| [BACKGROUND]Coronary heart disease (CHD) can seriously jeopardize our health, and was the leading cause of the death in developed countries.While in our country, with the improvement of living standard and the change of the life style,CHD is becoming the top danger factor affecting our health, the main pathologic basic of which is the atheroscletosis (AS). It is recognized that the abnormal plasma lipid is the vital risk factor contributing to the CHD. In recent half century, multiple researches on epidemicology, clinical pathology and the trials have validated the relationship between the cholesterol and AS. The Framingham Heart Study beginning from the 1948 investigated the pathological factors affecting the CHD among the 28,000 residents and their offspring, and initially introduced the "risk factor" concept in 1961. The follow-up visits conducted within the later thirty years had revealed that the total cholesterol (mainly the LDL-C) level in serum had a positive relationship with the incidence of the CHD. Lots of investigations had identified that with the level of LDL-C decrease 1 percent, the risks of CHD incidence decreased 2 percent. HDL is the key factor for AS. Available data show that among people suffering from the CHD, almost the half had accompanied with the low-HDL. The protection effect of HDL on heart and vessels had been clarified. The possible mechanism is that the cholesterol in the periphery cells is transported to the hepatocyte, namely the reverse cholesterol transport (RCT). Now it is belived that the initiation step of RCT was that cholesterol in periphery tissue cells was transported to blood plasma through cell membrane. Atleast two pathways are involved in the transport process, one is the passive diffusion and the other is the active transport way.However, the gene charging the cholesterol transported from endocyte to extracellular was confirmed as ATP-Binding Cassette Al (ABCA1) gene until 1999. Consequently, the famous magazine Science sang highly of the gene: we knew how the cholesterol entry the cell via the LDL receptor 25 years ago, now we firstly make out how the cholesterol release from the cell. The determination of the function of ABCA1 gene originated from the study of the Tangier disease. Tangier is certain scarce genetic degenerative disease, whose pathological characteristic is the nearly or complete disappearance of the HDL in circulation, and the cholesterol is deposited in the reticuloendothelial cell, the increase incidence of coronary artery disease, accompanying the extremely low and even disappearance of the HDL in plasma. Article published in Nature Genetics in August 1999 initially revealed that the defect of the ABCA1 gene contributed to the Tangier disease. More than 50 ABCA1 gene mutation sites have been reported to be disease-related. The research about ABCA1 gene knock-out mice show that the inactivation of ABCA1 gene can lead to the increase absorption of the cholesterol in ordinary forage, cholesterol-rich forage and the grease-rich forage, which can cause the similar pathological characteristic to the Tangier disease. The study on cell level reveal that there's an obvious dysfunction with the ABCA1 in Tangier's patients. Cultured fibroblasts extracted from Tangier patients and health people separately, then marked by the cholesterol, the result show that the cholesterol outflow rate of Tangier patients mediated by the HDL was much lower than that of the normal ones.The update papers show that the ABCA1 not only mediates the intracellular cholesterol outflow, but also is much related to the formation of the AS and foam cells. On the AHA conference hold in New Orleans in October of 2000, ABC Transporters was addressed and discussed as a special topic. Wilcox observed the artery tissues of health people and the AS patients by using in situ hybridization method, and found that in atherosclerotic area the expression of ABCA1 gene is up-regulated obviously. Panousis further validated that in the macrophage-derived foam cells, ABCA1 mRNA level is three times higher than that of the normal macrophage. Now it is validated that ABCA1 mRNA is expressed in tissues including liver, lung, adrenal gland,placenta, fetus tissue and etc.AS is a chronic inflammation