 µM) induced VEGF mRNA expression in ASMC cultures, suggesting an effect at transcriptional level. Crotonaldehyde and 4-hydroxy-2-nonenal, an endogenous α,β-unsaturated aldehyde, stimulated VEGF release, as did H2O2. CSE-evoked VEGF release was accompanied by rapid and lasting phosphorylation of p38 MAPK (mitogen-activated protein kinase), which was abolished by NAC and mimicked by acrolein. Both CSE- and acrolein-evoked VEGF release were blocked by selective inhibition of p38 MAPK signalling.
α,β-Unsaturated aldehydes and possibly reactive oxygen species contained in cigarette smoke stimulate VEGF expression and release from pulmonary cells through p38 MAPK signalling.
PMCID: PMC3101625  PMID: 21306579
COPD; airway smooth muscle cells; lung fibroblasts; acrolein; 4-hydroxy-2-nonenal; VEGF; p38 MAPK
20.  In vitro and clinical studies examining the expression of osteopontin in cigarette smoke-exposed endothelial cells and cigarette smokers 
Cigarette smoking is a leading cause of mortality and morbidity and is associated with cardiovascular disease via contributory processes such as endothelial dysfunction, inflammation and thrombosis. Cigarette smoke both contains and stimulates the production of cellular oxidants and it may also promote vascular inflammation. Osteopontin is a non-collagenous matrix protein first identified in bone and there is increasing evidence for its role in inflammation and cardiovascular disease via its action as a soluble cytokine.
In this study we have examined the mechanisms underlying the expression of osteopontin in human vascular endothelial cells in vitro following exposure to cigarette smoke particulate matter (PM), using PCR, electrochemiluminescence, immunostaining and Western blotting. We further determined if serum osteopontin levels changed in humans who quit smoking.
Non-cytotoxic concentrations of PM increased osteopontin levels in cultured human endothelial cells and this effect was reduced in the presence of ascorbate, suggesting a role for oxidants in the response to PM. However, oxidant production played no role in the PM-evoked induction MMP-3, an enzyme which cleaves osteopontin. In smokers who quit smoking for 5 days, serum osteopontin levels were significantly lowered compared to those measured prior to smoking cessation.
In vitro cigarette smoke extract exposure induced osteopontin expression in human endothelial cells in an oxidative stress-dependent manner, which may involve MMP-3 cleavage. In humans, serum osteopontin was decreased with short-term smoking cessation. Endothelial-derived osteopontin may contribute to inflammation in smokers, and may also contribute to atherosclerosis and cardiovascular disease-related processes.
PMCID: PMC3465212  PMID: 22978720
Osteopontin; In vitro; Endothelial cells; MMP-3; Smoking; Atherosclerosis
21.  Increased C-C Chemokine Receptor 2 Gene Expression in Monocytes of Severe Obstructive Sleep Apnea Patients and under Intermittent Hypoxia 
PLoS ONE  2014;9(11):e113304.
Obstructive sleep apnea (OSA) is known to be a risk factor of coronary artery disease. The chemotaxis and adhesion of monocytes to the endothelium in the early atherosclerosis is important. This study aimed to investigate the effect of intermittent hypoxia, the hallmark of OSA, on the chemotaxis and adhesion of monocytes.
Peripheral blood was sampled from 54 adults enrolled for suspected OSA. RNA was prepared from the isolated monocytes for the analysis of C-C chemokine receptor 2 (CCR2). The effect of intermittent hypoxia on the regulation and function of CCR2 was investigated on THP-1 monocytic cells and monocytes. The mRNA and protein expression levels were investigated by RT/real-time PCR and western blot analysis, respectively. Transwell filter migration assay and cell adhesion assay were performed to study the chemotaxis and adhesion of monocytes.
Monocytic CCR2 gene expression was found to be increased in severe OSA patients and higher levels were detected after sleep. Intermittent hypoxia increased the CCR2 expression in THP-1 monocytic cells even in the presence of TNF-α and CRP. Intermittent hypoxia also promoted the MCP-1-mediated chemotaxis and adhesion of monocytes to endothelial cells. Furthermore, inhibitor for p42/44 MAPK or p38 MAPK suppressed the activation of monocytic CCR2 expression by intermittent hypoxia.
This is the first study to demonstrate the increase of CCR2 gene expression in monocytes of severe OSA patients. Monocytic CCR2 gene expression can be induced under intermittent hypoxia which contributes to the chemotaxis and adhesion of monocytes.
PMCID: PMC4239065  PMID: 25411969
22.  The Toxic Effects of Cigarette Additives. Philip Morris' Project Mix Reconsidered: An Analysis of Documents Released through Litigation 
PLoS Medicine  2011;8(12):e1001145.
