The Regulation Mechanism Of ABCA1 On The Inflammatory Cytokine And Its Clinical Significance

【Background】The history of studying atherosclerosis (AS) was over 100 years. As early as 1914,Antischkow first suggested that there was a relationship between cholesterol andcholesterol-rich foam cells in the development of AS. As a major cause of myocardialinfarction and cerebral infraction currently, coronary artery disease (CAD) caused bycoronary atherosclerosis has become the top cause of death in some countries andregions. CAD and other cardiovascular diseases serious harm to human health, and ASis the main pathological basis. There were a lot of evidences to support that AS is aninflammatory disease, and oxidized low-density lipoprotein (Ox-LDL) can increaseinflammatory reaction of vascular macrophage.In the start-up phase, the cell toxicity and the role of free radicals of Ox-LDL candamage vascular endothelial cell function and promote cells to secrete adhesionmolecules and chemoattractant factors such as intercellular adhesion molecule-1(ICAM-1), which can mediate monocytes to adhesion to activated endothelial cells.The damaged vascular endothelial cells can also secrete monocyte chemoattractantprotein-1 (MCP-1) that promotes adhesion of monocytes to the vessel wall and differentiate into macrophages, which secrete some inflammatory cytokines includinginterleukin-1β(IL-1β), ICAM-1, MCP-1 and so on. It also increases infiltration ofinflammatory cells, lipid accumulation, and formation of foam cells and plaque in thevascular wall, and finally causes AS. Epidemiological and clinical studies indicated thatplasma low-density lipoprotein cholesterol (LDL-C) level and coronary artery diseaseprevalence rate was positively correlated and primary prevention indicated that risk ofsuffering from coronary artery disease lowered 2% when the LDL-C decreased by 1%.Statin lipid-lowering drug application enables us to have an effective way to lowerLDL-C. But the cholesterol-lowering therapy can only reduce the one third of cardiacevents, how to further reduce the incidence of coronary artery disease and cardiacevents? U.S. Framingham Heart Study and other studies have shown that high-densitylipoprotein cholesterol (HDL-C) levels were negatively correlated with the incidence ofcoronary artery disease. So, how to increase HDL-C has become a hot spot of concernto people.A variety of anti-AS functions of HDL were confirmed, including inhibition oflow-density lipoprotein (LDL) oxidation, improving endothelial cell dysfunction,anti-inflammatory, anti-thrombotic and promoting fibrinolysis. However, HDLexercises its anti-AS functions mainly through participation reverse cholesteroltransport (RCT) in vivo. RCT is refer to that free plasma lipoprotein cholesterol of theperipheral organizations (including the arterial wall) is transported to the liver andexcreted through the liver, which includes two processes: (1) removal of freecholesterol in peripheral cells; (2) transit free cholesterol of HDL to the liver.ATP binding cassette transporter A1 (ABCA1) was a recently identified HDLreceptor and discovered as a gene of active transport cholesterol from intracellular toextracellular in 1999. The ABCA1 in normal peripheral membrane of the cells transitsfree cholesterol and phospholipid to the extracellular, and it combines the apolipoprotein A-I (apo A-I) to form disc HDL that receives free cholesterol andphospholipid to form mature HDL, which starts RCT process.Human ABCA1 gene has 149 kb nucleotides, which contains 50 exons and itscDNA contains 6783 nucleotide. It can code a protein estimated molecular mass of 254kDa and 2261 amino acids. The protein has the following features: There are twoconservative Walker A and B nucleotide-binding domain and two transmembraneregions, and each transmembrane region include six transmembrane helixs, theuniqueness of ABCA1 is decided by the transmembrane region and the energy oftransport activity is provided by the ATP of