course, the formation of the AS involves vascular endothelial cell, vascular smooth muscle cell and macrophage. It is now believed that in the development of AS, vascular endothelial cell can release some anti-AS and/or AS-promoting cytokines, which can cause cellular dysfunction, but endothelial cell itself does not be foam-like change. The cytokines referred are also released by vascular smooth muscle cells, which can be changed into foam cells by swallowing cholesterol. Affected by chemotatic factor, the monocyte in the blood can enter into the vessel wall and transform into macrophage by swallowing the choleserol. In the initial phase of the AS, Ox-LDL can damage the vascular endothelial cell and thus promote secreting of adhension molecule and chemotatic factors. The ICAM-1 produced by vascular endothelial cell can mediate the monocyte in the blood adhering to the activated vascular endothelial cell. The damaged vascular endothelial cell can also produce MCP-1 which can drive the adherent monocyte to enter into the vessel wall and differentiate to the macrophage, which can then swallow the Ox-LDL and change into the foam cell. Besides, the vascular smooth muscular cell can also produce the ICAM-1 ^ MCP-1 and assistant in the transforming the foam cell. MCP-1 also promotes the differentiation of the vascular smooth muscular cell, leading to its transform from the contraction form to the synthesis form. Consequently the ICAM-1 is excreted increasingly to participate in the formation of the foam cell.It is unclear about the direct relationship between ABCA1 and ICAM-1 and MCP-1. Therefore, targeting the vascular endothelial cell, vascular smooth muscle cell and monocyte, the research is intended to investigate the expression of the ABCA1, ICAM-1, MCP-1 mRNA and their proteins and the expression of IL-1 ￡ in these cells under the stimulation of AS-contributing facor-OxLDL/ABCAl-inducing factor-8-r-cAMP and the change of above index after the cells were cultured with ABCA1 antisense oligonucleotide to clarify the possible mechanism related with the ABCA1 in the form of the AS. [METHODS]Human umbilical vein endothelial cells (ECV304), Human mononuclear cells (THP1), and Human aorta smooth muscle cell (CC2571) were cultured as the targetcells. The following experiments were performed to investigate the effect of 8-Br-cAMP and Ox-LDL on the expressions of ABCAl, ICAM-1, MCP-1 and IL-1 P in these cells. 1. Effects of Ox-LDL, 8-Br-cAMP on the expressions of ABCAl, ICAM-1, andMCP-1 mRNAin ECV304, THP1, and CC2571 cells.ECV304, THP1, and CC2571 cells were cultured, 3-4 passage cells were cultured, with serum-free medium RPMI 1640 or DMEM for 12 hours and then incubated with Ox-LDL(30ng/ml), 8-Br-cAMP (0.5mM) for 3, 6, 12, 24 hours, unstimulated cells cultured 24 hours were as the control. Total RNA was extracted from cells with Promega Extract KIT. Total RNA (5^1) was reverse-transcribed using Promega Reverse Transcription kit, RT React condition: 42 °C 60 minutes, 95°C 5 minutes. Use the SYBR Green kit of the MJ fluorescent quantitation PCR reaction system with the fluorescent quantitation RT~PCR device : SYBR Green regant 5ju\, H2O 3fi\, up and down stream primer (lOpmol/^l) 0.5^1, the reverse-transcript product 1/ul.The primer sequence of ICAM-1: Sense Primer 5' CGA GGT GAC CGT GAA TGT GCT , 3'Anti-sense Primer 5' TGG CTT GTG TGT TCG GTT TCA 3', product length 185 bp. PCR React condition: 95°C 5minutes, 96°C20second, 55°C20second, 72°C20second, 79 °Cl second, 45cycles, 72°C5minutes, melting curve from 65 "C to 98°C, every 0.2°C lsecond plate read.The primer sequence of MCP-1: Sense Primer 5' TCA GCC AGA TGC AAT CAA TGC 3', Anti-sense Primer5' TCC TGA ACC CAC TTC TGC TTG 3', product length 186 bp. PCR React condition: 95 °C Sminutes, 96°C25second, 55°C25second, 72 °C 25second, 79°Clsecond, 45cycles, 72°C5minutes, melting curve from 65°C to 98°C, every 0.2°C lsecond plate read.The primer sequence of ABCAl: Sense Primer 5' GAT GGC AAT CAT GGT CAA TGG 3', Anti-sense Primer 5' AGC TGG TAT TGT AGC ATG TTC CG 3', product length 201 bp. PCR React condition: 95°C 5minutes, 96°C30second, 55°C30second, 72°C30second, 79°Clsecond, 45cycles, 72°C5minutes, melting curve from 65°C to98°C, every 0.2°C lsecond plate read.8-Actin