ABSTRACT
Title
Pharmacological and genetic modulation of macrophage Reverse Cholesterol Transport
Authors
D. Greco
PhD student 2° Sperimental Pharmacology and toxicology XX ciclo
Dept. of Pharmacological and Biological Sciences and Applied Chemistries. University of Parma.
PhD student 2° Sperimental Pharmacology and toxicology XX ciclo
Dept. of Pharmacological and Biological Sciences and Applied Chemistries. University of Parma.
Abstract
The majority of peripheral cells can not catabolize cholesterol and the only way to eliminate it is the uptake by extracellular acceptors. This process, called lipid efflux, represents the first step of the reverse cholesterol transport (RCT), the physiological process by which excess cholesterol is removed from peripheral tissues and transported by lipoproteins (HDL) to the liver for elimination into the feces. RCT from macrophages, that are the cells mainly involved in the atherosclerosis development,is recognized as a protective pathway against the progression of atherosclerosis and can be affected by genetic and pharmacological factors.
In this study we measured RCT with anapproach that allows to specifically trace macrophage-derived cholesterol along the single steps of the process. In order to measure the macrophage-specific RCT we traced the transport of radiolabeled cholesterol (3H-cholesterol) from lipid loaded macrophages to feces. For this purpose 3H-cholesterol-loaded peritoneal macrophages were intraperitoneally injected into recipient mice. After 48 h animals were sacrificed and the amount of radiolabeled cholesterol was measured in plasma, liver and feces.
Firstly, we investigated the role of systemic and macrophage apolipoprotein E in macrophage RCT.Injecting apoE-/- macrophages into WT mice, we established that the absence of macrophage apoE impairs RCT compared to WT mice receiving WT macrophages (-29% p<0.05). Conversely, the expression of apoE in macrophages can promote a normal RCT in apoE-/- mice.In vitro experiments of cholesterol efflux demonstrated that apoE-/- plasma showed a similar cholesterol efflux potential compared to WT: 14.29% ±6.41 vs 12.40% ± 0.61 respectively. On the contrary, the deficiency of apoE in macrophages reduced cholesterol efflux in comparison with WT macrophages: ApoE-/- 8.5% ± 1.0 vs 12.40% ± 0.5 (p<0.01) respectively. These results indicate that the expression of apoE in macrophages affects RCT in vivo because it modulates cholesterol efflux from cells, whereas systemic apoE, despite the impact on plasma lipoprotein profile, does not affect their capacity to act as cholesterol acceptors.
Secondly, we analysed the effects of two pharmacological agents, T0901317 and cyclosporine (CsA) on RCT. T0901317, a synthetic agonist of the liver X receptors (LXR) that regulates the lipid metabolism, was administrated at 100mg/kg/day to BALB/c mice for 8 days. Animals treated with T0901317 showed an increased amount of labeled cholesterol in plasma (1.55 % ± 0.48 vs control 1.00%± 0.46 p< 0.05 ) and increased HDL plasma concentration (110.5 mg/dl ±2.1 vs control 41.0 mg/dl ± 9.9 p<0.05). In vitro cholesterol efflux experiments showed that plasma from treated animals promotes cellular cholesterol efflux more efficiently than untreated plasma. This results suggested that in vivo stimulation with LXR agonist increased macrophage RCT to feces (9.71% ±1.08 vs treated 18.32 ± 6.08), by increasing the ability of plasma to promote the release of cholesterol from macrophages. Cyclosporine A, an immunosuppressive drug that causes hyperlipidemia, was administrated to C57/BL6 mice at 50mg/kg/day for 14 days. The short treatment with CsA did not affect lipoprotein plasma profile (control 73.5 mg/dl ± 17.4 vs treated 85.0 mg/dl ± 14), while it reduces the fecal excretion of macrophage-derive cholesterol compared with untreated animals (0.7 % ± 0.1 vs1.1 % ± 0.2 p<0.01). This reduced lipid elimination may account for CsA-induced atherosclerosis.
In conclusion, we further confirmed that the extent of macrophage RCT inversely correlates with the progression of atherosclerosis. In fact T0901317, that was previously demonstrated to reduce atherosclerosis in mice, increased the rate of RCT, whereas, CsA that was associated with a pro-atherosclerotic effect was showed to reduce the process.
In this study we measured RCT with anapproach that allows to specifically trace macrophage-derived cholesterol along the single steps of the process. In order to measure the macrophage-specific RCT we traced the transport of radiolabeled cholesterol (3H-cholesterol) from lipid loaded macrophages to feces. For this purpose 3H-cholesterol-loaded peritoneal macrophages were intraperitoneally injected into recipient mice. After 48 h animals were sacrificed and the amount of radiolabeled cholesterol was measured in plasma, liver and feces.
Firstly, we investigated the role of systemic and macrophage apolipoprotein E in macrophage RCT.Injecting apoE-/- macrophages into WT mice, we established that the absence of macrophage apoE impairs RCT compared to WT mice receiving WT macrophages (-29% p<0.05). Conversely, the expression of apoE in macrophages can promote a normal RCT in apoE-/- mice.In vitro experiments of cholesterol efflux demonstrated that apoE-/- plasma showed a similar cholesterol efflux potential compared to WT: 14.29% ±6.41 vs 12.40% ± 0.61 respectively. On the contrary, the deficiency of apoE in macrophages reduced cholesterol efflux in comparison with WT macrophages: ApoE-/- 8.5% ± 1.0 vs 12.40% ± 0.5 (p<0.01) respectively. These results indicate that the expression of apoE in macrophages affects RCT in vivo because it modulates cholesterol efflux from cells, whereas systemic apoE, despite the impact on plasma lipoprotein profile, does not affect their capacity to act as cholesterol acceptors.
Secondly, we analysed the effects of two pharmacological agents, T0901317 and cyclosporine (CsA) on RCT. T0901317, a synthetic agonist of the liver X receptors (LXR) that regulates the lipid metabolism, was administrated at 100mg/kg/day to BALB/c mice for 8 days. Animals treated with T0901317 showed an increased amount of labeled cholesterol in plasma (1.55 % ± 0.48 vs control 1.00%± 0.46 p< 0.05 ) and increased HDL plasma concentration (110.5 mg/dl ±2.1 vs control 41.0 mg/dl ± 9.9 p<0.05). In vitro cholesterol efflux experiments showed that plasma from treated animals promotes cellular cholesterol efflux more efficiently than untreated plasma. This results suggested that in vivo stimulation with LXR agonist increased macrophage RCT to feces (9.71% ±1.08 vs treated 18.32 ± 6.08), by increasing the ability of plasma to promote the release of cholesterol from macrophages. Cyclosporine A, an immunosuppressive drug that causes hyperlipidemia, was administrated to C57/BL6 mice at 50mg/kg/day for 14 days. The short treatment with CsA did not affect lipoprotein plasma profile (control 73.5 mg/dl ± 17.4 vs treated 85.0 mg/dl ± 14), while it reduces the fecal excretion of macrophage-derive cholesterol compared with untreated animals (0.7 % ± 0.1 vs1.1 % ± 0.2 p<0.01). This reduced lipid elimination may account for CsA-induced atherosclerosis.
In conclusion, we further confirmed that the extent of macrophage RCT inversely correlates with the progression of atherosclerosis. In fact T0901317, that was previously demonstrated to reduce atherosclerosis in mice, increased the rate of RCT, whereas, CsA that was associated with a pro-atherosclerotic effect was showed to reduce the process.