the nucleotide binding domain. HumanABCA1 mRNA is expressed most in placenta, liver, lung, adrenal and fetal tissues, andleast in the kidney, pancreas, pituitary, bone marrow and breast. Cytological studieshave shown that ABCA1 exists in peripheral cells, including endothelial cells, vascularsmooth muscle cells, monocytes/macrophages, and so on.The determination of the ABCA1 gene function starts from the study of Tangierdisease. Tangier disease is a rare autosomal dominant inherited disease, which isperformance to be enlarged yellow tonsil, hepatosplenomegaly, plasma HDL-C andapoA-I reducing extremely. Because of ABCA1 functional deficiencies, Tangierpatients could not transfer intracellular free cholesterol to extracellular and could notcombine apoA-I to form the disc HDL. The free apoA-I is removed from the plasmasoon, and the apoA-I and HDL in the plasma significantly reduced. There are apparentreverse cholesterol transport dysfunction and low HDL-C in patients of Tangierdisease.The activity of ABCA1 can be regulated by a number of factors. ABCA1 is alipoprotein receptor gene that is induced by cyclic adenosine monophosphate (cAMP).8-Bromo-cAMP and cAMP increase its mRNA, protein expression and cholesterolefflux rate in parallel. Cholesterol, acetylated low density lipoprotein (Ac-LDL) and Ox-LDL can increase ABCA1 mRNA and protein expression, but HDL and apoA-Iinhibite ABCA1 mRNA and protein expression. The mechanism of cholesterol andcAMP to induce ABCA1 is alone and synergies.In normal cells, ABCA1 is rate limited protein in the lipid transport pathwaymediated by the apolipoprotein. It affects not only plasma HDL-C levels and reversecholesterol transport, but also closely links with AS. In situ hybridization method,ABCA1 gene expressions were more in the patient of atherosclerosis than that ofnormal. ABCA1 mRNA amounts in macrophage-derived foam cells were three timeshigher than normal. Research showed that ABCA1 eliminates excessive cholesterol inmacrophages and plays a role in stopping progress of atherosclerosis. But there werealso some additional information that ABCA1 could augment monocyte/macrophagecell expressing IL-1βand oxygen free radicals, as well as their clinical significance isunknown.At present, we have recognized that ABCA1 is the important gene, which canaffect HDL-C levels and reverse cholesterol transport. However, the role of ABCA1 onAS and its mechanism has not yet confirmed entirely. This study intends to investigatethe regulation of ABCA1 on inflammatory factors and effect of ABCA1 on AS in boththe level of cells and human being. The cell level researchs include the effect ofABCA1 on inflammatory cytokines mRNA, protein expression and signal transductionmechanism induced by Ox-LDL or 8-Br-cAMP in human THP-1 macrophage, andthen observe whether there is any pathological significance in cellular histologicalchanges by electron microscope. The human level studies include the impacts ofABCA1 gene polymorphism on inflammatory cytokines and ABCA1 on HDL-C andinflammatory cytokines and their mutual relations in patients with coronary arterydisease. It will be cleared that regulation mechanism of ABCA1 on the inflammationcytokine and its clinical significance. 【Methods, groups and objectives】Cell level studyObjectives: To investigate the impact on inflammatory factor and the signaltransduction mechanism of ABCA1 under the induction of Ox-LDL or 8-Br-cAMP, andto confirm whether Ox-LDL or 8-Br-cAMP causes the pathological morphologicalchanges of the cellular structure.Methods: To observe the effects of Ox-LDL or 8-Br-cAMP on the expressions ofABCA1, ICAM-1, MCP-1 and IL-1βmRNA and protein in THP-1 human macrophagethrough fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR), Western Blot and enzyme-linked immunosorbent assay (ELISA) method,and also to observe changes of these cytokines with ABCA1 antisense oligonucleotide.The signal transduction mechanisms of ABCA1 on cytokines stimulated byOx-LDL or 