Effects Of PPAR-agonists On ATP-Binding Cassette Transporter A1 Expression And Cholesterol Efflux In THP-1 Macrophages

Atherosclerosis (AS) is one of the leading causes of death in the developed and developing country worldwide, in which lipid accumulation and foam cells formation are the key steps of disease progression. As an antiatherogenesis gene, ATP-binding cassette transporter A1 (ABCA1) can induce cholesterol efflux from cells and inhibit development of AS. The human ABCA1 gene has been mapped to chromosome 9q3.1 and is composed of 50 exons and 49 introns, which encode 2261 amino acid residues. It is expressed in multitude of human tissues with highest expression levels in placenta, liver, lung, adrenal glands and fetal tissues. ABCA1 is upregulated by cholesterol influx in macrophages and it is suppressed by HDL3 mediated cholesterol efflux. ABCA1 deficiency causes both Tangier disease and familial HDL deficiency, characterized by accumulation of cholesterol esters in various tissues. ABCA1 is dramatically induced in lipid-loaded macrophages and facilitates the efflux of cellular cholesterol to extracellular apoAI or HDL. ABCA1 activation is to be the first step of the reverse cholesterol transport (RCT) pathway and is therefore important in the control of plasma level of HDL, which is an important factor in the development of antiatherogenesis.Peroxisome proliferators-activated receptors (PPARs) are nuclear receptors, which are involved lipid and glucose metabolism. PPARs isoforms are PPARα, PPARβ(σ), and PPARγ. PPARαis highly expressed in liver, heart, muscle, kidney and cells of arterial wall, such as endothelial cells and macrophages. PPARβis expressed broadly in many tissues, which is known little and have no specific ligands till now. PPARγis expressed highly in white adipose tissue, immune system and small intestine. PPARs bind up ligand upon heterodimerization with the retinoid X receptor (RXR), which function is as ligand-activated transcriptional regulators of genes. It is reported that PPARs have been exerted antiatherogenic effect.Liver X receptors (LXRs) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism. LXRs function as nuclear cholesterol sensors that are activated in response to elevated intracellular cholesterol levels. Once activated, LXRs induce the expression of an array of genes involved in cholesterol absorption, efflux, transport, and excretion. In addition to their function in lipid metabolism, LXRs have also been found to modulate immune and inflammatory responses in macrophages. LXRs have two isoforms: LXRαand LXRβ. LXRs form obligate heterodimers with retinoid X receptors (RXRs), and LXR/RXR heterodimers recognize and bind specific DNA response elements. Analysis of gene expression have confirmed that LXRαregulates a number of target genes involved in both cholesterol and fatty acid metabolism in liver, macrophages and intestine.The observation that LXRαis responsive to fatty acids and is expressed in metabolic tissues suggests that it also plays a general role in lipid metabolismPPARαagonist clofibrate and PPARγagonist troglitazone were investigated if they influenced cholesterol efflux through stimulatory of ABCA1 pathway in our research.The major results were summarized as follows:1. From human fresh serum LDL and HDL were separated and identified by one step density gradient ultracentrifugation. And LDL was oxidated to ox-LDL using Cu2+, and identified by 0.5% agarose gel electrophoresis.2.Using RT-PCR LXRαmRNA expression was induced by PPARαagonist clofibrate in human THP-1 derived macrophages by 200nmol/L PMA for 48 hours at the different times of 0, 4, 8, 12 and 24 hours. Using Western Blot analysis LXRαprotein expression was upregulated by clofibate in THP-1 derived macrophages at the different times of 0, 4, 8, 12 and 24 hours. And the expressions were all in the time-dependent manners.3.Using RT-PCR ABCA1 mRNA expression were induced by PPARαagonist clofibrate and PPARγagonist troglitazone respectively in human THP-1 derived macrophages at the different times of 0, 4, 8, 12 and 24 hours. Using Western Blot analysis ABCA1 protein expression was upregulated by clofibrate and troglitazone respectively in THP-1 derived macrophages at the different times of 0, 4, 8, 12 and 24 hours.4. In human macrophage-derived foam cells induced by 50mg/L ox-LDL lipid-loaded for 24 hours, using cholesterol efflux experiment we concluded that clofibrate and troglitazone respectively promoted cholesterol removal, and the removal are statistical significance in time-dependent manner. It is demonstrated that PPARαagonist clofibrate can induce LXRαexpression at the mRNA level and protein level. And it is proved that PPARαagonist clofibrate and PPARγagonist troglitazone induced ABCA1 expression in macrophages at mRNA level and protein level, resulting in an enhanced apoAI-mediated cholesterol removal from human macrophage-derived foam cells.