Advanced glycation end products increase lipids accumulation in macrophages through upregulation of receptor of advanced glycation end products: increasing uptake, esterification and decreasing efflux of cholesterol
The maintain of macrophage cholesterol homeostasis is of great importance in the prevention of atherosclerosis. Dysregulation of the balance of cholesterol influx, endogenous synthesis, esterification/hydrolysis, and cholesterol efflux leads to excessive accumulation of cholesterol in macrophages and their transformation into foam cells and death [15]. In the present study, we elucidated the underlying mechanisms of AGEs-RAGE regulated cellular influx, intracellular esterification/hydrolysis and efflux of cholesterol. Our results provide strong evidence that AGEs-RAGE interaction may regulate the processes of cholesterol homeostasis from influx to efflux by increasing the expression of SRA2, CD36, ACAT1, HMGCR and decreasing expression of ABCG1 in macrophages.
There is increasing evidence that AGEs and their interaction with RAGE play a pivotal role in atherosclerosis, in particular in the setting of diabetes. AGEs binding to RAGE activates various signalling pathways, including NADPH oxidases, mitogen-activated protein kinases (MAPKs), p21ras, ERK p38 and protein kinase C (PKC), and finally leads to sustained cellular dysfunction driven by long-term activation of the nuclear factor-kB (NF-kB) [27, 28]. The importance of AGEs as downstream mediators of hyperglycaemia in diabetes has been amply demonstrated by animal studies using inhibitors of advanced glycation to retard the development of vascular disease without directly influencing plasma glucose levels [29, 30]. Furthermore, dietary excess of AGEs has been shown to accelerate atherosclerosis without affecting glycaemic control [31]. Studies in vivo showed that administration of soluble RAGE (sRAGE), a truncated form of RAGE acting as a decoy for AGE, completely suppressed diabetic atherosclerosis in glycemia- and lipid-independent manners [13]. In diabetes-associated atherosclerosis models, RAGE overexpression in transgenic mice was associated with increased vascular injury, while RAGE deletion conferred partial vascular protection [32].
Despite the important contribution of AGE to the accelerated atherosclerosis in diabetes, the specific molecular mechanisms in response to AGE within macrophages (a central player in atherogenesis) remain unclear. In this study, we discovered that exposure of THP-1 macrophages to AGEs was concentration-dependently associated with lipid accumulation observed by oil O stain and intracellular CE/TC determination. At the same time, the expression of RAGE elevated in parallel with the increase of lipid accumulation. RAGE specific antibody led to reversions in lipid contents in macrophages, suggesting that AGE-elicited atherogenic effects in macrophages were, at least partly, RAGE-dependent. These observations suggest that excessive formation of AGEs could render diabetic patients under high risk of developing atherosclerosis.
The Dil-oxLDL binding assay in our study showed that AGEs increased the binding of Dil-oxLDL to macrophages, indicating that AGEs promote cholesterol uptake. Cholesterol uptake is a pathway by which extracellular modified LDL are ingested by macrophages via receptors-mediated phagocytosis and pinocytosis. SRs such as SR-A and CD36 have been implicated in this process. In vitro studies have shown that CD36 and SR-A account for 75–90 % of ox-LDL internalization by macrophages, whereas other SRs cannot compensate for their absence [33]. In present study we found AGEs can upregulated SRA2 and CD36 mRNA and protein levels, indicating that AGEs may promote cholesterol uptake by increasing SRA2 and CD36 expression. It has been reported that AGEs only without interacting with RAGE can up-regulate the expression of SRA2 and CD36 [34, 35]. However, It was not the same in our study. After blockade of RAGE by specific antibody, we found that SRA2 and CD36 expression decreased significantly accompanied with the reduction of cholesterol uptake, suggesting that AGEs increase cholesterol uptake of macrophages mainly through binding with RAGE. A unique feature of CD36 is that expression of its gene could be regulated by ligands via PPAR-? dependent signaling pathway, and SRA-2 is mainly regulated by PPAR-? and NF-kB signaling pathway [33]. Activation of RAGE with AGEs leads to the production of reactive oxygen (ROS) and nitrogen species (RNS) by a variety of mechanisms, which may activates the PPAR-? and NF-kB signaling pathway [24] [36]. It may be the molecular mechanism for upregulation of CD36 and SRA2 by AGEs-RAGE interaction.
The results of cholesterol efflux assay in our study showed that AGEs can reduce cholesterol efflux through HDL but not apo AI in macrophages. When blocking AGEs-RAGE interaction by anti-RAGE antibody, the decrease of cholesterol efflux would recover. It is confirmed that AGEs have deleterious effects on cholesterol efflux in macrophages through binding with RAGE. Then we determined the transporters associated with cholesterol efflux and found that AGEs decreased ABCG1 mRNA and protein levels in macrophages in a RAGE-dependent manner.
