**1. Introduction**

90 Lipid Metabolism

PM:22415873

PM:21157483

Yu, L., York, J., von, B.K., Lutjohann, D., Cohen, J.C., & Hobbs, H.H. (2003). Stimulation of cholesterol excretion by the liver X receptor agonist requires ATP-binding cassette transporters G5 and G8. *J.Biol.Chem.,* Vol. 278, No. 18, pp. 15565-15570, PM:12601003 Zhao, L.F., Iwasaki, Y., Nishiyama, M., Taguchi, T., Tsugita, M., Okazaki, M., Nakayama, S., Kambayashi, M., Fujimoto, S., Hashimoto, K., Murao, K., & Terada, Y. (2012). Liver X receptor alpha is involved in the transcriptional regulation of the 6-phosphofructo-2 kinase/fructose-2,6-bisphosphatase gene. *Diabetes,* Vol. 61, No. 5, pp. 1062-1071,

Zoncu, R., Efeyan, A., & Sabatini, D.M. (2011). mTOR: from growth signal integration to cancer, diabetes and ageing. *Nat.Rev.Mol.Cell Biol.,* Vol. 12, No. 1, pp. 21-35,

#### **1.1. Apolipoprotein E, inflammation and atherosclerosis**

The inflammatory disease atherosclerosis is characterized by plaque formation in the cardiovascular system, which together with thrombosis can lead to obstruction of blood vessels, potentially leading to ischemia, stroke, and heart failure (Libby et al., 2009; Chen et al., 2010; Drake et al., 2011). Atherosclerosis is triggered and sustained by inflammation related cytokines, chemokines, adhesion molecules and by the cellular components of the immune system (Ross, 1999; Epstein et al., 2004). Cholesterol, most of it transported as a low density lipoprotein (LDL) particle in the bloodstream, supports foam cell formation in atherosclerotic plaques. In parallel, cholesterol plays an important role in steroidogenesis and bile production (Lacapere and Papadopoulos, 2003), which have been correlated with mitochondrial 18 kDa Translocator Protein (TSPO) and apolipoprotein E (apoE) expression (Fujimura et al., 2008; Gaemperli et al., 2011). Lipoproteins are lipid transport vehicles that ensure the solubility of lipids within aqueous biological environments. Apolipoproteins stabilize the surface of lipoproteins, serve as cofactors for enzymatic reactions, and present themselves as ligands for lipoprotein receptors. The soluble apolipoprotein gene family, which includes apoE, encodes proteins with amphipathic structures that allow them to exist at the water-lipid interface (Chan, 1989). ApoE is a polymorphic 229-aa, 34-kDa protein, which is present in the cell nucleus and cytosolic compartments (Mahley & Huang, 1999). The human gene, located on chromosome 19, encodes three alleles: apoE2 (frequency in the human population, 5–10%), apoE3 (60–70%), and apoE4 (15–20%). The isoforms differ only at residues 112 and 158 (Cedazo-Minguez & Cowburn, 2001). However, there is only one isoform of apoE in mouse and it behaves like human apoE3 (Strittmatter & Bova Hill, 2002). It is suggested that apoE deficiency in mice mimics the human apoE4 status, which implies reduced apoE3 levels relative to apoE4 levels (Buttini et al., 1999; Sheng et al., 1998).

