The Impact Of Smoking And Smoking Cessation On Coronary Heart Disease In Patients With High-density Lipoprotein Function

BACKGROUND:Smoking is an important risk factor for the development of cardiovascular disease. A large number of epidemiological investigations and clinical studies have shown that the increase in cardiovascular events was associated with cigarette smoking dose-and time-dependently. For patients with existing coronary heart disease (CHD), smoking cessation can reduce the risk of myocardial infarction.Tobacco contains more than4000kinds of harmful substances, among which more than40kinds of chemicals are carcinogenic, and many of them are harmful for cardiovascular disease and lung disease, including nicotine, tar, nitrosamines, polycyclic aromatic hydrocarbons, cyanide of hydrogen, aldehydes, carbon monoxide and heavy metals (including cadmium, aluminum, lead, mercury, etc).To date, the potential mechanisms of deterioration of atherosclerosis by smoking and vascular benefit by smoking cessation have not yet been clarified. Smoking can affect all stages of atherosclerosis from endothelial dysfunction to acute clinical events. Quitting smoking can increase the compliance of small arteries, improve endothelial function, thereby, improve arterial stiffness, and reduce platelet aggregation and platelet redox imbalance in long-term smoking patients. But almost no research focuses on the functional link of smoking and lipoprotein. There is no data to study the impact of smoking on high density lipoprotein (HDL) function and reverse cholesterol transport. As we know that the composition of tobacco, including nicotine, carbon monoxide and tar etc. may impact on lipid metabolism, however, no research on the effects of such ingredient on lipid function was carried out.OBJECTIVES:The aim of our study was to investigate whether the anti-oxidant action, anti-chemotatic property of HDL and HDL-induced macrophages cholesterol efflux in healthy chronic smokers and coronary heart disease smokers would change after 3-month smoking cessation, and to explore the effects of nicotine and carbon monoxide on HDL functionality.METHODS:1. Healthy chronic smokers and chronic smokers with coronary artery disease (CAD) were enrolled and randomized into smoking continue group and smoking cessation group. Participants in smoking continue group would keep on smoking in the next3months, and participants in smoking cessation group would completely quit smoking for3months. Blood samples were drawn before and after follow-up period and stored in-80℃for further use. Healthy nonsmokers were also enrolled as normal controls.2. HDL was isolated by density gradient centrifugation, its concentration was measured by Lowry method and its purity was approved by agarose gel electrophoresis.3. Optical density of conjugated dienes, the products of lipoprotein oxidation induced by Cu2+, was detectd at234nm, to investigate the effects of HDL protecting LDL from oxidation.4. Migration of THP-1derived macrophages were counted in Transwell chamber to investigate the anti-chemotatic effects of HDL.5. NBD cholesterol was tested by fluorescence spectrometry to calculate HDL-induced cholesterol efflux.6. Plasma level of matrix metalloproteinases-9(MMP-9), interleukin-6(IL-6) and high sensitive C reactive protein (hs-CRP) were measured by enzyme linked immunosorbent assay (ELISA).7. Plasma activity of paraoxonase-1(PON-1) was tested by fluorescence spectrometry.8. The anti-oxidant ability, anti-chemotatic ability of HDL and HDL-mediated cholesterol efflux in healthy participants were investigated with nicotine or carbon monoxide-coincubated HDL (methods mentioned above).9. Statistical analysis:SPSS17.0software was used. Data were presented as mean± SD if date were normally distributed. If not, data were transformed logarithmicly into normal distribution. Differences among groups were determined using ANOVA methods followed by the Student-Newman-Keuls test for comparisons across multiple groups. Paird samples were determined by t-test. Statistical significance was assigned for P<0.05.RESULTS:1. Compared with healthy non-smokers, total bilirubin (P=0.018) and HDL-c (P=0.037) were lower in smokers. γ-glutamyltranspeptidase (GGT, P=0.011), triglyceride (P=0.001) and apolipoprotein B (P=0.041) were higher in CAD smokers. GGT and apoipoprotein B were higher (P=0.007and0.015, respectively) in healthy smokers. After3-month follow-up, ApoAl in CAD smoking cessation group was increased (P=0.005) compared with CAD smoking continue group. No other significant changes were observed among groups.2. LDL baseline OD was lower in non-smokers than