Stanton Glantz and colleagues analyzed previously secret tobacco industry documents and peer-reviewed published results of Philip Morris' Project MIX about research on cigarette additives, and show that this research on the use of cigarette additives cannot be taken at face value.
In 2009, the promulgation of US Food and Drug Administration (FDA) tobacco regulation focused attention on cigarette flavor additives. The tobacco industry had prepared for this eventuality by initiating a research program focusing on additive toxicity. The objective of this study was to analyze Philip Morris' Project MIX as a case study of tobacco industry scientific research being positioned strategically to prevent anticipated tobacco control regulations.
Methods and Findings
We analyzed previously secret tobacco industry documents to identify internal strategies for research on cigarette additives and reanalyzed tobacco industry peer-reviewed published results of this research. We focused on the key group of studies conducted by Phillip Morris in a coordinated effort known as “Project MIX.” Documents showed that Project MIX subsumed the study of various combinations of 333 cigarette additives. In addition to multiple internal reports, this work also led to four peer-reviewed publications (published in 2001). These papers concluded that there was no evidence of substantial toxicity attributable to the cigarette additives studied. Internal documents revealed post hoc changes in analytical protocols after initial statistical findings indicated an additive-associated increase in cigarette toxicity as well as increased total particulate matter (TPM) concentrations in additive-modified cigarette smoke. By expressing the data adjusted by TPM concentration, the published papers obscured this underlying toxicity and particulate increase. The animal toxicology results were based on a small number of rats in each experiment, raising the possibility that the failure to detect statistically significant changes in the end points was due to underpowering the experiments rather than lack of a real effect.
The case study of Project MIX shows tobacco industry scientific research on the use of cigarette additives cannot be taken at face value. The results demonstrate that toxins in cigarette smoke increase substantially when additives are put in cigarettes, including the level of TPM. In particular, regulatory authorities, including the FDA and similar agencies elsewhere, could use the Project MIX data to eliminate the use of these 333 additives (including menthol) from cigarettes.
Please see later in the article for the Editors' Summary
Editors' Summary
The tobacco industry in the United States has recognized that regulation of its products was inevitable as early as 1963 and devoted increasing attention to the likelihood of regulation by the US Food and Drug Administration in the mid-1990s, which finally became law in 2009. In addition, the World Health Organization (WHO) Framework Convention on Tobacco Control (WHO FCTC), which came into force in June 2003, includes provisions addressing the regulation of the contents of tobacco products and the regulation of tobacco product disclosures. Although these steps represent progress in tobacco control, the events of the past few decades show the determination of the tobacco industry to avoid regulation, including the regulation of additives. In the United States, executives of the tobacco company Philip Morris (PM) recognized the inevitability of regulation and responded by initiating efforts to shape legislation and regulation by reorganizing its internal scientific activities and conducting scientific research that could be used to shape any proposed regulations. For example, the company conducted “Project MIX,” a study of chemical constituents in and toxicity of smoke produced by burning cigarettes containing three different combinations of 333 cigarette additives that “were constructed to resemble typical commercial blended cigarettes.” The resulting four papers published in Food and Chemical Toxicology in January 2002 concluded that there was no evidence of substantial toxicity attributable to the cigarette additives studied.
Why Was This Study Done?
The use of cigarette additives is an important concern of the WHO, FDA, and similar national regulatory bodies around the world. Philip Morris has used the published Project MIX papers to assert the safety of individual additives and other cigarette companies have done similar studies that reached similar conclusions. In this study, the researchers used documents made public as a result of litigation against the tobacco industry to investigate the origins and design of Project MIX and to conduct their own analyses of the results to assess the reliability of the conclusions in the papers published in Food and Chemical Toxicology.
What Did the Researchers Do and Find?
The researchers systematically examined tobacco industry documents in the University of California San Francisco Legacy Tobacco Documents Library (then about 60 million pages made publicly available as a result of litigation) and used an iterative process of searching, analyzing, and refining to identify and review in detail 500 relevant documents.
The researchers found that in the original Project MIX analysis, the published papers obscured findings of toxicity by adjusting the data by total particulate matter (TPM) concentration. When the researchers conducted their own analysis by studying additives per cigarette (as was specified in the original Project MIX protocol), they found that 15 carcinogenic chemicals increased by 20%. The researchers also reported that, for unexplained reasons, Philip Morris deemphasized 19 of the 51 chemicals tested in the presentation of results, including nine that were substantially increased in smoke on a per cigarette basis of additive-added cigarettes, compared to smoke of control cigarettes.