8-Br-cAMP were observed by ELISA method.Simultaneously, the cellular structure changes of macrophage were observed underthe electron microscope.Clinical level studyObjectives: There were the reporters that suggested the role of ABCA1 on cell differfrom that on the individual. Our research has proved ABCA1 on cell level can regulatethe express of inflammatory factor. To further confirm the relationship between theABCA1 in individual level and inflammatory factor, the effects of ABCA1 singlenucleotide polymorphism (SNP), M883I and R1587K, on HDL-C will be sifted amongthe CAD patients. The effects of ABCA1 genotypes on inflammatory factor,relationship between HDL-C and inflammatory factor, and the regulation role ofABCA1 on inflammatory factor were investigated.Methods: The 112 cases with CAD and the 108 normal cases were investigated. Therewas no significant difference of age and gender between two groups. The exons of 18 and 35 of ABCA1 from the extracted DNA of two groups were amplified throughPrimer-introduced restriction analysis PCR (PIRA-PCR). The M883I of the exon of 18and the R1587K of the exon of 35 were analyzed through restriction endonucleaseEcoR V and BssS I respectively. At the same time, IL-1β,ICAM-1,MCP-1 and highsensitive C reactive protein (Hs-CRP) among the CAD group were tested. Therelationship and relativity among inflammatory factor, genotype, HDL-C were handledstatistically.Procedure of Cell level study1. Culture of THP-1 macrophageAfter revitalization of THP-1 monocyte cell strain, 3-4 passage cells were culturedwith phorbol 12-myristate 13-acetate (PMA, final concentration 100 nmol/L) for 72hours, then changed the cell to THP-1 macrophage and lately cultured with serum-freemedium RPMI 1640 for 12 hours to keep resting state. Add bovine serum albumin(BSA, 2 mg/ml) as surrogate of serum before the addition of stimulators.2. Effects of Ox-LDL or 8-Br-cAMP on the expressions of ABCA1, ICAM-1 andMCP-1mRNA in THP-1 macrophageThe cells were cultured as above, then incubated with Ox-LDL (30 mg/L) or8-Br-cAMP (0.5 mmol/L) for 3, 6, 12, 24 hours respectively and un-stimulated cellscultured 24 hours were as the control. Total RNA was extracted from cells in time andthen ABCA1, ICAM-1 and MCP-1 mRNA were detected by fluorescent quantitationRT-PCR.β-actin was the inner control.3. Effects of Ox-LDL or 8-Br-cAMP on the expressions of ABCA1, ICAM-1 andMCP-1 proteins in THP-1 macrophageThe cells were cultured as above, then incubated with Ox-LDL (30 mg/L) or 8-Br-cAMP (0.5 mmol/L) for 3, 6, 12, 24 hours respectively and un-stimulated cellscultured 24 hours were as the control. Total protein was extracted from cells and thenproteins of ABCA1, ICAM-1 and MCP-1 were detected by immunoblot assay. Everyprotein of ICAM-1, MCP-1 and IL-1βwere also detected using ELISA as instruction.4. Effects of Ox-LDL or 8-Br-cAMP on the expressions of ABCA1, ICAM-1 andMCP-1 mRNA in THP-1 macrophage with ABCA1 antisense oligonucleotideThe antisense oligonucleotide of ABCA1 was added to the cells and incubated for5 hours. Then the cells were cultured in complete RPMI 1640 with BSA (2 mg/ml),stimulated by Ox-LDL (30 mg/L) or 8-Br-cAMP (0.5 mmol/L) for 3, 6, 12 and 24hours, un-stimulated cells cultured 24 hours were as the control. At the time, ABCA1,ICAM-1 and MCP-1 mRNA were detected using fluorescent quantitation RT-PCR.5. Effects of Ox-LDL or 8-Br-cAMP on the expressions of ABCA1, ICAM-1,MCP-1 and IL-1βprotein in THP-1 macrophage with ABCA1 antisenseoligonucleotideThe antisense oligonucleotide of ABCA1 were added to the cells and incubated for5 hours. Then the cells were cultured in complete RPMI 1640 with BSA (2 mg/ml),stimulated Ox-LDL (30 mg/L) or 8-Br-cAMP (0.5 mmol/L) for 3, 6, 12, and 24 hours,un-stimulated cells cultured 24 hours were as the control. The total protein wasextracted at time and ABCA1, ICAM-1 and MCP-1 protein were detectedbyimmunoblot assay. Every protein changes of ICAM-1, MCP-1 and IL-1βwere alsodetected using ELISA