© 2013 Dimitrova-Shumkovska et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ApoE is synthesized in several areas of the body, including the liver, where it is produced by hepatic parenchymal cells, and becomes a component in the surface of circulating triglyceride-rich lipoproteins [very low density lipoprotein (VLDL) and chylomicrons, or their remnants], and certain high density lipoprotein (HDL) particles (Mahley, 1988). ApoE plays a major role in the transport of lipids in the bloodstream, where it participates in the delivery and clearance of serum triglycerides, phospholipids, and cholesterol (Mahley, 1988). ApoE is also synthesized in the spleen, lungs, adrenals, ovaries, kidneys, muscle cells, and macrophages (Mahley, 1988). ApoE-containing lipoproteins are bound and internalized via receptor-mediated endocytosis by a number of proteins of the LDL receptor (LDLR) and LDLR-related protein (LRP) families (Davignon et al., 1998). ApoE is considered to be a ligand that binds to 27 clusters of negatively charged cysteine-rich repeats in the extracellular domains of all LDLR gene family members. It has been suggested that apoE made its entrance on the evolutionary stage long after the receptors to which it binds (Beffert et al., 2004). This also indicates that the original primordial functions of the LDLR family did not involve interactions with apoE. The original functions of the LDLR family may have been on the one hand transporting macromolecules between increasingly specialized cells and on the other hand serving as sensors for intercellular communication and environmental conditions (Beffert et al., 2003).

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 93

apoE functions as an important carrier protein in the redistribution of lipids among cells (by incorporation into HDL (as HDL-E); 2) it plays a prominent role in the transport of cholesterol (by incorporating into intestinally synthesized cholymicrons); and 3) it takes part metabolism of plasma cholesterol and triglyceride (by interaction with the LDLR and the receptor binding of apoE lipoproteins (Krul & Tikkanen, 1988; Quinn et al., 2004; Elliott et

ApoE has an established immune modulatory function in the peripheral immune response to bacteria and viruses (Mahley & Rall, 2000). It also modulates inflammatory responses in cell culture models *in vitro* and in *in vivo* models of brain injury, where an apoE mimetic therapeutic peptide has been shown to reduce CNS inflammation (Lynch et al., 2003; McAdoo et al., 2005; Aono et al., 2003). Involvement of apoE in injurious and inflammatory processes in the brain has attracted intensive attention (Drake et al., 2011; Potter and Wisniewski, 2012). In the brain, as well as in the cerebrospinal fluid, non-neuronal cell types, most notably astroglia and microglia, are the primary producers of apoE (Boyles et al., 1985; Quinn et al., 2004), while neurons preferentially express the receptors for apoE (Beffert et al., 2003). Regarding pathological conditions, it has been shown that human neuroblastoma cells, such as SK-N-SH, express apoE mRNA and apoE protein (Elliott et al., 2007). ApoE expression in neurons can be induced during nerve regeneration after injury and in growth and development of the CNS (Quinn et al., 2004). Moreover, Harris et al. (2004) showed that neuron-generated apoE tends to accumulate intracellularly, whereas astrocyte-generated apoE tends to be secreted. ApoE present in neurons is found in the cytoplasm (Han et al., 1994; Xu et al., 1996). The appearance of apoE in neurons may be due to neuronal synthesis under particular conditions, or by insertion into the cytoplasm of extracellular apoE (Dupont-Wallois et al., 1997). As neurons, human fibroblasts express low level of apoE under normal conditions, but under specific circumstances, such as apoptosis and nerve injury, they can

produce increased levels of apoE (Do-Carmo et al., 2002; Quinn et al., 2004).

**1.2. The 18kDa Translocator Protein (TSPO) and apolipoprotein E** 

Recent studies by us and others have indicated that the mitochondrial 18kDa Translocator Protein (TSPO), also known as peripheral-type benzodiazepine receptor (PBR) is present throughout the cardiovascular system and may be involved in cardiovascular disorders including atherosclerosis (Veenman and Gavish, 2006). The primary intracellular location of the TSPO is the outer mitochondrial membrane. Various studies over the course of the last 3 decennia have indicated that mitochondrial TSPO, potentially in relation to cardiovascular disease, is involved in the regulation of cholesterol transport into mitochondria in relation to bile production and steroidogenesis (Krueger and Papadopoulos, 1990; Papadopoulos et al., 2006). In particular, TSPO regulates cholesterol transport from the outer to the inner mitochondrial membrane which is the rate-limiting step in steroid and bile acid biosyntheses (Krueger and Papadopoulos, 1990; Lacapère and Papadopoulos, 2003; Veenman et al., 2007). Three-dimensional models of the channel formed by the five α-helices of the TSPO indicate that it can accommodate a cholesterol molecule in the space delineated by the five helices. According to these models, the inner surface of the channel formed by

al., 2007).

