development
Pennings, M.
Citation
Pennings, M. (2008, September 16). Cholesterol and phospholipid transporters in
atherosclerotic lesion development. Division of Biopharmaceutics of the Leiden/Amsterdam Center for Drug Research|Leiden University Medical Center (LUMC), Leiden University.
Retrieved from https://hdl.handle.net/1887/13099
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License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Introduction
Introduction
The research described in this thesis is focused on the role and interaction of different cholesterol and phospholipid transporters. This introduction gives a short overview of cholesterol metabolism and its transporters, the pathogenesis of the development of atherosclerosis, and an outline of the thesis. A specific review on the regulation of cholesterol homeostasis in macrophages and its consequences for atherosclerotic lesion development is provided in chapter 2.
Cholesterol metabolism
Mammalian cells need cholesterol as well as phospholipids for their survival since these are essential structural components of membranes. Also biologically important compounds, such as hormones, are synthesized from cholesterol (1). Therefore cholesterol homeostasis is of great importance for the body, but also for cells individually.
Cholesterol homeostasis is accomplished via a tightly regulated balance between the intake of dietary cholesterol and de novo cholesterol synthesis. Dietary cholesterol is taken up in the intestine after emulsification and hydrolyses in the lumen. After absorption by enterocytes, dietary lipids are assembled into triglyceride-rich chylomicrons. The triglycerides in the chylomicrons are subject to lipolysis by lipoprotein lipase (LPL) in the blood circulation. Progressive hydrolysis of the core triglycerides of the chylomicron is accompanied by the transfer of phospholipids by phospholipid transfer protein (PLTP) to HDL. The resulting cholesteryl ester-enriched chylomicron remnants travel to the liver, where they are taken up and processed. The liver excretes very-low-density lipoprotein (VLDL) particles into the circulation.
Upon extrahepatic lipolysis of triglycerides in the core of the VLDL particle, so-called intermediate-density lipoproteins (IDL) are formed and eventually low-density lipoprotein (LDL) particles. The LDL particles can be taken up by peripheral tissues, but the majority is reabsorbed by the liver.
The liver and intestine also excrete lipid-poor Apolipoprotein AI (ApoAI), the precursor of high-density lipoproteins (HDL). Circulating lipid-poor ApoAI takes up the excess cholesterol and phospholipids from peripheral cells and from the liver, to
grow into a mature HDL particle, which is the facilitator of reverse cholesterol transport (RCT). This process brings cholesterol back from the peripheral sites to the liver. After delivery of the HDL cholesterol esters to the liver, the excess cholesterol can be converted to bile acids and released from the body by excretion into the faeces (2).
In summary, five major classes of lipoproteins can be distinguished, including chylomicrons, VLDL, IDL, LDL, and HDL. In table 1 the characteristics of the different lipoproteins are summarized.
A dysbalance in cholesterol metabolism may, among other consequences, lead to elevated plasma cholesterol levels and an increased risk for cardiovascular disease, which is the main cause of death in the Western World and Japan (3). Epidemiologic studies have identified numerous risk factors for the development of cardiovascular disease, including hypertension, diabetes mellitus, smoking, and family history. In addition, hyperlipidemia due to increased levels of LDL cholesterol is an important marker for increased risk for atherosclerosis. On the other hand high levels of HDL have shown to be atheroprotective (4-6).
Table 1: Summary of humanlipoprotein characteristics
Adapted from Ginsberg (Ginsberg H.N. Lipoprotein physiology. Endocrinol Metab Clin North Am 1998;27:503-519)
Chylomicrons VLDL LDL HDL
Density (g/ml) <0.96 0.96-1.006 1.019-1.063 1.063-1.210
Mw (x106 Da) 400 10-80 2.3 0.17-0.36
Diameter (nm) 75-1200 30-80 19-25 5-12
Lipid composition (% of total weight)
Triglyceride 80-95 45-65 18-22 2-7
Free Cholesterol 1-3 4-8 6-8 3-5
Cholesterol Ester 2-4 6-22 45-50 5-20
Phospholipid 3-6 5-20 18-24 26-32
Apolipoproteins AI, AII, AIV B48
CI, CII, CIII E
B100 CI, CII, CIII E
B100 AI, AII, AIV
CI, CII, CIII E
Atherosclerosis
A disruption in the cholesterol homeostasis can result in elevated plasma cholesterol levels and an increase in the risk for the development of cardiovascular disease (4-6).