assay.6. Signal transduction mechanism of ABCA1 on expression of inflammatory factorin THP-1 macrophage induced by Ox-LDL or 8-Br-cAMPOx-LDL: The study includes control group, Ox-LDL group, antisenseoligonucleotide group and p38 blocker group. The cells were cultured as abovementioned, the ABCA1 antisense oligonucleotide and p38 signal transduction blocker were respectively added into antisense oligonucleotide group and p38 blocker group for5 hours, and then cells were cultured with Ox-LDL (30 mg/L) including BSA (2 mg/ml)for 24 hours. THP-1 macrophage cells in Ox-LDL group were incubated withOx-LDL (30 mg/L) including BSA (2 mg/ml) for 24 hours and un-stimulated cellscultured 24 hours were as the control. IL-1βprotein was detected using ELISA assay.8-Br-cAMP: The study includes control group, 8-Br-cAMP group, antisenseoligonucleotide group and p38 blocker group. The cells were cultured as above, theABCA1 antisense oligonucleotide and p38 blocker signal transduction blocker wererespectively added into antisense oligonucleotide group and p38 blocker group for 5hours, and then cells were cultured with 8-Br-cAMP (0.5 mmol/L) including BSA (2mg/ml) for 24 hours. THP-1 macrophage cells in 8-Br-cAMP group were incubatedwith 8-Br-cAMP (0.5 mmol/L) including, BSA (2 mg/ml) for 24 hours andun-stimulated cells cultured 24 hours were as the control. IL-1βprotein was detectedusing ELISA assay.7. Effect of Ox-LDL or 8-Br-cAMP stimulation on microstrucre of macrophageand protective effect of ABCA1 antisense oligonucleotide and p38 blockerCell culture and groups were the same as that of signal transduction mechanismstudy. Transmission electron microscope (TEM) sample preparation: After 24 hoursincubation, cells were centrifuged at 1500 rpm for 5 minntes. This was followed by theaddition of 4℃2.5% glutaraldehyde to pre-fix for 48 hours, and then postfix foranother 1.5 hours by osmic acid after poaching. After dried out step by step, the samplewas embedded, sliced and dyed by uranyl acetate and lead citrate prior to observationunder HITACHI H-600 transmission electron microscope.Procedure of Clinical level study1. The determination of the observed cases The cases were divided into two groups: the coronary artery disease group inwhich there were 112 cases at the average age of 60.76±11.27 years old, included 79males and 33 females; the normal group in which there were 118 cases at the averageage of 59.89±7.92 years old, included 79 males and 33 females. There was nosignificant difference of age and gender between two groups.2. The test of inflammatory factor, biochemical index and plasma lipidThe plasma was separated from the venous blood that was drawn in the morning.Then the IL-1β, ICAM-1, MCP-1, Hs-CRP, total cholesterol (TC), triglyceride (TG),HDL-C, LDL-C and very low density lipoprotein cholesterol (VLDL-C) were tested byELISA, immunoenzyme technique and direct one-step method respectively.3. PCR amplification and analysis of restriction endonucleaseThe snatches of R1587K of the exon 35 and that of M883I of the exon 18 ofABCA1 were amplified by PIRA-PCR. The PIRA-PCR system is of 20μl, amongwhich included PCR products of 8μl, 10×NEB Buffer of 2μl,deionized water of 9μl,the restriction endonuclease of BssS I 4U for R1587K or of EcoR V 4U for M883I.Water-bath it at 37℃overnight and analyze the results with 2% agarose gelelectrophoresis.【Results】1. Effects of ABCA1 on the mechanism and significance of the expression ofmRNA and protein of inflammatory cytokines in THP-1 macrophage induced byOx-LDLThe expression of ABCA1, ICAM-1 and MCP-1 mRNA and protein increased asOx-LDL stimulation for three hours and the peak period of mRNA expression was 6hours, while the peak period of protein expression was 12 hours. After transfection of antisense oligonucleotide and stimulation of Ox-LDL for 3, 6 hours, the expression ofABCA1, ICAM-1 and MCP-1mRNA decreased, while the expression of proteindecreased at 12, 24 hours.After adding ABCA1 antisense oligonucleotides