Mechanism of an indirect effect of aqueous cigarette smoke extract on the adhesion of monocytic cells to endothelial cells in an in vitro assay revealed by transcriptomics analysis.

The adhesion of monocytic cells to the "dysfunctional" endothelium constitutes a critical step in the initiation of atherosclerosis. Cigarette smoke (CS) has been shown to contribute to this process, the complex mechanism of which still needs to be unraveled. We developed an in vitro adhesion assay to investigate the CS-induced adhesion of monocytic MM6 cells to human umbilical vein endothelial cells (HUVECs) following exposure to an aqueous CS extract (smoke-bubbled phosphate buffered saline: sbPBS), reasoning that in vivo monocytes and endothelial cells are exposed primarily to soluble constituents from inhaled CS absorbed through the lung alveolar wall. MM6 cell adhesion was increased exclusively by the conditioned medium from sbPBS-exposed MM6 cells, not by direct sbPBS exposure of the HUVECs within a range of sbPBS doses. Using a transcriptomics approach followed by confirmation experiments, we identified different exposure effects on both cell types and a key mechanism by which sbPBS promoted the adhesion of MM6 cells to HUVECs. While sbPBS provoked a strong oxidative stress response in both cell types, the expression of E-selectin, VCAM-1 and ICAM-1, responsible for the adhesion of MM6 cells to HUVECs, was induced in the latter through a proinflammatory paracrine effect. We confirmed that this effect was driven mainly by TNFα produced by MM6 cells exposed to sbPBS. In conclusion, we have elucidated an indirect mechanism by which sbPBS increases the adhesion of monocytic cells to endothelial cells in this in vitro assay that was designed for tobacco product risk assessment while mimicking the in vivo exposure conditions as closely as possible.

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.

Modulation of atherogenic lipidome by cigarette smoke in apolipoprotein E-deficient mice.

OBJECTIVE: Although relationships between smoking and cardiovascular diseases (CVD), and between CVD and lipids are established, the direct impact of smoking on lipidomes is not well understood. We investigated the effect of mainstream cigarette smoke (CS) exposure on plasma, liver, and aorta molecular lipid profiles, and liver transcriptome in the ApoE(-/-) mouse, a well-established mouse model for human atherogenesis. METHODS: Plasma, liver, and aorta samples from ApoE(-/-) mice exposed to CS or fresh air (sham) for six months were extracted for lipids using robotic-assisted method and analyzed by mass spectrometry. Gene expression in the liver was obtained on microarrays. Development of atherosclerosis in the aorta was further assessed by plaque size in the aortic arch and lipoprotein concentration in plasma and plaque. RESULTS: CS increased most lipid classes and molecular lipid species. In plasma, free cholesterol, ceramides, cerebrosides, and most phospholipids were increased in CS-exposed mice. In the liver, several lipid species including free and esterified cholesterol, triacylglycerols, phospholipids, sphingomyelins, and ceramides were elevated. In the aorta, more than 2-fold higher cholesteryl ester (CE), lysophosphatidylcholine, and glucosyl/galactosylceramide levels were seen. Moreover, CS exposure induced a significant decrease in several plasma CE and phosphatidylcholine species that contained polyunsaturated fatty acids. Genes involved in amino acid and lipid metabolism showed perturbed transcription profiles in the liver. CONCLUSION: We have quantified some of the molecular changes that accompany the increase of plaque size that is accelerated by CS exposure in the aortae of ApoE(-/-) mice. These results suggest that specific changes in the lipidome and transcriptome, for example in ceramide and polyunsaturated fatty acid species, may be associated with atherosclerosis.

Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase.

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS+nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS+nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions ((·)O(2)(-)) and peroxynitrite (ONOO(-)) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS+nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS+nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation.

A computable cellular stress network model for non-diseased pulmonary and cardiovascular tissue.

