Phospholipid Transfer Protein
Transfer of cholesterol esters generated by LCAT to the core of the HDL particles and the subsequent maturation of the HDL particles require the action of phospholipid transfer protein (PLTP), which supplies phospholipids allowing surface expansion of the shell of the particles. The phospholipids are liberated during the lipolysis of triglycerides in the core of apoB-containing lipoproteins by the action of lipoprotein lipase (Tall et al. 1985). In addition, PLTP facilitates the fusion of HDL3 particles to enlarged particles (Lusa et al. 1996). During this process lipidpoor apoA-I particles are released that can act as substrate for ABCA1-mediated cholesterol efﬂux from macrophages. In humans, common variants of PLTP have been identiﬁed that are associated with alterations in serum HDL cholesterol and the accumulation of small HDL particles (Albers et al. 2012). Furthermore, PLTP expression and activity is regulated during several diseases, including sepsis, multiple sclerosis, cancer, and cardiovascular disease (Albers et al. 2012), but it is largely unknown if the regulation of PLTP is a causative factor or simply a consequence of the processes underlying the diseases. The generation of PLTP knockout mice by the group of Alan Tall 15 years ago has greatly contributed to the general understanding of the role of PLTP in HDL metabolism. PLTP was inactivated in mice by replacing exon 2 containing the translation initiation codon, the signal peptide, and the ﬁrst 16 amino acids of mature PLTP with a neomycin-resistant gene (Jiang et al. 1999). The plasma transfer activity of the major phospholipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin into HDL, was completely blocked, while also the transfer of free cholesterol was impaired in PLTP knockout mice. Deletion of PLTP in mice led to markedly decreased levels of HDL phospholipids and cholesterol (65–70 %), illustrating the importance of transfer of surface phospholipids from apoB-containing lipoproteins by PLTP for maintaining HDL levels (Jiang et al. 1999). Furthermore, similarly as described for LCAT knockout mice the decrease in HDL cholesterol coincided with a signiﬁcant decrease in plasma apoA-I levels. Hepatocytes isolated from PLTP knockout mice synthesized normal amounts of apoA-I, albeit with reduced amounts of phosphatidylcholine (Siggins et al. 2007), indicating that the reduced plasma apoA-I levels were not due to impaired production. Qin et al. showed by in vivo turnover studies using autologous HDL that the reduced apoA-I levels are likely the consequence of increased catabolism of HDL in the PLTP knockout mice (Qin et al. 2000).
On regular chow diet, PLTP-deﬁcient mice absorb less cholesterol in the intestine (Liu et al. 2007), while hepatic phospholipids are increased and triglycerides are reduced in mice lacking PLTP (Siggins et al. 2007).
Feeding the mice a high saturated fat diet, containing 20 % hydrogenated coconut oil and 0.15 % cholesterol, led to the accumulation of surface components of apoB-containing lipoproteins as evidenced by a massive increase in VLDL and LDL phospholipids and cholesterol in the absence of changes in apoB (Jiang et al. 1999). Subsequent studies showed that the animals accumulated phospholipid and free cholesterol-rich lamellar particles containing apoA-IV and apoE (Qin et al. 2000). The accumulation of the lamellar particles speciﬁcally in coconut oil-fed PLTP knockout mice has been attributed to markedly reduced removal of these particles via scavenger receptor BI (SR-BI) by parenchymal liver cells (Kawano et al. 2002) and impaired secretion of biliary cholesterol and phospholipid (Yeang et al. 2010). Interestingly, these particles did not accumulate in the circulation when the mice were fed regular chow diet or a Western diet, containing 20 % milk fat and 0.15 % cholesterol (Kawano et al. 2002). In contrast, while feeding a Western diet, containing milk fat and 0.15 % cholesterol, PLTP deﬁciency was associated with an attenuated diet-induced hypercholesterolemia due to twofold lower concentrations of cholesterol transported by apoB-containing lipoproteins and HDL. Hepatic and intestinal lipid levels were not affected under these conditions, but cholesterol absorption in the intestine was reduced (Shelly et al. 2008).
Both on chow and while feeding the milk fat Western diet, PLTP knockout mice display reduced systemic inﬂammation as evidenced by lower levels of interleukin 6 and reduced expression of intercellular adhesion molecule 1 (ICAM1) and vascular adhesion molecule 1 (VCAM1) in aorta, indicating that mice lacking PLTP might have a reduced atherosclerosis susceptibility (Shelly et al. 2008).