Atherosclerosis is defined as a slow ongoing process of thickening of the vascular wall. As a consequence of atherosclerosis, the deprivation of oxygen and nutrition of distally located tissues leads to the development of cardiovascular disease (7).
Figure 1: Schematic overview of the development of atherosclerosis
(Adapted from Grahams Child Stages of endothelial dysfunction in atheroscerosis wikipedia.org)
Development of atherosclerosis is initiated by modulation of the endothelial cell layer lining the vessel wall, and as a consequence the endothelial cells increase the expression of selectins (P- and E-selectin) and intracellular and vascular adhesion molecules (ICAM and VCAM). This induces the adhesion of monocytes and an increased permeability of the endothelial cell layer. In response to different growth stimuli the monocytes differentiate into macrophages which subsequently take up the excess of cholesterol which is present in the form of modified LDL. This process is
mediated by different scavenger receptors. Phagocytosis of large amounts of cholesterol will lead to the transformation of macrophages into so-called foam cells.
These foam cells are the hallmark of atherosclerosis (8). The accumulation of foam cells and intracellular lipid in the vascular wall characterize a fatty streak, which does not lead to a significant obstruction of the arterial lumen. (9). Once cholesterol cannot be efficiently stored anymore, it becomes cytotoxic and the lipid-laden macrophages go either into apoptosis or necrosis, resulting in the evolution of the fatty streak into an atherosclerotic plaque with a necrotic core with extracellular lipid accumulation in the form of cholesterol crystals. Furthermore, smooth muscle cells form a collagen- rich fibrous cap overlying the atherosclerotic lesion. This results in the narrowing of the vessel, which is usually compensated for by outward remodelling of the vascular wall, and thereby initially preserving the blood flow (9). Macrophages and macrophage-derived foam cells of the atherosclerotic lesion release metalloproteinases (MMP) and other proteolytic enzymes that cause degradation of the matrix leading to a reduction of thickness of the fibrous cap of the lesion (10).
When the fibrous cap ruptures, the lipid core of the atherosclerotic plaque is exposed to the blood, resulting in the recruitment of platelets, coagulation, and the formation of a thrombus, which causes most acute coronary syndromes (9). Figure 1 represents a schematic overview of the development of atherosclerosis.
Atherosclerosis and Macrophages
Macrophages play an essential role in all stages of atherosclerotic lesion development.
Increasing the knowledge of macrophage function in the pathogenesis of atherosclerosis is therefore highly important for the development of new therapeutic intervention strategies to prevent cardiovascular disease.
The last decade bone marrow transplantation has become a commonly used technique to study the role of macrophage genes in atherosclerosis (11). Mice that are susceptible to atherosclerosis, such as the LDL receptor knock out (LDLr KO) mice (11), are lethally irradiated to destroy all endogenous bone marrow cells and thus the progenitor cells of macrophages. The next day the irradiated recipient mice are transplanted with murine donor bone marrow lacking or over-expressing the gene of interest, depending on the research question (12-14). In the following 8-week recovery period all circulating endogenous bone marrow-derived cells, including
monocytes, which are the precursors of macrophages, are replaced with cells derived from the donor bone marrow, and thus the gene of interest will be knocked out or over-expressed on the reconstituted cells. The length of the recovery period is of great importance for the study design since it takes 8 weeks to replace the bone marrow- derived cells, including most tissue macrophages, like the Kupffer cells in the liver (15). To induce atherosclerotic lesion development, the transplanted LDLr KO mice are subsequently fed a so-called high cholesterol/ high fat diet Western-type diet.
Figure 2 shows a schematic representation of the bone marrow transplantation procedure.