BACKGROUND: Humans and other organisms are equipped with a set of responses that can prevent damage from exposure to a multitude of endogenous and environmental stressors. If these stress responses are overwhelmed, this can result in pathogenesis of diseases, which is reflected by an increased development of, e.g., pulmonary and cardiac diseases in humans exposed to chronic levels of environmental stress, including inhaled cigarette smoke (CS). Systems biology data sets (e.g., transcriptomics, phosphoproteomics, metabolomics) could enable comprehensive investigation of the biological impact of these stressors. However, detailed mechanistic networks are needed to determine which specific pathways are activated in response to different stressors and to drive the qualitative and eventually quantitative assessment of these data. A current limiting step in this process is the availability of detailed mechanistic networks that can be used as an analytical substrate. RESULTS: We have built a detailed network model that captures the biology underlying the physiological cellular response to endogenous and exogenous stressors in non-diseased mammalian pulmonary and cardiovascular cells. The contents of the network model reflect several diverse areas of signaling, including oxidative stress, hypoxia, shear stress, endoplasmic reticulum stress, and xenobiotic stress, that are elicited in response to common pulmonary and cardiovascular stressors. We then tested the ability of the network model to identify the mechanisms that are activated in response to CS, a broad inducer of cellular stress. Using transcriptomic data from the lungs of mice exposed to CS, the network model identified a robust increase in the oxidative stress response, largely mediated by the anti-oxidant NRF2 pathways, consistent with previous reports on the impact of CS exposure in the mammalian lung. CONCLUSIONS: The results presented here describe the construction of a cellular stress network model and its application towards the analysis of environmental stress using transcriptomic data. The proof-of-principle analysis described here, coupled with the future development of additional network models covering distinct areas of biology, will help to further clarify the integrated biological responses elicited by complex environmental stressors such as CS, in pulmonary and cardiovascular cells.

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.

Involvement of JAM-A in mononuclear cell recruitment on inflamed or atherosclerotic endothelium: inhibition by soluble JAM-A.

OBJECTIVE: The junctional adhesion molecule (JAM)-A on endothelium contributes to the inflammatory recruitment of mononuclear cells involving engagement of its integrin receptor lymphocyte function-associated antigen (LFA)-1. It is unknown whether these functions can be inhibited by soluble forms of JAM-A, whether JAM-A is expressed on atherosclerotic endothelium, and whether it participates in atherogenic recruitment of mononuclear cells. METHODS AND RESULTS: Adhesion assays revealed that LFA-1-mediated binding of mononuclear cells to intercellular adhesion molecule (ICAM)-1 or cytokine-costimulated endothelium was dose-dependently inhibited by soluble JAM-A.Fc (sJAM-A.Fc). Similarly, sJAM-A.Fc reduced stromal cell-derived factor (SDF)-1alpha-triggered transendothelial chemotaxis of activated T cells and their SDF-1alpha-triggered arrest on cytokine-costimulated endothelium under flow conditions. Immunofluorescence analysis revealed an upregulation of JAM-A on early atherosclerotic endothelium of carotid arteries from apolipoprotein E-deficient (apoE-/-) mice fed an atherogenic diet. In ex vivo perfusion assays, pretreatment of mononuclear cells with sJAM-A.Fc inhibited their very late antigen (VLA)-4-independent accumulation on atherosclerotic endothelium of these arteries. CONCLUSIONS: Soluble forms of JAM-A can be effectively applied to inhibit distinct steps of mononuclear cell recruitment on inflamed or atherosclerotic endothelium. In conjunction with its expression on atherosclerotic endothelium, this suggests a functional contribution of JAM-A to atherogenesis.

How mammalian transcriptional repressors work.

Research on the regulation of transcription in mammals initially focused on the mechanism of transcriptional activation and 'positive control' of gene regulation. In contrast, transcriptional repression and 'negative control' of gene transcription was viewed rather as part of the 'prokaryotic book of biology'. However, results obtained in recent years have shown convincingly that transcriptional repression mediated by repressor proteins is a common regulatory mechanism in mammals and may play a key role in many biological processes. In particular, the fact that human diseases, such as Rett and ICF syndromes as well as some human forms of cancer, are connected with the activities of human repressor proteins indicates that transcriptional repression and gene silencing is essential for maintenance of the cellular integrity of a multicellular organism. The wide range of diseases caused by aberration in transcriptional repression sheds light on the importance of understanding how mammalian transcriptional repressor proteins work.