is the inner control. The result from fluorescent quantitation system, Ct is analyzed and compared after dealing with 2"AACt method.2. Effects of Ox-LDL, 8-Br-cAMP on the expressions of ABCAl, ICAM-1, MCP-1, and IL-1 ￡ protein in ECV304, THP1, and CC2571 cells.2.1 ABCAl, ICAM-1, and MCP-1 protein were detected by Western blot:Cells culture was the same as above, total protein were extracted using cell lysis buffer, the total protein (5Qug) were separated on 6% (ABCAl), 7.5% (ICAM-1), 13.5% (MCP-1) SDS-PAGE, and then transferred onto a nitrocellulose membrane. Goat-anti-human ABCAl, Rabbit-anti-human ICAM-1, Mouse-anti-human MCP-1 antibody were added, HRP marked antibody was added , the protein was detected by western blot.2.2 ICAM-1, MCP-1, IL-1B proteins were detected by ELISA:The cells were done the same as above, total protein was extracted and protein changes of ICAM-1, MCP-1, IL-1B were deteced using ELISA as instruction.3. Effects of Ox-LDL, 8-Br-cAMP on the expressions of ABCAl, ICAM-1, MCP-1 mRNA in ECV304, THP1, and CC2571 cells with ABCAl antisense oligonucleotide.The cells were cultured as above, 3-4 passage cells were cultured with serum-free medium RPMI 1640 or DMEM for 12 hours, the antisense oligonucleotide of ABCAl was added to the wells and incubated for 5 hours. Then the cells were washed with D-Hank's, the cells were cultured in complete RPMI1640 or DMEM, eight hours later, incubated with Ox-LDL(30ng/ml), 8-Br-cAMP (0.5mM) for 3, 6,12, and 24 hours, unstimulated cells cultured 24 hours were as the control. The total RNA extraction, reverse transcription, and fluorescent quantitation PCR as protocol 1.4. Effects of Ox-LDL, 8-Br-cAMP on the expressions of ABCAl, ICAM-1, MCP-1, and IL-1 P protein in ECV304, THP1, and CC2571 cells with ABCAl antisense oligonucleotide.The cells were cultured as above, the antisense oligonucleotide of ABCAl wereadded to the wells and incubated for 5 hours. Then the cells were washed with D-Hank's, the cells were cultured in complete RPMI1640 or DMEM, eight hours later, incubated with Ox-LDL(30ng/ml), 8-Br-cAMP (0.5mM) for 3, 6, 12, and 24 hours, unstimulated cells cultured 24 hours were as the control. The total protein was extracted and protein changes of ABCAl, ICAM-1, MCP-1 and IL-1 P were detected using Western blot and ELISA assay. [RESULTS]1. In Human blood vessel endothenial cell ECV304, the mRNA and protein amounts of ABCAl, ICAM-land MCP-1 and also IL-18 protein amount were increased after the incubated with Ox-LDL and 8-Br-cAMP for 6 and 12 hours. After transfection with antisense oligonucleotides of ABCAl, the expressions of ABCAl, ICAM-1, and MCP-1 mRNA were decreased at 3 and 6 hours, and ABCAl, ICAM-1, MCP-1, and IL-18 protein were decreased at 12 and 24 hours.2. In THP1 cells, the mRNA and protein amounts of ABCAl, ICAM-land MCP-1 and also IL-16 protein amount were increased after the incubated with Ox-LDL and 8-Br-cAMP for 6 and 12 hours. After transfection with antisense oligonucleotides of ABCAl, the expressions of ABCAl, ICAM-1, and MCP-1 mRNA were decreased at 3 and 6 hours, and ABCAl, ICAM-1, MCP-1, and IL-1B protein were decreased at 12 and 24 hours.3. In Human blood vessel smooth muscle cell CC2571, the mRNA and protein amounts of ABCAl, ICAM-land MCP-1 and also IL-1B protein amount were increased after the incubated with Ox-LDL for 6 and 12 hours. After transfection with antisense oligonucleotides of ABCAl, the expressive amounts of ABCAl, ICAM-1, and MCP-1 mRNA levels were decreased at 3 and 6 hours, and ABCAl, ICAM-1, MCP-1, and IL-18 protein were decreased at 12 and 24 hours. [CONCLUSIONS]1. ABCAl was expressed in human vascular endothelial cell ECV304, monocyte THP1, and aortic smooth muscle cell CC2571.2. Ox-LDL, 8-Br-cAMP can increase the expressions of IL-1 P , ICAM-1, MCP-1 in human vascular endothelial cell ECV304, monocyte THP1, and aortic smooth muscle cell CC2571.3. The new mechanisms of ABCA1 on atherosclerogenosis may exist: ABCA1 not only introduce cholesterol out-flow from inner cells, but also may increase the expressions of inflammatory cyrokines, ABCA1 may increase ICAM-1 and MCP-1 through up-regulating the expression of IL-1 P , and then promote the development of AS.
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