Macrophage phospholipid transfer protein contributes significantly to total plasma phospholipid transfer activity and its deficiency leads to diminished atherosclerotic lesion development.

OBJECTIVE: Systemic phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, whereas overexpression of human PLTP in mice increases atherosclerotic lesion development. PLTP is also expressed by macrophage-derived foam cells in human atherosclerotic lesions, but the exact role of macrophage PLTP in atherosclerosis is unknown. METHODS AND RESULTS: To clarify the role of macrophage PLTP in atherogenesis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP(-/-)) mice into irradiated low-density lipoprotein receptor knockout mice. Selective deficiency of macrophage PLTP (PLTP(-M/-M)) resulted in a 29% (P<0.01 for difference in lesion area) reduction in aortic root lesion area as compared with mice possessing functional macrophage PLTP (384+/-36*10(3) microm2 in the PLTP(-M/-M) group (n=10), as compared with 539+/-35*10(3) microm2 in the PLTP(+M/+M) group (n=14)) after 9 weeks of Western-type diet feeding. The decreased lesion size in the PLTP(-M/-M) group coincided with significantly lower serum total cholesterol, free cholesterol, and triglyceride levels in these mice. Furthermore, plasma PLTP activity in the PLTP(-M/-M) group was 2-fold (P<0.001) lower than that in the PLTP(+M/+M) group. CONCLUSION: Macrophage PLTP is a significant contributor to plasma PLTP activity and deficiency of PLTP in macrophages leads to lowered atherosclerotic lesion development in low-density lipoprotein receptor knockout mice on Western-type diet.

Serum, but not monocyte macrophage foam cells derived from low HDL-C subjects, displays reduced cholesterol efflux capacity.

The main antiatherogenic function of HDL is to promote the efflux of cholesterol from peripheral cells and transport it to the liver for excretion in a process termed reverse cholesterol transport. The aim of this study was to evaluate the cholesterol efflux capacity in low- and high-HDL subjects by utilizing monocytes and serum from 18 low-HDL and 15 high-HDL subjects. Low and high HDL levels were defined, respectively, as HDL < or =10(th) and HDL > or =90(th) Finnish age/sex-specific percentile. Cholesterol efflux from [(3)H]cholesterol-oleate-acetyl-LDL-loaded monocyte-derived macrophages to standard apolipoprotein A-I (apoA-I), HDL(2), and serum was measured. In addition, cholesterol efflux from acetyl-LDL-loaded human THP-1 macrophages to individual sera (0.5%) derived from the study subjects was evaluated. Cholesterol efflux to apoA-I, HDL(2), and serum from macrophage foam cells derived from low- and high-HDL subjects was similar. The relative ABCA1 and ABCG1 mRNA expression levels in unloaded macrophages, as well as their protein levels in loaded macrophage foam cells, were similar in the two study groups. Cholesterol efflux from THP-1 foam cells to serum recovered from high-HDL subjects was slightly higher than that to serum from low-HDL subjects (P = 0.046). Cholesterol efflux from THP-1 macrophages to serum from study subjects correlated with serum apoB (P = 0.033), apoA-I (P = 0.004), apoA-II (P < 0.0001), and the percentage of apoA-I present in the form of prebeta-HDL (P = 0.0001). Our data reveal that macrophages isolated from either low- or high-HDL subjects display similar cholesterol efflux capacity to exogenous acceptors. However, sera from low-HDL subjects have poorer cholesterol acceptor ability as compared with sera from high-HDL subjects.

Association of cholesteryl ester transfer protein with HDL particles reduces its proteolytic inactivation by mast cell chymase.