Harvest donor bone marrow
Lethally irradiated recipient mice
Transplant recipient mice with donor bone marrow
Western Type diet feeding
Atherosclerotic development determiniation Harvest donor bone marrow
Lethally irradiated recipient mice
Transplant recipient mice with donor bone marrow
Western Type diet feeding
Atherosclerotic development determiniation
Figure 2: Schematic representation of a bone marrow transplantation study.
Cholesterol and Phospholipid transporters
The accumulation of lipid-laden foam cells in the arterial wall plays an essential role in the onset of atherosclerotic lesion development. Efflux of lipids from macrophages is an important natural protective mechanism to prevent macrophage foam cell formation. Several transporters are involved in the intracellular trafficking and efflux of cholesterol and phospholipids, among which the ATP-binding cassette transporter (ABC)-transporters are of special interest. In this short introduction, I will focus on the transporters which are specifically studied in this thesis including ABCA1, ABCB4, and scavenger receptor BI (SR-BI).
ATP-binding cassette transporter A1
ATP-binding cassette transporter A1 (ABCA1) belongs to the super family of ABC- transporters. All family members are involved in the energy dependent transport of a wide variety of substrates. ABC-transporters are evolutionary highly conserved and characteristically composed of two transmembrane domains formed by six membrane spanning helixes and two ATP-binding sites containing two sequence motifs (Walker A and Walker B) that bind ATP and provide the energy for transport (16). In addition to these so-called full transporters, some ABC transporters consist of only one transmembrane domain and one ATP-binding site, and are therefore called ABC half transporters (16).
ABCA1 mediates the transport of cholesterol from peripheral cells to lipid-poor ApoAI, and is thought to be the rate-limiting step in the RCT process. Mutations in the human ABCA1 gene lead to Tangier disease, a disease which is characterized by an almost complete absence of HDL (17-19). ABCA1 deficient mice display the same drastically decreased serum cholesterol levels, mainly due to the loss of HDL cholesterol (20). In addition, the serum levels of both ApoAI and phospholipid are diminished in both humans and mice lacking ABCA1 (20).
Liver-specific ABCA1 deficient mice show a 80% reduction in HDL levels, indicating that hepatic ABCA1 is most critical for the maintenance of plasma HDL levels (21). Singaraja et al showed that although hepatic ABCA1 is crucial for the initiation of HDL formation, other extrahepatic tissues play an important role in the maturation of HDL particles by mediating the cholesterol transfer to the nascent HDL particles (22). Specific disruption of ABCA1 on macrophages leads to an increase in atherosclerosis (23), while overexpression of ABCA1 in bone marrow-derived cells inhibits the progression of atherosclerotic lesion development (24). Macrophage ABCA1 thus protects against atherosclerotic lesion development, probably via the transport of cholesterol from the macrophage to lipid-poor ApoAI (20).
Scavenger receptor class B type I
The second transporter that is important in reverse cholesterol transport is the scavenger receptor class B type I (SR-BI). SR-BI transfers cholesteryl esters from the HDL particle to the cytoplasm of the hepatocyte where the cholesterol can be processed and secreted into the bile directly or as bile acids (25, 26). Disruption of the SR-BI gene in mice results in the absence of functional SR-BI protein and as a result
impaired metabolism of HDL particles, hampered HDL depletion of cholesteryl esters, and an increase in size of the HDL particles (27, 28). Due to this defect in the RCT pathway a higher vulnerability to atherosclerosis is observed in mice lacking SR-BI (29). In humans the physiological role of CLA-1, the human ortholog of SR- BI, is not yet definitively defined, although expression patterns and tissue distribution closely resemble those observed in mice. Several studies on common polymorphisms of human SR-BI have shown that variants of the SR-BI gene interfere with the metabolism of ApoB lipoproteins. However, the effect differs in men and women and is also affected by age (30). Human SR-BI deficiency has thus far not been identified.
Heterozygosity for a single nucleotide (proline to serine at position 297) was recently demonstrated to result in markedly increased HDL cholesterol levels, suggesting that in humans SR-BI may be equally important in controlling HDL cholesterol levels (31).
