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 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.

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.

How do dietary flavanols improve vascular function? A position paper.

Epidemiological and clinical studies revealed that high-flavanol diet or isolated (-)-epicatechin improves the function of the vascular endothelium, as assessed by flow-mediated dilation, through elevation of bioavailability and bioactivity of NO*. We have demonstrated that exposure of human endothelial cells to (-)-epicatechin elevates the cellular levels of NO* and cyclic GMP and protects against oxidative stress elicited by proinflammatory agonists. (-)-Epicatechin acts like a prodrug, since these effects involve O-methylation of the flavanol and are attributed to apocynin-like inhibition of endothelial NADPH oxidase. Thus, generation of superoxide and peroxynitrite is diminished and, consequently, the cellular NO* level is preserved or augmented. We propose therefore that endothelial NO* metabolism rather than general antioxidant activity is a major target of dietary flavanols and that NADPH oxidase activity is a crucial site of action. Moreover, flavonoid glucuronides appear to serve as plasma transport metabolites to target cells rather than solely as excretion products. Implications for the interpretation of the role of dietary polyphenols for cardiovascular health are discussed.

Mono-O-methylated flavanols and other flavonoids as inhibitors of endothelial NADPH oxidase.

The dietary flavan-3-ol (-)-epicatechin improves the bioactivity of nitric oxide in arterial vessels in vivo. Moreover, it effectively protects cultured vascular endothelial cells from signs of oxidative stress and elevates intracellular nitric oxide in vitro. We addressed the effects of (-)-epicatechin, its metabolic conversion products and structurally related compounds on NADPH oxidase activity in intact human umbilical vein endothelial cells (HUVEC) and in cell lysates. (-)-Epicatechin proved to be an O2*(-)-scavenger but did not inhibit NADPH oxidase activity, whereas the converse pattern was observed for the metabolites 3'- and 4'-O-methyl epicatechin. The dimer procyanidin B2 and (-)-epicatechin glucuronide were O2*(-)-scavengers and inhibited NADPH oxidase. Analysis of structure-activity relations with 45 compounds suggests an apocynin-like mode of NADPH oxidase inhibition. Notably, HUVEC converted (-)-epicatechin to NADPH oxidase-inhibitory methyl ethers. These data identify endothelial NADPH oxidase as candidate target of dietary flavonoids and particularly of their metabolites.

(-)-Epicatechin elevates nitric oxide in endothelial cells via inhibition of NADPH oxidase.

Dietary (-)-epicatechin is known to improve bioactivity of (*)NO in arterial endothelium of humans, but the mode of action is unclear. We used the fluorophore 4,5-diaminofluorescein diacetate to visualize the (*)NO level in living human umbilical vein endothelial cells (HUVEC). Untreated cells showed only a weak signal, whereas pretreatment with (-)-epicatechin (10 microM) or apocynin (100 microM) elevated the (*)NO level. The effects were more pronounced when the cells were treated with angiotensin II with or without preloading of the cells with (*)NO via PAPA-NONOate. While (-)-epicatechin scavenged O2(*-), its O-methylated metabolites prevented O2(*-) generation through inhibition of endothelial NADPH oxidase activity, even more strongly than apocynin. From the effect of 3,5-dinitrocatechol, an inhibitor of catechol-O-methyltransferase (COMT), on HUVEC it is concluded that (-)-epicatechin serves as 'prodrug' for conversion to apocynin-like NADPH oxidase inhibitors. These data indicate an (*)NO-preserving effect of (-)-epicatechin via suppression of O2(*-)-mediated loss of (*)NO.

Protein modification elicited by oxidized low-density lipoprotein (LDL) in endothelial cells: protection by (-)-epicatechin.

The action of oxidatively modified low-density lipoprotein (oxLDL) on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. OxLDL was shown here to elicit oxidative stress in bovine aortic endothelial cells or human umbilical vein endothelial cells, as judged by an increase in 2',7'-dichlorofluorescein fluorescence and elevated levels of carbonylated, nitrated, and 2-hydroxynonenal-coupled proteins. These effects were sensitive to apocynin, indicating involvement of NADPH oxidase. A 170-kDa polypeptide carbonylated upon exposure of cells to oxLDL was identified by immunoprecipitation as EGF receptor. Immunocytochemical visualization by confocal microscopy revealed the highest levels of modified proteins in the perinuclear region. Exposure of endothelial cells to oxLDL led to modulation of the expression levels of *NO synthases; the endothelial isoform (eNOS) was down-regulated via proteasomal degradation, whereas the inducible isoform (iNOS) was up-regulated in an enzymatically active state. eNOS protein was found to be both carbonylated and nitrated upon exposure of cells to oxLDL. iNOS contributed to the generation of modified proteins as judged by the effects of the selective inhibitor L-NIO. These oxLDL-elicited changes in vascular endothelial cells described were suppressed by (-)-epicatechin, a dietary polyphenol, which inhibited NADPH oxidase activity in these cells.