The researchers explored the possibility that the failure of Project MIX to detect statistically significant changes in the toxicity of the smoke from cigarettes containing the additives was due to underpowered experiments rather than lack of a real effect by conducting their own statistical analysis. This analysis suggests that a better powered study would have detected a much broader range of biological effects associated with the additives than was identified in Philip Morris' published paper, suggesting that it substantially underestimated the toxic potential of cigarette smoke and additives.
The researchers also found that Food and Chemical Toxicology, the journal in which the four Project MIX papers were published, had an editor and 11 of its International Editorial Board with documented links to the tobacco industry. The scientist and leader of Project MIX Edward Carmines described the process of publication as “an inside job.”
What Do These Findings Mean?
These findings show that the tobacco industry scientific research on the use of cigarette additives cannot be taken at face value: the results demonstrate that toxins in cigarette smoke increase substantially when additives are put in cigarettes. In addition, better powered studies would probably have detected a much broader range of adverse biological effects associated with the additives than identified to those identified in PM's published papers suggesting that the published papers substantially underestimate the toxic potential combination of cigarette smoke and additives.
Regulatory authorities, including the FDA and similar agencies elsewhere who are implementing WHO FCTC, should conduct their own independent analysis of Project MIX data, which, analyzed correctly, could provide a strong evidence base for the elimination of the use of the studied additives (including menthol) in cigarettes on public health grounds.
Additional Information
Please access these Web sites via the online version of this summary at
For PLoS Medicine's own policy on publishing papers sponsored by the tobacco industry see
The World Health Organization (WHO) provides information on the Framework Convention on Tobacco Control (FCTC)
The documents that the researchers reviewed in this paper can be found at the Legacy Tobacco Documents Library
PMCID: PMC3243707  PMID: 22205885
23.  Monocytic Adhesion Molecule Expression and Monocyte-Endothelial Cell Dysfunction is Increased in Patients with Peripheral Vascular Disease versus Patients with Abdominal Aortic Aneurysms 
The Journal of surgical research  2012;177(2):373-381.
Statin therapy is utilized in the medical management of patients with peripheral vascular disease (PVD) and abdominal aortic aneurysm (AAA) for the pleiotropic and anti-inflammatory benefits. We hypothesize that the inflammatory mechanisms of monocyte-endothelial cell interactions in endothelial barrier dysfunction is more significant in patients with PVD compared to AAA. The purpose of this study is to assess patient peripheral blood monocyte adhesion molecules by flow cytometry and monocyte-induced endothelial barrier dysfunction by using an in vitro endothelial cell layer and electric cell-substrate impedance sensing system (ECIS).
Peripheral blood was collected from patients with either PVD (ABI<0.9, toe-arm index <0.8, or required lower extremity vascular intervention) or AAA (aortic diameter >3.0 cm). Monocytes were isolated from fresh whole blood using an accuspin-histopaque technique. The separated monocytes underwent flow cytometry analysis to evaluate expression levels of the cell membrane adhesion molecules: CD18, CD11a/b/c, and VLA-4. Endothelial cell function was assessed by adding monocytes to an endothelial monolayer on ECIS arrays and co-culturing overnight. Peak changes in trans-endothelial resistance were measured and compared between patient groups.
Twenty-eight monocyte samples were analyzed for adhesion molecules (PVD: 19, AAA: 9) via flow cytometry and 11 patients were evaluated for endothelial dysfunction (PVD: 7, AAA: 4) via ECIS. There was no significant difference between risk factors among PVD and AAA patients except for age, where AAA patients were significantly older than PVD patients in both flow cytometry and ECIS groups (P=0.02, 0.01 respectively). There were significantly higher levels of adhesion molecules CD11a, CD18, and CD11c (averaged MFI P-values: 0.047, 0.038, 0.014, respectively) in PVD patients compared to AAA patients. No significant difference was found for CD11b and VLA-4 expression (P=0.21, 0.15 respectively). There was significantly more monocyte-endothelial cell dysfunction as a result of monocytes obtained from patients with PVD than from AAA, with a maximal effect seen at 15 hours after monocyte addition (P=0.032).
Patients with PVD have increased expression levels of certain monocyte adhesion molecules and greater monocyte-induced endothelial layer dysfunction when compared to patients with AAA. This may lead to other methods of targeted therapy to improve outcomes of these vascular patients.
PMCID: PMC4441414  PMID: 22809707
24.  Cigarette Smoke Enhances the Expression of Profibrotic Molecules in Alveolar Epithelial Cells 
PLoS ONE  2016;11(3):e0150383.