Reverse cholesterol transport (RCT) is the primary pathway for the removal of excess cholesterol and involves lipid transporters such as ABCA1 and ABCG1 that mediate the transfer of cholesterol from peripheral cells to selected extracellular acceptors [37, 38]. The full ABC transporter ABCA1 appears most effective at mediating cholesterol efflux to apoAI as acceptor [39, 40]. The “half transporter” ABCG1 also facilitates cholesterol efflux from macrophages, preferring HDL as acceptor [41, 42]. LXR can control the expression of both ABCA1 and ABCG1 [43], and several recent reports suggest that both transporters share similar transcriptional control mechanisms [44, 45]. The effects of AGEs-RAGE axis on ABC transporters reported in previous studies are contradictory. The results of Passarelli M et al. [46] have shown that AGEs can impair cholesterol efflux from cultured human fibroblasts and murine macrophages through suppressing ABCA1 expression [46]. However, Isoda K et al. [47] reported that AGEs reduced macrophage cholesterol efflux to HDL through decreasing ABCG1 expression in a LXR-? independent way [47]. Recent study from Daffu G et al. [48] have demonstrated that RAGE suppressed macrophage cholesterol efflux in diabetic animal models and several different cell lines by moderately upregulating ABCA1 expression and significantly upregulating ABCG1 expression in a LXR?-independent way [48]. Our results were more like Daffu G’s that ABCA1 expression was slightly changed without statistical significance after treatment, while ABCG1 expression was dramatically changed with obviously statistical significance. LXR-? expression was contrary to both ABCA1 and ABCG1. So we report here that AGEs mainly reduces the expression of ABCG1 but not ABCA1 in a LXR?-independent manner in THP-1 macrophages, supporting the notion that ABCG1 may be especially important in diabetic atherosclerosis and providing a novel mechanistic insight into the relationship between HDL and atherosclerosis risk in diabetic patients. Our results also provide evidence that there are other signaling pathway to regulate expression of ABCG1 except LXR-?. Peroxisome proliferator response elements (PPRE) in the promoter regions of target genes is recognized as a important regulator which can binging with PPAR-? to activate transcription [49]. It has been demonstrated that RAGE ligands suppressed ABCG1 and ABCA1 promoter luciferase activity and transcription of ABCG1 and ABCA1 through PPRE but not LXR elements [48]. So it can be speculated from our results that AGEs-RAGE axis may regulate ABCG1 expression mainly through PPRE binding with PPAR-?, while ABCA1 expression may be mainly controlled by classical PPAR-?/LXR-? signaling pathway which is less affected by AGEs-RAGE axis. Besides PPRE, previous findings suggest a hitherto unsuspected degree of complexity in the regulation of the ABCG1 gene. The human ABCG1 gene spans 97 kb comprising 20 exons, potentially giving rise to multiple transcripts, and further contains two promoters with binding sites for multiple transcription factors, including NF-kB and sterol regulatory element-binding protein (SREBP) [50]. Both SREBP and NF-kB have been shown to be involved in the coactivation of transcription of genes involved in cholesterol metabolism [51]. Such structural complexity raise speculation about the potential mechanism that AGEs-RAGE axis interfere with the expression of ABCG1 through NF-kB and SREBP signaling pathway. Further studies will be required to elucidate the details of AGEs-RAGE-NF-kB/ SREBP-ABCG1 regulating pathway.
In the process of foam cell formation, ACAT1 re-esterifies excess FC to promote the biosynthesis of CE that is stored in lipid droplets [52]. In this study, we found that AGEs increased cellular CE levels. Furthermore, we demonstrated that high concentration of AGEs targeted expression of ACAT1, regulated the mRNA and protein levels of ACAT1, and increased CE formation in macrophage-derived foam cells. In contrast, low concentration of AGEs increased the protein expression of ACAT1 but had no effect on its mRNA expression, which can’t be explained by present study. However, it should be noted that ACAT1 protein increased similarly after treatment with 300 and 600 ?g/ml AGEs, but the CE levels didn’t increase over control at 300 ?g/ml AGEs. The possible reseason for that may be the difference of internalization of lipoproteins by macrophages between 300 and 600 ?g/ml AGEs group. Besides, we didn’t detect the expression of nCEH which hydrolyzes CE to cholesterol for efflux out of the cells. The changes of nCEH caused by different concentration of AGEs can also affect the levels of CE. In addition to uptake of extracellular lipoprotein, another main sources of intracellular FC are endogenous synthesis, which is regulated by HMGCR, a rate-limiting enzyme in the pathway for cholesterol synthesis [53]. Our study have demonstrated that AGEs can upregulate expression of HMGCR which can increase intracellular cholesterol and promote CE formation. Both ACAT1 expression and HMGCR expression decreased when using anti-RAGE antibody to pretreat, which indicated that the role of AGEs in upregulating ACAT1 and HMGCR needed to bind with RAGE. SREBP2 is probably the key factor connecting AGEs/RAGE with ACAT1/HMGCR [54].