To study the effect of PLTP deﬁciency on atherosclerotic lesion development, PLTP knockout mice were crossbred with human apoB transgenic mice, apoE knockout mice, and LDLr knockout mice (Jiang et al. 2001). Both in the apoB transgenic and the apoE knockout background, PLTP deﬁciency led, on top of the reduction in HDL lipids, to substantially lower VLDL lipid levels. Interestingly, this effect on VLDL lipids was not found in the LDLr knockout background, indicating the involvement of the LDLr in the lowering of VLDL lipids. In line, apoB production was reduced in the apoB transgenic and apoE knockout background, but not in the LDLr knockout background (Jiang et al. 2001). The content of the antioxidant vitamin E was signiﬁcantly increased in all three backgrounds in the absence of PLTP, while autoantibodies to oxidized LDL were largely decreased (Jiang et al. 2002). HDL isolated from PLTP knockout mice crossbred to both the apoB transgenic and the LDLr knockout background displayed improved antiinﬂammatory properties and reduced the ability of LDL to induce monocyte chemotaxis (Yan et al. 2004). After 6 months of feeding apoB transgenic mice Western diet and after feeding apoE knockout mice chow for 3 months, atherosclerotic lesion size was reduced ﬁvefold in mice lacking PLTP. In contrast, in LDLr knockout mice PLTP deﬁciency led to a twofold reduction in atherosclerotic lesion size after 8 weeks Western diet, whereas no signiﬁcant effects were observed after 12 weeks Western diet feeding (Jiang et al. 2001). Thus, while PLTP deﬁciency can reduce early atherosclerotic lesion development in mice lacking the LDLr, the pro-atherogenic effects of PLTP should be primarily attributed to its effects on the production of apoB-containing lipoproteins in mice fed regular chow or a Western diet. Notably, when apoE knockout mice lacking PLTP were challenged with a coconut oil-enriched, high fat diet for 7 weeks plasma levels of free cholesterol were 23 % higher due to the accumulation of the lamellar free cholesterol and phospholipid-rich particles. Under these conditions no effects on atherosclerotic lesion development were found (Yeang et al. 2010).
PLTP is widely distributed with expression in placenta > pancreas > lung > kidney > heart > liver > skeletal muscle > brain (Day et al. 1994). In addition, PLTP is found in endothelial cells (Day et al. 1994) and in smooth muscle cell and
macrophage foam cells in atherosclerotic lesions (Desrumaux et al. 2003; Lafﬁtte et al. 2003; O'Brien et al. 2003). Interestingly, Vikstedt and colleagues showed that the expression of PLTP is sixfold higher in Kupffer cells, macrophages of the liver as compared to hepatocytes (Vikstedt et al. 2007). Mice with a hepatocyte-speciﬁc deletion of PLTP were generated by injection of PLTP-ﬂox/ﬂippase animals with adenovirus-associated virus expressing Cre-recombinase under control of the thyroxine binding globulin promoter (Yazdanyar et al. 2013). PLTP activity was reduced by approximately 25 % in these animals with a hepatocyte-speciﬁc deletion of PLTP, leading to a 20 % reduction of HDL cholesterol. In addition, non-HDL cholesterol was 29 % lower due to an impaired production of apoB-containing lipoproteins (Yazdanyar et al. 2013). Conversely, liver-speciﬁc expression of PLTP in a PLTP knockout background promoted the secretion of apoB-containing lipoproteins by the liver (Yazdanyar and Jiang 2012). The contribution of macrophage PLTP to plasma PLTP activity was determined by bone marrow transplantation studies in LDLr knockout mice. Selective deletion of PLTP in bone marrowderived cells led to a decrease in plasma PLTP activity on chow and Western diet (Vikstedt et al. 2007). Vikstedt et al. showed that after 9 weeks Western diet feeding atherosclerotic lesion size was 29 % smaller in LDLr knockout mice transplanted with PLTP-deﬁcient bone marrow, which coincided with decreased serum VLDLcholesterol and phospholipid levels, while HDL phospholipid and apoA-I were increased (Vikstedt et al. 2007). In contrast, Valenta et al. also found lower levels of cholesterol in apoB-containing lipoproteins upon disruption of PLTP in bone marrow-derived cells of LDLr knockout mice, but surprisingly atherosclerotic lesion development was increased (Valenta et al. 2006). No effect on atherosclerosis was observed upon transplantation of PLTP-deﬁcient bone marrow into LDLr knockout mice overexpressing human apoA-I (Valenta et al. 2006). The studies by Valenta et al. suggest that locally in the arterial wall PLTP produced by macrophages can be anti-atherogenic. A possible explanation is that PLTP production by macrophages promotes ABCA1-mediated cholesterol efﬂux, as evidenced by decreased efﬂux to apoA-I from PLTP-deﬁcient macrophages (Lee-Rueckert et al. 2006). In agreement, macrophages isolated from PLTP knockout mice were shown to accumulate more cholesterol upon incubation with native or acetylated LDL (Ogier et al. 2007). Importantly, the higher levels of lipid-poor apoA-I in LDLr knockout mice overexpressing human apoA-I can overcome the pro-atherogenic effects of deletion of PLTP in macrophages, probably by stimulating the cholesterol efﬂux capacity of the macrophages.
In summary, the effects of PLTP on atherosclerotic lesion development are determined by a balance between systemic effects inﬂuencing the levels of antiatherogenic HDL and pro-atherogenic apoB-containing lipoproteins and the antioxidant vitamin E and local effects of PLTP produced by macrophages in the arterial wall inﬂuencing macrophage apoE production and ABCA1-mediated cholesterol efﬂux.