Neointimal smooth muscle cells display a proinflammatory phenotype resulting in increased leukocyte recruitment mediated by P-selectin and chemokines.

Leukocyte recruitment is crucial for the response to vascular injury in spontaneous and accelerated atherosclerosis. Whereas the mechanisms of leukocyte adhesion to endothelium or matrix-bound platelets have been characterized, less is known about the proadhesive role of smooth muscle cells (SMCs) exposed after endothelial denudation. In laminar flow assays, neointimal rat SMCs (niSMCs) supported a 2.5-fold higher arrest of monocytes and "memory" T lymphocytes than medial SMCs, which was dependent on both P-selectin and VLA-4, as demonstrated by blocking antibodies. The increase in monocyte arrest on niSMCs was triggered by the CXC chemokine GRO-alpha and fractalkine, whereas "memory" T cell arrest was mediated by stromal cell-derived factor (SDF)-1alpha. This functional phenotype was paralleled by a constitutively increased mRNA and surface expression of P-selectin and of relevant chemokines in niSMCs, as assessed by real-time PCR and flow cytometry. The increased expression of P-selectin in niSMCs versus medial SMCs was associated with enhanced NF-kappaB activity, as revealed by immunofluorescence staining for nuclear p65 in vitro. Inhibition of NF-kappaB by adenoviral IkappaBalpha in niSMCs resulted in a marked reduction of increased leukocyte arrest in flow. Furthermore, P-selectin expression by niSMCs in vivo was confirmed in a hypercholesterolemic mouse model of vascular injury by double immunofluorescence and by RT-PCR after laser microdissection. In conclusion, we have identified a NF-kappaB-mediated proinflammatory phenotype of niSMCs that is characterized by increased P-selectin and chemokine expression and thereby effectively supports leukocyte recruitment.

Crucial role of stromal cell-derived factor-1alpha in neointima formation after vascular injury in apolipoprotein E-deficient mice.

BACKGROUND: Recent evidence indicates that stromal cell-derived factor-1alpha (SDF-1alpha) is expressed in human atherosclerotic plaques, whereas high plasma levels are clinically associated with stable coronary artery disease. Herein, we investigate the involvement of SDF-1alpha in neointimal formation after vascular injury. METHODS AND RESULTS: SDF-1alpha was detected by immunohistochemistry in carotid arteries of apolipoprotein E-deficient (apoE-/-) mice at various stages of neointima formation after wire-induced injury. Double immunofluorescence revealed that SDF-1alpha staining was mostly confined to smooth muscle cells (SMCs). Furthermore, SDF-1alpha plasma levels peaked 1 day after vascular injury. Treatment of apoE-/- mice after carotid injury with a neutralizing SDF-1alpha monoclonal antibody for 3 weeks reduced neointimal lesion area by 44% (n=5, P<0.05) compared with isotype control. In SDF-1alpha antibody-treated apoE-/- mice, neointimal SMC content was decreased (21.7+/-2% versus 39.4+/-4%, n=5, P=0.005), whereas the relative content of neointimal macrophages remained unchanged. As shown by flow cytometry, carotid injury resulted in a marked expansion of circulating Sca-1+lineage- progenitor cells (PBPCs) in the peripheral blood of apoE-/- mice after 1 day, which was mediated by SDF-1alpha. Systemic injection of isolated PBPCs after vascular injury demonstrated their recruitment to neointimal lesions, where they can adopt an SMC-like phenotype. CONCLUSIONS: SDF-1alpha plays an instrumental role in neointimal formation after vascular injury in apoE-/- mice by regulating neointimal SMC content. This contribution appears to be attributable to SDF-1alpha-dependent recruitment of circulating SMC progenitor cells.

Suppression of endothelial adhesion molecule up-regulation with cyclopentenone prostaglandins is dissociated from IkappaB-alpha kinase inhibition and cell death induction.