Cholesterol accumulation within atherosclerotic plaque occurs when cholesterol influx into the arterial wall (from apoB-containing lipoproteins) exceeds cholesterol efflux. Early in atherogenesis circulating monocytes are recruited to the arterial sub-endothelium where they differentiate into macrophages, ingest cholesterol, and develop into "foam cells" (Ross, 1973; 1999; Ross et al., 2001). Initially, monocytes adhere to activated endothelium on which up-regulated cell adhesion molecules (CAMs) are displayed, a dynamic process sensitive to inflammatory cytokines, shear stress, and oxidative insults (Chia, 1998). Induction of vascular cell adhesion molecule-1 (VCAM-1), a member of the immunoglobulin superfamily of CAMs, is increasingly described as the key factor in monocyte infiltration (Nakashima et al., 1998; Truskey et al., 1999). ApoE-knockout mice (apoE KO) have been extensively used to study the relation of hypercholesterolemia and lipoprotein oxidation to atherogenesis (Hoen et al., 2003; Yang et al., 2009; Kunitomo et al., 2009). ApoE-deficient mice have elevated VCAM-1 in aortic lesions (Nakashima et al., 1998), which enhances monocyte recruitment and adhesion (Ramos & Partridge, 2005), while apoE expression in the artery wall reduces early foam cell lesion formation (Hasty et al., 1999). These findings imply that apoE may influence early inflammatory responses by suppressing endothelial activation and CAM expression (Stannard et al., 2001). ApoE helps protect against atherosclerosis, in part by mediating hepatic clearance of remnant plasma lipoproteins (Weisgraber et al., 1994). When apoE is absent or dysfunctional, severe hyperlipidemia and atherosclerosis ensue (Kashyap et al., 1995; Linton & Fazio, 1999). ApoE is also abundant in atherosclerotic lesions, secreted by resident cholesterol-loaded macrophages (Linton & Fazio, 1999). This locally produced apoE is atheroprotective by contributing to reverse cholesterol transport and by inhibiting smooth muscle cell proliferation (Mahley et al., 1999; Mahley and Ji, 2006). ApoE exerts several functions regarding lipid and cholesterol transport and metabolism: 1) apoE functions as an important carrier protein in the redistribution of lipids among cells (by incorporation into HDL (as HDL-E); 2) it plays a prominent role in the transport of cholesterol (by incorporating into intestinally synthesized cholymicrons); and 3) it takes part metabolism of plasma cholesterol and triglyceride (by interaction with the LDLR and the receptor binding of apoE lipoproteins (Krul & Tikkanen, 1988; Quinn et al., 2004; Elliott et al., 2007).

92 Lipid Metabolism

ApoE is synthesized in several areas of the body, including the liver, where it is produced by hepatic parenchymal cells, and becomes a component in the surface of circulating triglyceride-rich lipoproteins [very low density lipoprotein (VLDL) and chylomicrons, or their remnants], and certain high density lipoprotein (HDL) particles (Mahley, 1988). ApoE plays a major role in the transport of lipids in the bloodstream, where it participates in the delivery and clearance of serum triglycerides, phospholipids, and cholesterol (Mahley, 1988). ApoE is also synthesized in the spleen, lungs, adrenals, ovaries, kidneys, muscle cells, and macrophages (Mahley, 1988). ApoE-containing lipoproteins are bound and internalized via receptor-mediated endocytosis by a number of proteins of the LDL receptor (LDLR) and LDLR-related protein (LRP) families (Davignon et al., 1998). ApoE is considered to be a ligand that binds to 27 clusters of negatively charged cysteine-rich repeats in the extracellular domains of all LDLR gene family members. It has been suggested that apoE made its entrance on the evolutionary stage long after the receptors to which it binds (Beffert et al., 2004). This also indicates that the original primordial functions of the LDLR family did not involve interactions with apoE. The original functions of the LDLR family may have been on the one hand transporting macromolecules between increasingly specialized cells and on the other hand serving as sensors for intercellular communication