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology. A growing body of evidence indicates that it may result from an aberrant activation of alveolar epithelium, which induces the expansion of the fibroblast population, their differentiation to myofibroblasts and the excessive accumulation of extracellular matrix. The mechanisms that activate the alveolar epithelium are unknown, but several studies indicate that smoking is the main environmental risk factor for the development of IPF. In this study we explored the effect of cigarette smoke on the gene expression profile and signaling pathways in alveolar epithelial cells. Lung epithelial cell line from human (A549), was exposed to cigarette smoke extract (CSE) for 1, 3, and 5 weeks at 1, 5 and 10% and gene expression was evaluated by complete transcriptome microarrays. Signaling networks were analyzed with the Ingenuity Pathway Analysis software. At 5 weeks of exposure, alveolar epithelial cells acquired a fibroblast-like phenotype. At this time, gene expression profile revealed a significant increase of more than 1000 genes and deregulation of canonical signaling pathways such as TGF-β and Wnt. Several profibrotic genes involved in EMT were over-expressed, and incomplete EMT was observed in these cells, and corroborated in mouse (MLE-12) and rat (RLE-6TN) epithelial cells. The secretion of activated TGF-β1 increased in cells exposed to cigarette smoke, which decreased when the integrin alpha v gene was silenced. These findings suggest that the exposure of alveolar epithelial cells to CSE induces the expression and release of a variety of profibrotic genes, and the activation of TGF-β1, which may explain at least partially, the increased risk of developing IPF in smokers.
PMCID: PMC4775036  PMID: 26934369
25.  Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts 
Human Reproduction (Oxford, England)  2014;29(6):1255-1270.
How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)?
Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death.
Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductive tract (FRT), which may participate in promoting reproductive success.
This is a cross sectional study involving control samples versus treatment. SP (pooled from twenty donors) was first tested for dose- and time-dependent cytotoxic effects on eEC and eSF (n = 4). As exposure of eEC or eSF to 1% SP for 6 h proved to be non-toxic, a second set of eEC/eSF samples (n = 4) was treated under these conditions for transcriptome, protein and functional analysis. With a third set of samples (n = 3), we further compared the transcriptional response of the cells to SP versus fresh semen.
eEC and eSF were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. RNA was isolated and processed for microarray studies to analyze global transcriptomic changes. Secreted factors in conditioned media from SP-treated cells were analyzed by Luminex and for the ability to stimulate migration of CD14+ monocytes and CD4+ T cells.
Pathway identifications were determined using the Z-scoring system in Ingenuity Pathways Analysis (Z scores ≥|1.5|). SP induced transcriptomic changes (P < 0.05) associated with promoting leukocyte and endothelial cell recruitment, and proliferation of eEC and eSF. Cell viability pathways were induced, while those associated with cell death were suppressed (P < 0.05). SP and fresh semen induced similar sets of pathways, suggesting that SP can model the signaling effects of semen in the endometrium. SP also induced secretion of pro-inflammatory and pro-chemotactic cytokines, as well as pro-angiogenic and proliferative growth factors (P < 0.05) in both eEC and eSF. Finally, functional assays revealed that conditioned media from SP-treated eEC and eSF significantly increased (P < 0.05) chemotaxis of CD14+ monocytes and CD4+ T cells.
This study is limited to in vitro analyses of the effects of SP on endometrial cells. In addition, the measured response to SP was conducted in the absence of the ovarian hormones estradiol and progesterone, as well as epithelial-stromal paracrine signaling. While this study focused on establishing the baseline cellular response of endometrial cells to SP, future work should assess how hormone signaling in the presence of appropriate paracrine interactions affects SP-induced genes in these cells.
The results of this study support previous findings that SP and semen contain bioactive factors capable of eliciting chemotactic responses in the uterus, which can lead to recruitment of leukocytes to the endometrium. Future directions will explore if similar changes in gene expression do indeed occur after coitus in vivo, and how the signaling cascades initiated by SP in the endometrium can affect reproductive success, female reproductive health and susceptibility to sexually transmitted diseases. The gene list provided by the transcriptome analysis reported here should prove a valuable resource for understanding the response of the upper FRT to SP exposure.
This project was supported by NIH AI083050-04 (W.C.G./L.C.G.); NIH U54HD 055764 (L.C.G.); NIH 1F32HD074423-02 (J.C.C.); DOD W81XWH-11-1-0562 (W.C.G.); NIH 5K12-DK083021-04, NIH 1K99AI104262-01A1, The UCSF Hellman Award (N.R.R.). The authors have nothing to disclose.
PMCID: PMC4017943  PMID: 24626806
semen; endometrium; reproduction; microarray; chemotaxis

Results 1-25 (1654033)