The cyclopentenone prostaglandins (cPG) 15-deoxy-Delta12,14-prostaglandin J2 (dPGJ2) and PGA1 can inhibit multiple steps in nuclear factor (NF)-kappaB signaling and can induce cell death. Here we characterized the effects of dPGJ2 and PGA1 on the inflammatory induction of endothelial cell adhesion molecules (CAM). Pretreatment of endothelial cells with dPGJ2 or PGA1 at low concentrations dose dependently inhibited the up-regulation of CAM expression and monocyte arrest by tumor necrosis factor (TNF)-alpha but not expression of inhibitor of apoptosis proteins. Only at high concentrations, cPG enhanced TNF-alpha-induced cell death and inhibited TNF-alpha-induced IkappaB-alpha kinase (IKK) activation, IkappaB-alpha degradation, and NF-kappaB/p65 translocation, while promoting AP-1/c-jun phosphorylation. Expression of an IKK-beta mutant (C179A) resistant to interaction with cPG impaired cell death induction but not inhibition of CAM up-regulation by cPG. Gel shift and reporter gene analysis revealed that cPG at low concentrations directly impaired DNA binding of NF-kappaB and NF-kappaB-dependent transactivation. The synthetic analogs dPGA1 or dPGA2 were ineffective, indicating structural specificity of cPG. Thus, the suppression of endothelial CAM up-regulation with cPG is dissociated from cell death sensitization and IKK inhibition above threshold concentrations and related to interference with NF-kappaB binding. Our findings define distinct mechanisms for anti-inflammatory and proapoptotic effects of cPG in endothelial cells.

RE-1 silencing transcription factor (REST) regulates human synaptophysin gene transcription through an intronic sequence-specific DNA-binding site.

Synaptophysin, one of the major proteins on synaptic vesicles, is ubiquitously expressed throughout the brain. Synaptophysin and synapsin I, another synaptic vesicle protein, are also expressed by retinoic acid-induced neuronally differentiated P19 teratocarcinoma cells. Here, we show that inhibition of histone deacetylase activity in P19 cells is sufficient to activate transcription of the synaptophysin and synapsin I genes, indicating that neuronal differentiation and impairment of histone deacetylases results in a similar gene expression pattern. The transcription factor REST, a repressor of neuronal genes in non-neuronal tissues, has been shown to function via recruitment of histone deacetylases to the transcription unit, indicating that modulation of the chromatin structure via histone deacetylation is of major importance for REST function and neuron-specific gene transcription. Furthermore, REST has been shown to be the major regulator of neuronal expression of synapsin I. Here, we have identified a functional binding site for REST in the first intron of the human synaptophysin gene indicating that REST blocks human synaptophysin gene transcription through an intronic neuron-specific silencer element. The synaptophysin promoter is, however, devoid of neuron-specific genetic elements and directs transcription in both neuronal and non-neuronal cells. Using a dominant-negative approach we have identified the transcription factor Sp1 as one of the regulators responsible for constitutive transcription of the human synaptophysin gene.

RE-1 silencing transcription factor-4 (REST4) is neither a transcriptional repressor nor a de-repressor.

The zinc finger protein RE-1 silencing transcription factor (REST) is a transcriptional repressor that represses neuronal genes in non-neuronal tissues. A neuronal splice form of REST, termed REST4, has been described in the rat. It encompasses the N-terminus of REST, including the N-terminal repressor domain and five of the eight zinc fingers of the DNA-binding domain. The biological function of REST4 is controversial. Transcriptional repression as well as transcriptional de-repression activity has been attributed to the REST4 protein of rat. Here, we have expressed a 'humanized' version of REST4 (hREST4) to facilitate a comparison of the biological functions of hREST4 and REST. The biological activity the human REST protein has been extensively studied in the past. Additionally, hREST4 has a high degree of homology with the REST4 protein of rat. An immunofluorescence analysis showed that hREST4 is expressed in the nucleus, indicating that the protein may have a potential impact on gene regulation. We analyzed the biological function of hREST4 in NS20Y neuroblastoma cells using human synapsin I promoter/reporter gene constructs. The human synapsin I gene is negatively regulated by REST. The results show that hREST4, in contrast to the full-length human REST protein, does not impair human synapsin I promoter activity. Moreover, co-transfection experiments with expression vectors encoding REST and hREST4 did not reveal any evidence that REST4 blocks the transcriptional repression activity of REST.