Cholesterol accumulation within atherosclerotic plaque occurs when cholesterol influx into the arterial wall (from apoB-containing lipoproteins) exceeds cholesterol efflux. Early in atherogenesis circulating monocytes are recruited to the arterial sub-endothelium where they differentiate into macrophages, ingest cholesterol, and develop into "foam cells" (Ross, 1973; 1999; Ross et al., 2001). Initially, monocytes adhere to activated endothelium on which up-regulated cell adhesion molecules (CAMs) are displayed, a dynamic process sensitive to inflammatory cytokines, shear stress, and oxidative insults (Chia, 1998). Induction of vascular cell adhesion molecule-1 (VCAM-1), a member of the immunoglobulin superfamily of CAMs, is increasingly described as the key factor in monocyte infiltration (Nakashima et al., 1998; Truskey et al., 1999). ApoE-knockout mice (apoE KO) have been extensively used to study the relation of hypercholesterolemia and lipoprotein oxidation to atherogenesis (Hoen et al., 2003; Yang et al., 2009; Kunitomo et al., 2009). ApoE-deficient mice have elevated VCAM-1 in aortic lesions (Nakashima et al., 1998), which enhances monocyte recruitment and adhesion (Ramos & Partridge, 2005), while apoE expression in the artery wall reduces early foam cell lesion formation (Hasty et al., 1999). These findings imply that apoE may influence early inflammatory responses by suppressing endothelial activation and CAM expression (Stannard et al., 2001). ApoE helps protect against atherosclerosis, in part by mediating hepatic clearance of remnant plasma lipoproteins (Weisgraber et al., 1994). When apoE is absent or dysfunctional, severe hyperlipidemia and atherosclerosis ensue (Kashyap et al., 1995; Linton & Fazio, 1999). ApoE is also abundant in atherosclerotic lesions, secreted by resident cholesterol-loaded macrophages (Linton & Fazio, 1999). This locally produced apoE is atheroprotective by contributing to reverse cholesterol transport and by inhibiting smooth muscle cell proliferation (Mahley et al., 1999; Mahley and Ji, 2006). ApoE exerts several functions regarding lipid and cholesterol transport and metabolism: 1)

and environmental conditions (Beffert et al., 2003).

ApoE has an established immune modulatory function in the peripheral immune response to bacteria and viruses (Mahley & Rall, 2000). It also modulates inflammatory responses in cell culture models *in vitro* and in *in vivo* models of brain injury, where an apoE mimetic therapeutic peptide has been shown to reduce CNS inflammation (Lynch et al., 2003; McAdoo et al., 2005; Aono et al., 2003). Involvement of apoE in injurious and inflammatory processes in the brain has attracted intensive attention (Drake et al., 2011; Potter and Wisniewski, 2012). In the brain, as well as in the cerebrospinal fluid, non-neuronal cell types, most notably astroglia and microglia, are the primary producers of apoE (Boyles et al., 1985; Quinn et al., 2004), while neurons preferentially express the receptors for apoE (Beffert et al., 2003). Regarding pathological conditions, it has been shown that human neuroblastoma cells, such as SK-N-SH, express apoE mRNA and apoE protein (Elliott et al., 2007). ApoE expression in neurons can be induced during nerve regeneration after injury and in growth and development of the CNS (Quinn et al., 2004). Moreover, Harris et al. (2004) showed that neuron-generated apoE tends to accumulate intracellularly, whereas astrocyte-generated apoE tends to be secreted. ApoE present in neurons is found in the cytoplasm (Han et al., 1994; Xu et al., 1996). The appearance of apoE in neurons may be due to neuronal synthesis under particular conditions, or by insertion into the cytoplasm of extracellular apoE (Dupont-Wallois et al., 1997). As neurons, human fibroblasts express low level of apoE under normal conditions, but under specific circumstances, such as apoptosis and nerve injury, they can produce increased levels of apoE (Do-Carmo et al., 2002; Quinn et al., 2004).

