Effect Of Overexpression And Silence Of Pentraxin3on Lipid Uptake And Efflux In Human And Mouse Macrophages

BackgroundRecently, atherosclerosis can be considered to represent an inflammatory response of macrophages and lymphocytes to ’invading’ pathogenic lipoproteins in the arterial wall. Inflammatory molecules involved in cross-talk between inflammation and lipid metabolism have attracted attention as markers of disease activity. Pentraxin3(PTX3) was the first long pentraxin discovered, and the prototypic marker of inflammation, appears to be an active participant in atherosclerosis. Much data are accumulating to report that modified atherogenic lipoproteins induce expression of PTX3by human vascular smooth muscle cells (SMCs). Moreover, there have several studies reported that the colocalization of PTX3and macrophages has been demonstrated in advanced human atherosclerotic plaque Furthermore, in ApoE-/-mice, increased PTX3expression was observed in advanced atherosclerotic lesions, confirming previous observations in human atherosclerotic lesions, and was associated with macrophages and endothelial cells. Despite its observed overexpression in macrophages, mainly foam cells in advanced human atherosclerotic plaque, little data existed on the mechanism of PTX3in lipid storage and efflux in these cells and its relevance to atherosclerosis.ObjectiveThe objective of this study was to determine the effects and potential mechanisms of Pentraxin3(PTX3) on human oxidized low density lipoprotein (ox-LDL) uptake and cholesterol efflux from human and mouse macrophage foam cells, which may play a critical role in atherogenesis.MethodsHuman THP-1macrophages and mouse RAW264.7macrophages were pre-transfected with pSG5hPTX3or PTX3siRNA plasmids, inhibitors (PD98059, LY294002, SB203580) were added to medium before transfection, then cells were either exposed to ox-LDL for uptake assay or ox-LDL and [3H]-cholesterol and apolipoprotein A-Ⅰ (apoA-I) for cholesterol efflux assay and analyzed the expression of several key molecules which are involved in cholesterol efflux.Results1. The data showed that the concentrations of TC, FC, and CE/TC were increased when THP-1and RAW264.7macrophages transfected with pSG5empty vectors in the presence of50μg/ml ox-LDL for24hours compared with untreated cells (P<0.05). Moreover, THP-1and RAW264.7macrophages transfected with pSG5hPTX3expression vectors in the presence of ox-LDL (50μg/ml) was a significant upregulation of TC, CE and CE/TC compared with pSG5empty vector tranfection+ox-LDL group (P<0.05). Moreover, such increase of the intracellular lipid accumulation was obviously inhibited by co-incubation with the specific ERK1/2inhibitor PD98059and p38/MAPK-2inhibitor SB203580, respectively (P<0.05).2. After pSG5hPTX3transfection, THP-1cells and RAW264.7cells showed markedly increase in PTX3mRNA and protein level, and the cholesterol efflux from THP-1cells and RAW264.7cells to apoA-I was reduced by33%and29%compared with controls (pSG5empty vector), respectively. In contrast, THP-1cells and RAW264.7cells showed significantly decrease in PTX3mRNA and protein level, and [3H] cholesterol efflux to apoA-I was markedly elevated by27%and21%when transfected with synthetic siRNA against PTX3compared with that in GFP RNAi empty vector, respectively. The specific ERK1/2inhibitor PD98059effectively abolished PTX3-induced inhibition of cholesterol efflux from THP-1cells by27%compared with pSG5hPTX3transfection group (P<0.05, n=3). Furthermore, specific p38/MAPK-2inhibitor SB203580effectively abolished PTX3-induced inhibition of cholesterol efflux from RAW264.7cells by34%compared with pSG5hPTX3transfection group (P<0.05, n=3).3. THP-1and RAW264.7macrophages overexpressing PTX3significantly inhibited the expression of peroxisome proliferator-activated receptor gamma (PPARy), liver X receptor alpha (LXRa) and ATP-binding membrane cassette transporter Al (ABCA1), while PTX3silence increased that expression.ConclusionPTX3promotes ox-LDL uptake and inhibits cholesterol efflux from human and mouse foam cells derived from THP-1cells and RAW264.7cells in vitro through ERK1/2activation or p38/MAPK and down-regulation of intracellular cholesterol transport molecules PPARy, LXRa and ABCA1. Therefore, PTX3might contribute to lipid accumulation in the intima of the arterial wall.