Human atherosclerotic intima contains mast cells that secrete the neutral protease chymase into the intimal fluid, which also contains HDL-modifying proteins, such as cholesteryl ester transfer protein (CETP), in addition to abundant amounts of nascent discoidal HDL particles. Here, we studied chymase-dependent degradation of a) CETP isolated from human plasma and b) CETP-HDL complexes as well as the functional consequences of such degradations. Incubation with chymase caused a rapid cleavage of CETP, yielding a specific proteolytic pattern with a concomitant reduction in its cholesteryl ester transfer activity. These chymase-dependent effects were attenuated after CETP was complexed with HDL. This attenuation was more effective when CETP was complexed with HDL(3) and HDL(2) than with discoidal reconstituted high density lipoprotein (rHDL). Conversely, rHDL, but not spherical HDLs, was protected in such CETP complexes against functional inactivation by chymase. Thus, in contrast to the complexes of CETP with spherical HDLs, the ability of the CETP-rHDL complexes to promote cholesterol efflux from macrophage foam cells remained unchanged, despite treatment with chymase. In summary, complexation of CETP and HDL modifies their resistance to proteolytic inactivation: spherical HDLs protect CETP, and CETP protects discoidal HDL. These results suggest that in inflamed atherosclerotic intima, CETP, via its complexation with HDL, has a novel protective role in early steps of reverse cholesterol transport.

Cholesterol efflux from macrophage foam cells is enhanced by active phospholipid transfer protein through generation of two types of acceptor particles.

Phospholipid transfer protein (PLTP) is expressed by macrophage-derived foam cells in human atherosclerotic lesions, suggesting a regulatory role for PLTP in cellular cholesterol homeostasis. However, the exact role of PLTP in the reverse cholesterol transport pathway is not known. PLTP is present in plasma as two forms, a highly active (HA-PLTP) and a lowly active (LA-PLTP) form. In this study we clarify the role of the two forms of PLTP in cholesterol efflux from [3H]cholesterol oleate-acetyl-LDL-loaded THP-1 macrophages. Incubation of HDL in the presence of HA-PLTP resulted in the formation of two types of acceptor particles, prebeta-HDL and large fused HDL. HA-PLTP increased prebeta-HDL formation and caused a 42% increase in [3H]cholesterol efflux to HDL, while LA-PLTP neither formed prebeta-HDL nor increased cholesterol efflux. Removal of the formed prebeta-HDL by immunoprecipitation decreased cholesterol efflux by 47%. Neither HA- nor LA-PLTP enhanced cholesterol efflux to lipid-free apoA-I. Importantly, also the large fused HDL particles formed during incubation of HDL with HA-PLTP acted as efficient cholesterol acceptors. These observations demonstrate that only HA-PLTP increases macrophage cholesterol efflux, via formation of efficient cholesterol acceptors, prebeta-HDL and large fused HDL particles.

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells.

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.

Active and low-active forms of serum phospholipid transfer protein in a normal Finnish population sample.

Human serum phospholipid transfer protein (PLTP) exists as a catalytically active (HA-PLTP) and a low-active (LA-PLTP) form. In this study, the association of PLTP activity and the concentrations of both forms with lipid and carbohydrate parameters were investigated. In a random Finnish population sample, serum PLTP concentration (n=250) was 6.56 +/- 1.45 mg/l, the mean lipoprotein-independent (PLTPexo) phospholipid transfer activity was 6.59 +/- 1.66 micromol/ml/h, and the mean lipoprotein-dependent (PLTPendo) activity was 1.37 +/- 0.29 micromol/ml/h. Of the serum PLTP concentration, approximately 46% was in a catalytically active form. HA-PLTP concentration correlated positively with serum PLTPexo activity (r=0.380, P <0.001), HDL cholesterol (r=0.291, P <0.001), and apolipoprotein A-I (r=0.187, P <0.01). Of the potential regulatory factors for PLTP, apolipoprotein E showed a weak positive correlation with serum PLTPexo (r=0.154, P <0.05) and PLTPendo (r=0.192, P <0.01) activity but not with PLTP concentration. Weak associations were also observed between PLTP parameters and determinants of glucose homeostasis (glucose, insulin, and homeostasis model assessment for insulin resistance). The present data on PLTP activity and concentration reveal novel connections of the two PLTP forms to lipid and carbohydrate metabolism.