Myeloperoxidase-mediated LDL oxidation and endothelial cell toxicity of oxidized LDL: attenuation by (-)-epicatechin.

Recent data suggest an inverse epidemiological association between intake of flavanol-rich cocoa products and cardiac mortality. Potential beneficial effect of cocoa may be attributed to flavanol-mediated improvement of endothelial function, as well as to enhancement of bioavailability and bioactivity of nitric oxide in vivo. ( - )-Epicatechin is one bioactive flavanol found in cocoa. This review deals with protective actions of ( - )-epicatechin on two key processes in atherogenesis, oxidation of LDL and damage to endothelial cell by oxidized LDL (oxLDL), with emphasis on data from this laboratory. ( - )-Epicatechin not only abrogates or attenuates LDL oxidation but also counteracts deleterious actions of oxLDL on vascular endothelial cells. These protective actions are only partially shared by other vasoprotective agents such as vitamins C and E or aspirin. Thus, ( - )-epicatechin appears to be a pleiotropic protectant for both LDL and endothelial cells.

Cytotoxicity of myeloperoxidase/nitrite-oxidized low-density lipoprotein toward endothelial cells is due to a high 7beta-hydroxycholesterol to 7-ketocholesterol ratio.

Oxygenated cholesterols (oxysterols) formed during oxidation of low-density lipoprotein (LDL) are associated with endothelial dysfunction and atherogenesis. We compared the profile of oxysterols in modified human LDL obtained on reaction with myeloperoxidase/H2O2 plus nitrite (MPO/H2O2/nitrite-oxLDL) with that on Cu2+ -catalyzed oxidation. The 7beta-hydroxycholesterol/7-ketocholesterol ratio was markedly higher in MPO/H2O2/nitrite-oxLDL than in Cu2+ -oxidized LDL (7.9 +/- 3.0 versus 0.94 +/- 0.10). Like MPO/H2O2/nitrite-oxLDL, 7beta-hydroxycholesterol was cytotoxic toward endothelial cells through eliciting oxidative stress. Cytotoxicity was accompanied by DNA fragmentation and was prevented by the NADPH oxidase inhibitor apocynin, suggesting stimulation of NADPH oxidase-mediated O2-* formation. 7-Ketocholesterol was only cytotoxic when added alone, whereas a 1:1-mixture with 7beta-hydroxycholesterol surprisingly was noncytotoxic. We conclude from our data that (i) 7beta-hydroxycholesterol is a pivotal cytotoxic component of oxidized LDL, (ii) 7-ketocholesterol protects against 7beta-hydroxycholesterol in oxysterol mixtures or oxLDL, (iii) the 7beta-hydroxycholesterol/7-ketocholesterol ratio is a crucial determinant for cytotoxicity of oxidized LDL species and oxysterol mixtures, and (iv) the low share of 7-ketocholesterol explains the higher cytotoxicity of MPO/H2O2/nitrite-oxLDL than other forms of oxidized LDL. The dietary polyphenol (-)-epicatechin inhibited not only formation but also cytotoxic actions of both oxLDL and oxysterols.

Epicatechin protects endothelial cells against oxidized LDL and maintains NO synthase.

Intake of flavanol-rich food or beverages was previously shown to ameliorate endothelial function and to enhance bioactivity of nitric oxide with individuals at risk for cardiovascular disease. Here, we examined whether the major dietary flavanol, (-)-epicatechin, counteracts the action of oxidized LDL on endothelial cells, an action considered pivotal for endothelial dysfunction in the pathogenesis of atherosclerosis. Oxidation by myeloperoxidase plus nitrite rendered human LDL cytotoxic towards endothelial cells, more so than oxidation by Cu2+. Oxidized LDL also caused a marked loss of endothelial NO synthase protein which did not occur in the presence of a proteasome inhibitor, lactacystin. Both actions of oxidized LDL, which were not evoked by native LDL, were effectively counteracted by (-)-epicatechin. We conclude that dietary flavanols contribute to protection of the integrity of endothelial cells not only by scavenging free radicals but also by maintaining endothelial NO synthase.