Figure 1.
Effects of nicotinic acid (NA) on mRNA transcription of MCP-1, TNFα, PPARγ, and CD36.
Basal THP-1 macrophages were pre-incubated with LPS prior to administration of NA versus vehicle controls. 1A LPS increased mRNA transcription of MCP-1 and TNFα, while NA treatment attenuated this LPS-induced up-regulation (n = 3 per group). 1B LPS suppressed mRNA transcription of PPARγ and its downstream effector CD36, yet treatment by NA effectively reversed it (n = 3 per group). *P<0.05, **P<0.01, ***P<0.001, ns = non significant.
Figure 2.
Foam cell induction and the effects of NA and GW1929 on foam cell cholesterol efflux.
2A Phase-contrast microscopy of Oil-red-O staining of THP-1 cells in basal (upper) and foam cells (lower) states, confirming foam cell induction. 2B Foam cells were treated with NA, GW1929, or vehicles only for 24 hours, followed by cholesterol efflux facilitated by HDL3 or ApoAI in the culture medium. Cholesterol efflux was studied by measuring cellular cholesterol content after hexane:isopropanol extraction. GW1929 treatment reduced extracted cellular cholesterol in GW1929+ HDL group versus with HDL alone; and in GW1929+ ApoAI group versus with ApoAI alone (n = 2 per group). NA treatment had no effect on extracted cellular cholesterol content at a range of NA concentration 1×10−3 to 10−6 M compared to either HDL3 or ApoAI alone groups. (Representative data at 1×10−4 M NA concentration shown). *P<0.05, **P<0.01, ***P<0.001, ns = non significant.
Figure 3.
Effects of NA and GW1929 on reverse cholesterol transport apparatus: LXRα, ABCA1 and ABCG1.
3A mRNA transcription of all three genes were significantly up-regulated immediately following cholesterol loading but returned to baseline or below-baseline level after resting in standard RPMI media for 24 hours. 3B In basal macrophages, NA significantly up-regulated ABCG1 mRNA transcription (n = 3 per group); this effect was not seen in foam cells. PPARγ activation using GW1929 significantly up-regulated mRNA transcription with both NR1H3 and ABCG1 in both basal macrophages and foam cells. *P<0.05, **P<0.01, ***P<0.001, ns = non significant.
Figure 4.
Effects of NA on PPARγ and cAMP pathways in basal versus foam cells.
4A PPARs nuclear binding ELISA showed that cholesterol loading reduced PPARγ transcription factor binding (n = 6 per group); and that NA significantly increased PPARγ binding in basal macrophages (n = 3 per group) but not in foam cells. 4B NA (1×10−4 M) rapidly reduced cAMP level and this effect persisted for 24 hours. 4C and 4D Pre-incubation with pertussis toxin (PTX) or vehicles for 18 hours followed by NA. In basal macrophages, cAMP level diminished in NA alone group compared to control (n = 3 per group) and this effect was abolished by PTX pre-incubation. In contrast, NA did not affect cAMP level in foam cells (n = 3 per group). *P<0.05, **P<0.01, ***P<0.001, ns = non significant.
Figure 5.
Down-regulation of GPR109A in human macrophage-derived foam cells.
5A GPR109A mRNA was not expressed in undifferentiated THP-1 cells but expression was seen after differentiation into basal macrophages. Treatment with NA for 24 hours caused significant down-regulation of GPR109A mRNA transcription (n = 3 per group). Foam cells induction down-regulated GPR109A mRNA transcription compared to basal macrophages (n = 3 per group). 5B Western blot showed the loss of protein expression of GPR109A in total cell lysates from foam cells compared to basal macrophages. 5C and 5D Confocal fluorescence immunocytochemistry demonstrated the expression of GPR109A on the cell surface in cultured THP-1 basal macrophages (5C), and the down-regulation of GPR109A expression after foam cell induction (5D).
Figure 6.
Expression of GPR109A in ex-vivo human carotid plaques.
6A Fluorescence immunohistochemistry staining with antibodies against CD68 and GPR109A. Immediately adjacent 10 µm cryosection was stained with Oil Red O for lipid distribution and visualized using Texas Red excitation filter (540–580 nm) in epifluorescence. CD68-positive cells were seen clustering at the interface between lipid-rich region and the overlying fibrous cap. GPR109A co-expression was seen in a sub-population of these CD68-positive cells outside of the lipid-rich region (yellow). Purple dotted line represents the boundary of the lipid-rich region as seen in the image above. 6B and 6C Loss of GPR109A expression in lipid-rich region. Confocal fluorescence images of CD68-positive cells outside of lipid-rich region are shown in 6B; and those within the lipid-rich region, likely to represent foam cells, are shown in 6C.
Figure 7.
Down-regulation of GPR109A in ex-vivo lipid-laiden plaque foam cells.
Adipophilin (perilipin 2) staining is specific to intra-cellular lipid-droplet associated protein and represents a sensitive marker of foam cells. 7A High-powered confocal image showed the outline of intra-cellular lipid droplets. 7B GPR109A is strongly co-expressed in CD68-positive plaque macrophages without extensive lipid-loading i.e. non-foam cells; whereas plaque foam cells, which showed strong co-expression of CD68 and adipophilin due to extensive lipid-loading as shown in 7C, do not co-express GPR109A.