Effects Of Smoking And Smoking Cessation In Patients With Coronary Heart Disease Macrophage Cholesterol Efflux

Background:Cigarette smoking is an independent risk factor of coronary heart disease and has been shown to induce significant cardiovascular changes characterized by endothelial dysfunction and vascular remodeling. Coronary Artery Disease (CAD) is not only the most common disease in our country, but also one of the most important culprits for morbidity and mortality. It was commen consent that plasma lipid profile was disturbed by chronic cigarette smoking, while the mechanism involved was not clear. Whether human macrophage was affected by cigarette smoking was not clear. In atherosclerotic lesions, the primary cell type is foam cells, which are macrophages overloaded with cholesterol ester. ATP-binding cassette transporters A1(ABCA1) and G1(ABCG1) play pivotal roles in mediated intercellular cholesterol efflux maintained the homeostasis of cholesterol. Many studies indicated dysfunction of ABCA1mediated cholesterol efflux to apolipoprotein A-1(apoA-1) was associated with atherosclerotic diseases. ABCG1mediated intercellular cholesterol efflux to HDL shown to be coordinated to ABCA1in removal of excessive cellular cholesterol, although whether ABCG1is atheroprotective factor remained to be discussed. So we investigated the impact of cigarette smoking and smoking cessation on human macrophage function of cholesterol metabolism by a new method NBD-cholesterol for assay macrophages intecelluar cholesterol efflux. Component of tobacco including nicotine and carbon monoxide was also examized in cultured THP-1derived macrophages.Objective:1. Anlysis plasma lipid profile, compared the lipid profile change of smoking cessation with of continued smoking and.2. Investigate the ABCA1and ABCG1expression and function of mediated intercellular efflux in macrophages from healthy non-smokers, healthy smokers and CAD smokers.3. Investigate the change of ABCA1and ABCG1expression and function in macrophages before and after smoking cessation.4. Study the effect of nicotine and CO on THP-1derived macrophage cultured in vitro.Methods:1. Established a new method to evaluated macrophage intercellular cholesterol efflux by NBD-cholesterol.2. PBMCs collected from all the subjects at the baseline were cultured and differentiated into macrophages. Determine macrophages intercellular cholesterol effluxe rate mediated by ABCA1and ABCG1respectively. Lipid profile of subjects was recorded.3. Smoking subjects were randomly assigned in a1:1ratio to either smoking cessation subgroup or continued smoking subgroup. Smokers randomized into smoking cessation subgroup were asked to stop smoking for at least90days while others follow their habit. After three months follow up, PBMCs were collected for assay of ABCA1and ABCG1funtion and expression. Lipid profile of subjects was recorded again.4. Investigate the effect of nicotine and CORM-2on THP-1derived macrophages.Results:1. Percentage efflux of NBD-cholesterol was significantly correlated to that of [3H]-cholesterol using either apoA-1or HDL as lipid acceptor in human macrophages. Fluorescent NBD-cholesterol can be used as a sensitive and specific probe for cholesterol efflux assay in macrophages.2. Lipid profile in different group of subjects at baseline:1) apoA-1:healthy non-smoker (1.501±0.064g/L)>healthy smoker (1.338±0.044g/L)>CAD smoker (1.349±0.043g/L)(p=0.022and p=0.048).2) HDL-cholesterol:Healthy non-smoker (1.389■0.065mmol/L)>healthy smoker (1.167±0.053mmol/L)> CAD smoker (1.098±0.051mmol/L)(p=0.008and p=0.001).3) TC and LDL-C:No significant different betweent all three group. TG:healthy non-smoker (0.996±0.165mmol/L)<healthy smoker (1.868±0.219mmol/L)(p=0.007). apoB:healthy non-smoker (0.773±0.063g/L)<healthy smoker (0.934■0.036g/L)(p=0.007).3. ABCA1protein expression was obviously inhibited in macrophages from both CAD smokers and healthy smokers compared with that from non-smokers, it was even lower in macrophages from CAD subjects than that from healthy smokers. ABCA1mRNA in macrophages from CAD smokers was significantly reduced compared with that from non-smokers while it was augmented dramatically in macrophages from healthy smokers compared with that from non-smokers. There was no difference in ABCG1protein expression in each group.4. ABCA1mediated cholesterol efflux to apoA-1was attenuated in macrophages from CAD smokers compared with that from non-smoke subjects (8.15%±1.5vs.15.30%±5.70, p<0.001). It was also diminished in healthy smokers compared with that in non-smokers (9.97%±2.00vs.15.30%±5.70, p<0.001). ABCA1mediated cholesterol efflux was even lower in CAD smokers compared with that in healthy smokers (8.15%±1.52vs.9.97%±2.00, p=0.017).5. Both protein and mRNA expression of ABCA1in macrophages from CAD smoking cessation subjects were recovered after3months smoking cessation. After three months follow up, ABCA1mediated cholesterol efflux to apoA-1was dramatically increased in macrophages from CAD smoking cessation subjects (8.14%±1.61vs.11.47%±3.61, p=0.004) but not from CAD continue smoking subjects (8.16%±1.55vs.8.62%±1.49, p=0.473). The net difference of ABCA1mediated cholesterol efflux between CAD smoking cessation group and smoking continue group after three months follow up was statistically significant (3.33%±3.72vs.0.64%±1.97, p=0.036). ABCG1mRNA expression was up-regulated dramatically (p<0.001) in macrophages from CAD smokers after3months tobacco quit, while ABCG1protein expression and function was not improved.6. In healthy smokers, there was no obvious improvement in ABCA1protein expression in macrophages, although ABCA1mRNA expression was increased significantly after smoking cessation (p<0.001). ABCA-1mediated cholesterol efflux to apoA-1was improved in macrophages from both healthy smoking cessation group (10.61%±1.84vs.11.53%■2.78, p=0.002) and continue smoking group (9.33%±2.01vs.11.10%±1.96, p=0.137) after3months follow-up. There was a tendency of increased cholesterol efflux in smoking cessation group compared with that in parallel control group (1.77%±1.57vs.0.92%±1.98, p=0.255). ABCG1expression and function appeared to be no changes in macrophages from healthy smokers after tobacco weaning.7. ABCA1and CD36were upregulated by the stimulation of nicotine in THP-1derived macrophages cultured in vitro. ABCA1was also upregulated in macrophages from non-smoker who smokerd10cigarettes within one hour. ABCA1expression was not affected by the stimulation of CORM-2released CO.Conculsions: Cigarette smoking is associated with impaired intercellular cholesterol efflux resulting from the inhibition of ABC A1expression and function, which may lead to the reduction of plasma HDL-cholesterol level. ABCA1expression as well as its function could be reversed by tobacco abstinence in CAD patients. ABCG1might not play a role in tobacco induced changes of macrophage cholesterol metabolism. Nicotine, as an addictive component in tobacco, upregulated ABCA1and CD36expression in cultured THP-1derived macrophages while ABCA1was not affected by the stimulation of carbon monoxide.