or p38 blocker for stimulationfollowed by Ox-LDL for induction, the level of IL-1βin the THP-1 was lowersignificantly than that in the cells of Ox-LDL induced group.Electron microscopy revealed that the number of organelle was decreased inOx-LDL induced cells. The structure of mitochondrion and endoplasmic reticulum wasnot clear, and some mitochondrial cristae was fuzzy. Bubble increased significantly.Impaired organelles that were wrapped in the endoplasmic reticulum can be seen in thecells. Nuclear around gap widened and matrix scattered. ABCA1 antisenseoligonucleotides and p38 blockers can significantly lightened the damage of cellstructure.2. Effects of ABCA1 on the mechanism and significance of the expression ofmRNA and protein of inflammatory cytokine in THP-1 macrophage cells inducedby 8-Br-cAMPThe expression of ABCA1, ICAM-1 and MCP-1 mRNA and protein increased as8-Br-cAMP stimulation for three hours and the peak period of mRNA expression was 6hours, while the peak period of protein expression was 12 hours. After transfection ofantisense oligonucleotide and stimulation of 8-Br-cAMP for 3, 6 hours, the expressionof mRNA decreased, while the expression of protein decreased at 12, 24 hours.After adding ABCA1 antisense oligonucleotides or p38 blocker for stimulation,followed by 8-Br-cAMP induction, the level of IL-1βin the THP-1 was lowersignificantly than that in the cells of 8-Br-cAMP induced group.Electron microscopy revealed that the cell membrane structure of THP-1 controlgroup, 8-Br-cAMP group, antisense oligonucleotide group and p38 blocker group was clear. Mitochondria were clearly seen in the cytoplasm. The rough endoplasmicreticulum, lysosomes, phagosome and nuclear membrane were clear, and the nucleuswas normal.3. The influence of ABCA1 on the expression of HDL-C and inflammatorycytokines in plasma of patients with coronary artery disease and their mutualrelationsThe HDL-C levels of K-Carrier in CAD group and the controls were significantlylower than that of non-K-Carrier for R1587K site. The influence of R1587K genotypeson the level of HDL-C was significant. However, the R1587K genotypes had notsignificant affect on inflammatory cytokines (IL-1β, ICAM-1, MCP-1, Hs-CRP). Therewas no correlation between the levels of HDL-C and inflammatory cytokines. M883Igenotypes had no significant effect on HDL-C level in patients with coronary arterydisease, and the inflammatory cytokines of each genotype had no significant change.The joint analysis of the polymorphism of R1587K and M883I on incidence risk ofcoronary artery disease showed that compared to those who carry the genotypes of1587RR and 883MM/MI, there was no significant decrease in the incidence risk ofcases in those with other genotypes combinations.【Conclusions】1. ABCA1 can increase the expression of inflammatory cytokines including IL-1β,ICAM-1 and MCP-1 induced by Ox-LDL in the THP-1 human macrophages andOx-LDL caused structural damage of macrophages.2. ABCA1 can increase the expression of inflammatory cytokines including IL-1β,ICAM-1 and MCP-1 induced by 8-Br-cAMP in the THP-1 human macrophages, butunlike Ox-LDL, 8-Br-cAMP did not cause structural damage of macrophages.3. ABCA1 in THP-1 human macrophages increased the expression both of ICAM-1and MCP-1 induced by Ox-LDL/8-Br-cAMP that might through the pathway of p38-ABCA1-IL-1β.4. The polymorphisms of R1587K and M883I in ABCA1 did not affect the expressionof inflammatory cytokines (IL-1β, ICAM-1, MCP-1, Hs-CRP). The R1587K regulatedthe levels of HDL-C in patients with coronary artery disease and control people, buthad no effect on the expression of inflammatory cytokines of plasma in patients withcoronary artery disease.5. At cell level, ABCA1 not only increased the outflow of cholesterol in cells, but alsoincreased the expression of inflammatory cytokines in THP-1 human macrophages. Athuman level, ABCA1 R1587K only affected the level of HDL-C, but did not influencethe expression of inflammatory cytokines.