that in smokers (P<0.001). It was higher in CAD smokers than that in healthy smokers before follow-up (P<0.001,). After3-month follow-up, it didn't change significantly in both CAD smoking cessation group (P=0.254) and healthy smoking cessation group (P=0.158). Lagtime of mixed lipoprotein (HDL+LDLpool, reflecting the protective effect of HDL on LDL oxidation) was longer in non-smoker than that in smokers (P=0.001). It was shortened in CAD smokers than that in healthy smokers before follow-up (P<0.001). Vmax was lower in non-smoker than that in smokers (P=0.001). It was significantly higher in CAD smokers than that in healthy smokers (P<0.001). After3-month follow-up, lagtime in both CAD smoking cessation group and healthy smoking cessatoin group were significantly prolonged (P<0.001and P=0.025). Vmax in both CAD smoking cessation group and healthy smoking cessation group were significantly reduced (both P<0.001). Maximum OD was lower in non-smokers than that in smokers (P<0.001). It was significantly higher in CAD smokers than that in healthy smokers (P<0.001). After3-month follow-up, Maximum OD in both CAD smoking cessation group and healthy smoking cessation group were significantly reduced (both P<0.001). 3. For LDL-coincubated THP-1derived macrophages, there were less migrated cells (passing through and attaching to the polycarbonate membrane) in non-smokers than that in smokers (P<0.001). There were more cells in CAD smokers than that in healthy smokers (P<0.001). After3-month follow-up, it didn't change significantly in both CAD smoking cessation group (P=0.166) and healthy smoking cessation group (P=0.572). For HDL+LDL-coincubated macrophages, there were less migrated cells in non-smokers than that in smokers (P<0.001). There were more cells in CAD smokers than that in healthy smokers (P<0.001). After3-month follow-up, it was significantly reduced in CAD smoking cessation group (P<0.001) but not obviously changed in healthy smoking cessation group (P=0.2).4. NBD cholesterol efflux in macrophages mediated by HDL in non-smokers was higher than that in smokers (P=0.001). It was significantly lower in CAD smokers than that in healthy smokers (P<0.001). After3month-follow-up, it didn't change significantly in both CAD smoking cessation group (P=0.19) and healthy smoking cessation group (P=0.768).5. The activity of PON-1was higher (P<0.001), the level of MMP-9, hs-CRP and IL-6was lower (all P<0.001) in non-smokers than that in smokers. There was no statistical difference in CAD smokers and healthy smokers in PON-1activity and level of MMP-9, hs-CRP, IL-6in plasma (P=0.494,0.710,0.359and0.698). After3-month follow-up, activity of PON-1was significantly enhanced (P<0.001), level of MMP-9was significantly reduced (P<0.001), level of hs-CRP was also significantly reduced (P<0.001) in healthy smoking cessation group. Level of hs-CRP in CAD smoking cessation group was significantly reduced (P=0.002).6. CORM-2(a release of carbon monoxide compounds) dose-dependently reduced the anti-oxidant ability of HDL (reflected in shorter lagtime,5μM CORM-2vs control: P=0.013;10μM CORM-2vs control:P=0.002), had little effect on the anti-chemotatic ability of HDL. Nicotine dose-dependently enhanced the anti-chemotatic ability of HDL (reflected in less migrated macrophages,75μM nicotine vs control:P=0.031;100μM nicotine vs control:P=0.007), had little effect on the anti-oxidant ability of HDL. Both of nicotine and CORM-2have little effects on cholesterol efflux induced by HDL in macrophages (data is not shown).CONCLUSIONS:1. Anti-oxidant ability, anti-chemotatic ability of HDL and HDL-mediated cholesterol efflux in chronic smokers were significantly lower than those in healthy non-smokers.2. Anti-oxidant ability, anti-chemotatic ability of HDL and HDL-mediated cholesterol efflux in chronic smokers with coronary artery disease were significantly lower than those in healthy chronic smokers.3. Anti-oxidant ability, anti-chemotatic ability of HDL, but not HDL-mediated cholesterol efflux in healthy chronic smokers were recovered after3-month's smoking cessation.4. Plasma activity of PON-1in chronic smokers was significantly lower than that in healthy non-smokers, level of MMP-9, IL-6and hs-CRP in chronic smokers were significantly higher than those in healthy non-smokers. Smoking cessation increased plasma activity of PON-1and dereased level of MMP-9and hs-CRP in healthy chronic smokers, while merely derease hs-CRP level in chronic smokers with coronary artery disease.5. Nicotine dose-dependently increased the anti-chemotatic ability of HDL, but carbon monoxide dose-dependently decreased the anti-oxidant ability of HDL.