Systems toxicology approaches enable mechanistic comparison of spontaneous and cigarette smoke-related lung tumor development in the A/J mouse model.

The A/J mouse is highly susceptible to lung tumor induction and has been widely used as a screening model in carcinogenicity testing and chemoprevention studies. However, the A/J mouse model has several disadvantages. Most notably, it develops lung tumors spontaneously. Moreover, there is a considerable gap in our understanding of the underlying mechanisms of pulmonary chemical carcinogenesis in the A/J mouse. Therefore, we examined the differences between spontaneous and cigarette smoke-related lung tumors in the A/J mouse model using mRNA and microRNA (miRNA) profiling. Male A/J mice were exposed whole-body to mainstream cigarette smoke (MS) for 18 months. Gene expression interaction term analysis of lung tumors and surrounding non-tumorous parenchyma samples from animals that were exposed to either 300 mg/m(3) MS or sham-exposed to fresh air indicated significant differential expression of 296 genes. Ingenuity Pathway Analysis(®) (IPA(®)) indicated an overall suppression of the humoral immune response, which was accompanied by a disruption of sphingolipid and glycosaminoglycan metabolism and a deregulation of potentially oncogenic miRNA in tumors of MS-exposed A/J mice. Thus, we propose that MS exposure leads to severe perturbations in pathways essential for tumor recognition by the immune system, thereby potentiating the ability of tumor cells to escape from immune surveillance. Further, exposure to MS appeared to affect expression of miRNA, which have previously been implicated in carcinogenesis and are thought to contribute to tumor progression. Finally, we identified a 50-gene expression signature and show its utility in distinguishing between cigarette smoke-related and spontaneous lung tumors.

Cigarette smoke induces molecular responses in respiratory tissues of ApoE(-/-) mice that are progressively deactivated upon cessation.

Cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD) and a risk factor for both lung and cardiovascular (CV) diseases, which are rarely investigated concomitantly. Although smoking cessation shows clear CV risk benefit, lung-related disease risk remains higher in former smokers than in never smokers. We sought to determine the differential molecular responses of murine respiratory tissues to better understand the toxicity pathways involved in smoking-related disease risk and those related to the benefits of smoking cessation. ApoE(-/-) mice were exposed to mainstream cigarette smoke (CS) or a smoking cessation-mimicking protocol for up to 6 months and transcriptomics analysis of nasal epithelium and lung parenchyma performed. We supported our gene expression profiling approach with standard lung histopathology and bronchoalveolar lavage fluid (BALF) analysis. Many BALF analytes involved in functions ranging from inflammation to cell proliferation and tissue remodeling were found elevated in BALF. Gene expression levels of these molecules were also increased in lung tissue, suggesting that the inflammatory response was the result of local tissue activation and the contribution of recruited inflammatory cells. Gene set enrichment analysis (GSEA) of expression data from murine lungs and nasal epithelium showed distinct activation patterns of inflammation, complement, and xenobiotic metabolism pathways during CS exposure that were deactivated upon smoking cessation. Pathways involved in cell proliferation and tissue remodeling were activated by CS and progressively deactivated upon smoke exposure cessation. Differential CS-mediated responses of pulmonary and nasal tissues reflect common mechanisms but also the varying degrees of epithelial functional specialization and exposure along the respiratory tract.

Cigarette-smoke-induced atherogenic lipid profiles in plasma and vascular tissue of apolipoprotein E-deficient mice are attenuated by smoking cessation.

Tobacco smoke exerts perturbations on lipid metabolism and arterial cell function that accelerate atherosclerosis. Lipidomics has emerged as a key technology in helping to elucidate the lipid-related mechanisms of atherosclerosis. In this study, we investigated the effects of smoking cessation on plaque development and aortic arch content of various lipid molecular classes and species. Apolipoprotein E-deficient mice were exposed to fresh air (sham) or to mainstream cigarette smoke (CS) for 6 months, or to CS for 3 months followed by sham for 3 months (cessation group). Lipids from plasma and aortic arches, plasma lipoprotein profiles and plaque morphometry measurements were analyzed. We already showed that CS exposure accelerated plaque size and total cholesterol content of the aortic arch at 3 and 6 months. Marked increases were seen in the relative enrichment of cholesteryl esters, phospholipids, sphingomyelins, and glycosphingolipids. Smoking cessation slowed plaque progression and resulted in lower levels of many lipid species in plasma and aortic arch. While CS exposure promoted rapid lipid accumulation in mouse aorta, smoking cessation translated into a slow removal of lipids from the vessel wall. Despite the smoking cessation-dependent metabolic changes leading to increased animal body weight, accumulation of proatherogenic lipids in the vessel was halted after exposure cessation, indicating that the clinical benefits of smoking cessation translate directly to the vessel wall and its lipid makeup.