#### **1.2. The 18kDa Translocator Protein (TSPO) and apolipoprotein E**

Recent studies by us and others have indicated that the mitochondrial 18kDa Translocator Protein (TSPO), also known as peripheral-type benzodiazepine receptor (PBR) is present throughout the cardiovascular system and may be involved in cardiovascular disorders including atherosclerosis (Veenman and Gavish, 2006). The primary intracellular location of the TSPO is the outer mitochondrial membrane. Various studies over the course of the last 3 decennia have indicated that mitochondrial TSPO, potentially in relation to cardiovascular disease, is involved in the regulation of cholesterol transport into mitochondria in relation to bile production and steroidogenesis (Krueger and Papadopoulos, 1990; Papadopoulos et al., 2006). In particular, TSPO regulates cholesterol transport from the outer to the inner mitochondrial membrane which is the rate-limiting step in steroid and bile acid biosyntheses (Krueger and Papadopoulos, 1990; Lacapère and Papadopoulos, 2003; Veenman et al., 2007). Three-dimensional models of the channel formed by the five α-helices of the TSPO indicate that it can accommodate a cholesterol molecule in the space delineated by the five helices. According to these models, the inner surface of the channel formed by

#### 94 Lipid Metabolism

the TSPO molecule would present a hydrophilic but uncharged pathway, allowing amphiphilic cholesterol molecules to cross the outer mitochondrial membrane (Papadopoulos et al., 1997, 2006; Veenman et al., 2007). At cellular levels TSPO is present in virtually all of the cells of the cardiovascular system, where they appear to take part in the responses to various challenges that an organism and its cardiovascular system face (Veenman & Gavish, 2006), including atherosclerosis and accompanying symptoms (Onyimba et al., 2011; Bird et al., 2010; Dimitrova-Shumkovska et al., 2010a,b,c, 2012).

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 95

cardiovascular diseases, including cardiac ischemia. It has also been shown that apoE is

Apparently as a consequence of its role in steroidogenesis, TSPO typically are very abundant in steroidogenic tissues (Benavides et al., 1983; De Souza et al., 1985). Steroid hormones can affect TSPO levels, while in turn TSPO provides a modulatory function for steroid hormone production by regulation of mitochondrial cholesterol transport (Veenman et al., 2007). It is known that cholesterol affects TSPO function (Falchi et al., 2007). Interestingly, apoE is also well expressed in steroidogenic organs such as adrenal gland, ovary, and testis (Blue et al., 1983; Elshourbagy et al., 1985; Law et al., 1997). Nonetheless, studies by us suggests that elevated cholesterol levels, such as found in apoE KO mice, do not appear to affect TSPO levels in steroidogenic organs (Inbar Roim, M.Sc. Thesis, Technion – Israel Institute of Technology, 2008), even though effects in the cardiovascular system can be observed (Dimitrova-Shumkovska et al., 2010a). As has been reported, TSPO levels can be regulated by steroid hormones, which may be part of an organism's response to stress and injury (Anholt et al., 1985; Weizman et al., 1992; Gavish & Weizman, 1997; Gavish et al., 1999; Veenman et al., 2007; Mazurika et al., 2009; Veenman and Gavish, 2012). This suggests that TSPO levels may be part of a feedback control system for steroid production (responding to alterations in steroid levels), rather than be regulated by a feed forward signal provided by cholesterol (i.e. TSPO levels in relation to steroidogenesis are not being

