The Effects Of Cigarette Smoking On Macrophages Reverse Cholesterol Transport In Vivo And In Vitro Studies

BackgroundCigarette smoking is an independent risk factor for atherosclerosis and coronary heart disease. Numerous studies have demonstrated that cigarette smoking influences all phases of atherosclerosis, which has an impact on endothelial dysfunction, platelet activation, increasing oxidative stress and inflammation as well as alterations in the lipid profile. However, the exact mechanism by which cigarette smoking increases the risk of atherosclerosis is not completely clarified. The formation of atherosclerotic lesion is a complex process. The accumulation of excess cholesterol in macrophages contributes to form foam cells, which is the hallmark of the atherosclerotic lesion. Reverse cholesterol transport is a process by which cholesterol in peripheral tissues is transported to the liver for excretion in the bile and feces, which can reduce the cholesterol deposition in the vascular wall and prevent atherosclerotic plaque formation. Therefore, reverse cholesterol transport is thought to be a protective mechanism against atherosclerosis. Cholesterol efflux is the first and also the rate-limiting step in macrophages reverse cholesterol transport. The rate of cellular cholesterol efflux is dependent on the expression of cholesterol transporters ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), scavenger receptor B1 (SR-B1) and the composition and concentration of extracellular cholesterol acceptor high density lipoprotein (HDL). The final step of macrophages reverse cholesterol transport involve liver uptake of HDL-derived cholesterol and excretion in the bile and feces. ATP-binding cassette transporters G5 (ABCG5) and ATP-binding cassette transporters G8 (ABCG8) heterodimerize into a functional complex ABCG5/G8 that is crucial for hepatobiliary and intestinal sterol excretion. Therefore, ABCG5/G8 also plays an important role in macrophages reverse cholesterol transport. Numerous studies have' demonstrated that cigarette smoking can decrease HDL cholesterol concentration and promote HDL to be oxidatively modified. In addition, cigarette smoking has an impact on ABCG5 and ABCG8. However, it is not clear whether cigarette smoking impairs macrophages reverse cholesterol transport in vivo.ObjectiveA surrogate approach was developed to measure macrophages reverse cholesterol transport after the C57BL/6J mice were exposed to cigarette smoke for 8 weeks. The aim of this study was to study the effects of cigarette smoking on macrophages reverse cholesterol transport in vivo and the potential mechanisms. We also investigated the effects of mice serum and cigarette smoke extract (CSE) on macrophages cholesterol efflux and the potential mechanisms. MethodsFourteen healthy male C57BL/6 mice were randomly divided into control group and cigarette smoking group (n=7 each group). C57BL/6J mice were exposed to cigarette smoke for 8 weeks. We injected the mice intraperitoneally with 3H-cholesterol labeled and acetylated LDL (Ac-LDL) loaded macrophages. After 48 hours, serum, liver, bile and feces were collected for detecting the amounts of 3H-cholesterol. ABCG5 and ABCG8 mRNA and protein expressions in liver and intestine were determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot respectively. RAW264.7 macrophages were labeled and loaded identically with media containing Ac-LDL (50μg/ml) and 3H-cholesterol (1μCi/ml) for 24 hours. RAW264.7 macrophages were treated with mice serum or different concentrations of CSE. We determined the rate of macrophages cholesterol efflux mediated by liquid scintillation counting. We also detected the protein expressions of ABCA1, ABCG1, SR-B1, liver X receptorα(LXRα) and peroxisome proliferator-activated receptorγ(PPARγ) in macrophages by western blot. We also determined the effect of N-acetylcysteine (NAC) or PPARγagonist 15d-PGJ2 on macrophages cholesterol efflux and cholesterol transporters.Results1. Compared with the control group, the amounts of 3H-cholesterol in serum, liver, bile and feces were significantly decreased in cigarette smoking group.2. The rate of macrophages cholesterol efflux to mice serum was significantly decreased in cigarette smoking group than that in control group.3. Compared with control group, ABCG5 and ABCG8 mRNA and protein expressions in liver and intestine were significantly decreased in cigarette smoking group.4. After the C57BL/6J mice were exposed to cigarette smoke for 8 weeks, the mice of cigarette smoking group had lower HDL cholesterol, while had higher serum triglycerides (TG) and maleic dialdehyde (MDA) than those of the control group.5. Pretreatment the antioxidant NAC with the serum from cigarette smoking group for 1 hour, NAC significantly increased the rate of macrophages cholesterol efflux mediated by serum from the cigarette smoking group.6. Treated with different concentrations of CSE for 24 hours, the macrophages cholesterol efflux mediated by HDL was dose-dependently decreased.7. CSE with different concentrations dose-dependently decreased the protein expressions of ABCA1, ABCG1, LXRαand PPARγin macrophages. However, CSE had no effect on the protein expression of SR-B1.8. Pretreatment with the PPARγagonist 15d-PGJ2 for 1 hour could significantly increased macrophages cholesterol efflux induced by 10% CSE. Compared with the 10% CSE group, PPARγagonist 15d-PGJ2 could significantly increase the protein expressions of ABCA1, ABCG1 and LXRαin macrophages. However,15d-PGJ2 had no effect on the protein expression of SR-B1.Conclusions1. Cigarette smoking impaired macrophages reverse cholesterol transport in mice.2. Cigarette smoking decreased the mRNA and protein expression of ABCG5 and ABCG8 in liver and intestine of mice.3. The effect of cigarette smoking decreasing macrophages cholesterol efflux mediated by mice serum was attributed to its effect on decreasing HDL cholesterol and its effect on promoting HDL to be oxidatively modified.4. The effect of CSE on PPARγ-LXRαpathway could decrease the protein expressions of ABCA1 and ABCG1 in macrophages, which contributed to decrease macrophages cholesterol efflux.5. The macrophages reverse cholesterol transport was decreased in cigarette smoking group, which was possibly attributed to the decreased macrophage cholesterol efflux mediated by mice serum and the decreased mRNA and protein expressions of ABCG5 and ABCG8 in liver and intestine.