Towards the validation of a lung tumorigenesis model with mainstream cigarette smoke inhalation using the A/J mouse.

A generally accepted and validated laboratory model for smoking-associated pulmonary tumorigenesis would be useful for both basic and applied research applications, such as the development of early diagnostic endpoints or the evaluation of modified risk tobacco products, respectively. The A/J mouse is susceptible for developing both spontaneous and induced lung adenomas and adenocarcinomas, and increased lung tumor multiplicities were also observed in previous cigarette smoke inhalation studies. The present study was designed to collect data useful towards the validation of an 18-month mainstream smoke (MS) inhalation model. Male and female A/J mice were exposed whole-body at three MS concentration levels for 6h/day, and the results were compared to a previous study in the same laboratory and with a similar design. A linear MS concentration-dependent increase in lung tumorigenesis was observed with similar slopes for both sexes and both studies and a maximal 5-fold increase in multiplicity beyond sham control. The minimal detectable difference in lung tumor multiplicity for the current study was 37%. In the larynx, papillomas were detectable in all MS-exposed groups in a non-concentration dependent manner. No other extra-pulmonary MS-dependent neoplastic lesions were found. Gene expression signatures of lung tumor tissues allowed a clear differentiation of sham- and high dose MS-exposed mice. In combination with data from previous smoke inhalation studies with A/J mice, the current data suggest that this model for MS inhalation-induced pulmonary tumorigenesis is reliable and relevant, two crucial requirements towards validation of such a model.

Cigarette smoke enhances abdominal aortic aneurysm formation in angiotensin II-treated apolipoprotein E-deficient mice.

Cigarette smoke, hyperlipidemia, and hypertension with the risk of development and progression of atherosclerosis and associated pathologies such as abdominal aortic aneurysm (AAA) are correlated. We examined the interaction of cigarette mainstream smoke (MS) and angiotensin-II (Ang II)-induced hypertension in the atherosclerotic process using hyperlipidemic apolipoprotein E-knockout (ApoE(-/-)) mice. ApoE(-/-) mice were treated with Ang II for 4 weeks and then further exposed to MS or to fresh air for 4 weeks. AAA formation was observed in all mice treated with Ang II, regardless of smoke exposure; however, smoke exposure increased the incidence of AAA in these mice. Ang II treatment resulted in higher gene expression of matrix metalloproteinases (MMP)-2, -3, -8, -9, and -12 in the abdominal aortas, which was further increased by MS exposure. The proteolytic activity of MMP-2 and MMP-9 was also enhanced in Ang II-treated mice exposed to MS, but only minor changes were seen with either smoke exposure or Ang II treatment alone. This study shows for the first time that both formation and severity of AAA in hypertensive ApoE(-/-) mice are accelerated by exposure to MS and that the proteolytic activity of MMPs is enhanced by the combination of Ang II and MS.

Cigarette smoke exposure promotes arterial thrombosis and vessel remodeling after vascular injury in apolipoprotein E-deficient mice.

BACKGROUND: Cigarette smoking is a major risk factor for the development of cardiovascular disease. However, in terms of the vessel wall, the underlying pathomechanisms of cigarette smoking are incompletely understood, partly due to a lack of adequate in vivo models. METHODS: Apolipoprotein E-deficient mice were exposed to filtered air (sham) or to cigarette mainstream smoke at a total particulate matter (TPM) concentration of 600 microg/l for 1, 2, 3, or 4 h, for 5 days/week. After exposure for 10 +/- 1 weeks, arterial thrombosis and neointima formation at the carotid artery were induced using 10% ferric chloride. RESULTS: Mice exposed to mainstream smoke exhibited shortened time to thrombotic occlusion (p < 0.01) and lower vascular patency rates (p < 0.001). Morphometric and immunohistochemical analysis of neointimal lesions demonstrated that mainstream smoke exposure increased the amount of alpha-actin-positive smooth muscle cells (p < 0.05) and dose-dependently increased the intima-to-media ratio (p < 0.05). Additional analysis of smooth muscle cells in vitro suggested that 10 microg TPM/ml increased cell proliferation without affecting viability or apoptosis, whereas higher concentrations (100 and 500 microg TPM/ml) appeared to be cytotoxic. CONCLUSIONS: Taken together, these findings suggest that cigarette smoking promotes arterial thrombosis and modulates the size and composition of neointimal lesions after arterial injury in apolipoprotein E-deficient mice.