Various studies have shown the presence of TSPO in all cell types of the immune system, thus proposed functional roles of the TSPO included modulation of stress-induced immunosuppression and immune cell activity (Lenfant et al., 1985; Ruff et al., 1985; Bessier et al., 1992; Marchetti et al., 1996; Bono et al., 1999; Veenman & Gavish, 2006). TSPO are present in platelets, lymphocytes, and mononuclear cells, and are also found in the endothelium, the striated cardiac muscle, the vascular smooth muscles, and the mast cells of the cardiovascular system (Veenman & Gavish, 2006). TSPO in the cardiovascular system appears to play roles in several aspects of the immune response, such as phagocytosis and the secretion of interleukin-2, interleukin-3, and immunoglobulin A (Veenman & Gavish, 2006). Mast cells are considered to be important for immune response to pathogens (Marshall, 2004) and they have also been implicated in the regulation of thrombosis and inflammation and cardiovascular disease processes such as atherosclerosis as well as in neoplastic conditions (Wojta et al., 2003). Studies have shown that benzodiazepines' inhibition of serotonin release in mast cells could reduce blood brain barrier permeability, influence pain levels, and decrease vascular smooth muscle contractions (Veenman and Gavish, 2006). Benzodiazepines have been found to bind to specific receptors constituted by the TSPO on macrophages and to modulate *in vitro* their metabolic oxidative responsiveness (Lenfant et al., 1985). TSPO in the cardiovascular system also has been associated with the development of atherosclerosis (Camici et al., 2012). It for example has been suggested that reductions in TSPO levels may act as a protective mechanisms against the development of

oxidative stress in aorta and liver (Dimitrova-Shumkovska et al., 2010a, b, c; 2012).

regulated by cholesterol levels *in vivo*) (Veenman and Gavish, 2012).

*1.2.1. Involvement of TSPO in inflammation* 

involved in cardiac ischemia (Mahley, 1988).

TSPO are located in various components of blood vessels, including endothelial cells where TSPO may take part in immunologic and inflammatory responses (Hollingsworth et al., 1985; Bono et al., 1999; Milner et al., 2004; Veenman & Gavish, 2006). To establish a factual correlation between atherogenic challenges and TSPO binding characteristics, we have previously assayed TSPO binding characteristics in different tissues of rats fed a high fat high cholesterol (HFHC) diet, in comparison to rats fed a normal diet (Dimitrova-Shumkovska et al., 2010a). It appeared that enhancement of oxidative stress in the aorta and liver due to the atherogenic HFHC diet was accompanied by significant reductions in TSPO binding density in these organs. Binding levels of the TSPO specific ligand [3H]PK 11195 in heart appeared not to be affected by the HFHC diet in this rat model.

Previous studies have shown that TSPO as well as apoE can be associated with processes such as: cholesterol metabolism, oxidative stress, apoptosis, glial activation, inflammation, and immune responses. As a ligand for cell-surface lipoprotein receptors, apoE can prevent atherosclerosis by clearing cholesterol-rich lipoproteins from plasma (Mahley and Huang, 1999). The TSPO protein has also been shown to be present in the plasma membrane of red blood cells, as well as in the plasma membrane of neutrofils, where it was shown to stimulate NADPH-oxidase activation of these cells. The plasma membrane forms of TSPO may be involved in heme metabolism, calcium channel modulation, cell growth, and immunomodulation. Furthermore, nucleus expulsion in mature erythrocytes is inhibited by excess cellular cholesterol (Fan et al., 2009). However, the involvement of the TSPO in this process has not been investigated. A recent study in cell culture showed that TSPO is important for the regulation of mitochondrial protoporphyrin IX and heme levels (Zeno et al., 2012). Thus, the TSPO appears to take part in various stages of red blood cell formation.