Figure 3: The role of ABCA1, SR-BI, and ABCB4 in cholesterol and phospholipid transport ABCA1 and SR-BI transport cholesterol from macrophages in the arterial wall to lipid poor ApoAI and HDL respectively. SR-BI is also implicated in the cholesterol uptake by macrophages.
The role of macrophage ABCB4 in the arterial wall is not yet known. In the liver, ABCA1 facilitates the generation of nascent HDL by lipidation of ApoAI, while hepatic SR-BI depletes the HDL from cholesteryl esters. The final step in the reverse cholesterol transport is mediated by hepatic ABCB4, through the efflux of phospholipids and cholesterol from the liver into the bile.
The role of macrophage SR-BI in atherosclerosis is complex. Bone marrow transplantation studies showed that macrophage SR-BI facilitates early lesion development, while it is protective in the latter stages of lesion development (32, 33).
This reflects the dual role of macrophage SR-BI. SR-BI can function in the uptake of atherogenic lipoproteins and act as an efflux transporter, transporting cholesterol from the macrophage to an acceptor like HDL (32).
ATP-binding cassette transporter B4
ATP-binding cassette transporter B4 (ABCB4), formerly known as multidrug resistant protein 2 (Mdr2) in mice or MDR3 in humans, facilitates the last step of the RCT process, the energy dependent transport of cholesterol and phospholipid from the hepatocyte into the bile, after which these molecules can be secreted into the faeces (34). In agreement, ABCB4 deficient mice show impaired phospholipid and cholesterol flux to the bile (34). Human subjects with a disruption in the ABCB4 gene develop progressive familial intrahepatic cholestasis (35). However, the role of this transporter in atherosclerotic lesion development is currently still unknown. In addition, the function of macrophage ABCB4 has not been elucidated yet, although it is known that macrophages do express ABCB4 mRNA (36), and that ABCB4 functions as a phospholipid and cholesterol transporter at other sites of the body (34).
A summary of the anticipated roles of SR-BI, ABCA1, and ABCB4 in reverse cholesterol transport is depicted in figure 3.
Outline of the thesis
The general aim of this thesis was to investigate the role of cholesterol and phospholipid transporters in lipoprotein metabolism, macrophage cholesterol homeostasis, and atherosclerotic lesion development. Chapter 2 represents a review of the regulation of cholesterol homeostasis in macrophages and the consequences for atherosclerotic lesion development. The importance of ABC transporters as key molecules for macrophage cholesterol efflux are described in detail. Furthermore, the role of ABCB4 in lipid homeostasis is portrayed. Since macrophages express ABCB4 mRNA (35) and the fact that ABCB4 functions as a phospholipid and cholesterol transporter at other sites of the body (34), it is likely that macrophage ABCB4 plays a role in atherosclerotic lesion development. In chapter 3 we explore the role of bone marrow-derived ABCB4 in atherosclerosis by means of transplantation of ABCB4 deficient bone marrow into the LDLr KO mice.
The key regulators of reverse cholesterol transport ABCA1 (17-19) and SR-BI (25) mediate cellular cholesterol efflux. Additionally, ABCA1 is important for the synthesis of HDL, mostly by liver and intestine, while SR-BI regulates serum HDL levels by mediating the selective uptake of cholesteryl esters from HDL by the liver and the adrenals. In chapter 4, the role of ABCA1 and SR-BI in reverse cholesterol
transport is reviewed. Since ABCA1 and SR-BI play an essential role at either end of the RCT pathway, it is conceivable that these proteins might act synergistically in this process. To study the RCT process under conditions in which both of these key mediators are absent, ABCA1/SR-BI double knockout (dKO) mice were generated by cross-breeding. The characterisation of these mice is described in chapter 5.
Facilitation of macrophage RCT is thought to be an important mechanism by which HDL protects against cardiovascular disease (5). To study the role of macrophage ABCA1 and SR-BI in atherosclerotic lesion development in absence of effects on HDL cholesterol levels, bone marrow transplantation studies were performed using the ABCA1/SR-BI double knockout mice as donors. In chapter 6 the results of a study on the role of bone marrow derived ABCA1 and SR-BI in atherosclerotic lesion development are presented. A final summary and future perspectives are found in chapter 7.
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