Time-dependent expression pattern of nitric oxide and superoxide after myocardial infarction in rats.

Previous experiments by our group have demonstrated a subacute increased contribution of nitric oxide (NO) to vasoreactivity after myocardial infarction in rats. However, the activation pattern of NO may be phasic after infarction and has been described to be strongly associated with superoxide production. Therefore, the present study evaluated the morphological distribution and time-dependent induction of NO and superoxide at the protein (via immunohistochemistry, chemiluminescence and spectrophotometry) and mRNA (via real-time RT-PCR) level after experimental induction of a myocardial infarction in rats. Myocardial infarction led to a modest but lasting upregulation of endothelial NO synthase (eNOS) in blood vessels and cardiomyocytes. In contrast, inducible NOS (iNOS) showed dramatic de novo formation 1 week after infarction, predominantly in the infarcted area and cardiomyocytes. In addition, a gradually increased myocardial production of superoxide was detected during remodelling, probably related by an upregulation of NADH oxidase in the peri-infarcted and remote myocardium. Furthermore, peroxynitrite formation was increased after myocardial infarction, indicating strong interaction between NO and superoxide. In conclusion, during the early remodelling phase after myocardial infarction mRNA concentrations and protein levels of eNOS and iNOS show an augmentation in a biphasic manner. Moreover, upregulation of NADH oxidase leads to increased presence of superoxide and peroxynitrite and thereby to a reduction of the bioavailability and compensatory effects of NO, as such influencing post-infarction remodelling.

Role of nitric oxide during coronary endothelial dysfunction after myocardial infarction.

This study aimed to investigate whether permanent ischaemia influences subacute vasodilatation responses of non-infarcted rat coronary vasculature, and to characterise these coronary changes. Ischaemia led to a significant impairment of the endothelium-dependent vasodilator response, while coronary vasodilatory capacity remained unaltered. In normal coronary circulation, nitric oxide (NO) and prostanoids contributed to vasodilatation, while basal involvement of endothelium-derived hyperpolarising factor was limited. Vasodilatory impairment following myocardial infarction did not originate from alterations in the prostanoid pathway, and only a slightly increased influence of K+ channels was observed. However, NO-mediated vasodilatation was significantly increased after ischaemia, as also confirmed by higher mRNA and protein levels of iNOS and eNOS. Additionally, the amount of superoxide was enhanced following infarction. We conclude that subacute postinfarction remodeling is accompanied by endothelial dysfunction in non-infarcted coronary arteries. Although the NO-mediated response is increased after ischaemia, its final action is restricted due to the presence of superoxide.

Role of nitric oxide and oxidative stress in ischaemic myocardial injury and preconditioning.

Nitric oxide (NO) plays an important role in the physiologic modulation of coronary artery tone and myocardial function. However, increased formation of NO within the myocardium can also have detrimental effects, contributing to the pathophysiology of myocardial dysfunction in ischaemic heart diseases. The role of reactive nitrogen species in the pathogenesis of myocardial dysfunction after ischaemia has been investigated in numerous studies. They reveal divergent and opposed effects of nitric oxide: from a cardioprotective action leading to ischaemic preconditioning after short ischaemic periods to a cardiodepressive action after severe ischaemia/reperfusion injury and heart failure. This review describes the determining role of reactive oxygen species on these opposite myocardial effects of NO. The final action of NO, whether cardioprotective or cardiodepressive, strongly depends on the level of oxidative stress in the myocardium. Nitric oxide disrupts free radical and oxidant-mediated reactions, due to a strong attraction and interaction with superoxide.The level of oxidative stress is positively related to the severity of the ischaemic injury, making the results in different myocardial syndromes more concordant. If the increased production of NO is well in balance with a moderate increase in oxygen radicals, then NO will exert beneficial effects. However, if the oxygen radicals are produced in excess of NO as in prolonged ischaemic injury, then deleterious effects will be induced. Consequently, the balance between NO and free oxygen radicals is crucial in modulating the outcome after an ischaemic insult.