Furthermore, TSPO takes part in the regulation of gene expression for proteins involved in adhesion, which potentially may play a role in platelet aggregation (Bode et al., 2012; Veenman et al., 2012). ApoE has also been found to be involved in platelet aggregation, while TSPO platelet levels have been found to be increased with various neurological disorders, in particular stress related disorders (Veenman and Gavish, 2000, 2006, 2012). It has been suggested that platelet aggregation may be affected by nitric oxide (NO) generation via apoE, while other studies suggest that NO requires the TSPO to induce collapse of the mitochondrial membrane potential (ΔΨm), mitochondrial reactive oxygen species (ROS) generation and cell death (Shargorodsky et al., 2012). Thus, the TSPO may present one pathway whereby NO does affect platelet aggregation. Furthermore, various alteration in TSPO density in the heart as a response to stress have been reported (Gavish et al., 1992; Veenman and Gavish, 2006), suggesting one aspect of involvement of TSPO in cardiovascular diseases, including cardiac ischemia. It has also been shown that apoE is involved in cardiac ischemia (Mahley, 1988).

Apparently as a consequence of its role in steroidogenesis, TSPO typically are very abundant in steroidogenic tissues (Benavides et al., 1983; De Souza et al., 1985). Steroid hormones can affect TSPO levels, while in turn TSPO provides a modulatory function for steroid hormone production by regulation of mitochondrial cholesterol transport (Veenman et al., 2007). It is known that cholesterol affects TSPO function (Falchi et al., 2007). Interestingly, apoE is also well expressed in steroidogenic organs such as adrenal gland, ovary, and testis (Blue et al., 1983; Elshourbagy et al., 1985; Law et al., 1997). Nonetheless, studies by us suggests that elevated cholesterol levels, such as found in apoE KO mice, do not appear to affect TSPO levels in steroidogenic organs (Inbar Roim, M.Sc. Thesis, Technion – Israel Institute of Technology, 2008), even though effects in the cardiovascular system can be observed (Dimitrova-Shumkovska et al., 2010a). As has been reported, TSPO levels can be regulated by steroid hormones, which may be part of an organism's response to stress and injury (Anholt et al., 1985; Weizman et al., 1992; Gavish & Weizman, 1997; Gavish et al., 1999; Veenman et al., 2007; Mazurika et al., 2009; Veenman and Gavish, 2012). This suggests that TSPO levels may be part of a feedback control system for steroid production (responding to alterations in steroid levels), rather than be regulated by a feed forward signal provided by cholesterol (i.e. TSPO levels in relation to steroidogenesis are not being regulated by cholesterol levels *in vivo*) (Veenman and Gavish, 2012).

#### *1.2.1. Involvement of TSPO in inflammation*

94 Lipid Metabolism

the TSPO molecule would present a hydrophilic but uncharged pathway, allowing amphiphilic cholesterol molecules to cross the outer mitochondrial membrane (Papadopoulos et al., 1997, 2006; Veenman et al., 2007). At cellular levels TSPO is present in virtually all of the cells of the cardiovascular system, where they appear to take part in the responses to various challenges that an organism and its cardiovascular system face (Veenman & Gavish, 2006), including atherosclerosis and accompanying symptoms

TSPO are located in various components of blood vessels, including endothelial cells where TSPO may take part in immunologic and inflammatory responses (Hollingsworth et al., 1985; Bono et al., 1999; Milner et al., 2004; Veenman & Gavish, 2006). To establish a factual correlation between atherogenic challenges and TSPO binding characteristics, we have previously assayed TSPO binding characteristics in different tissues of rats fed a high fat high cholesterol (HFHC) diet, in comparison to rats fed a normal diet (Dimitrova-Shumkovska et al., 2010a). It appeared that enhancement of oxidative stress in the aorta and liver due to the atherogenic HFHC diet was accompanied by significant reductions in TSPO binding density in these organs. Binding levels of the TSPO specific ligand [3H]PK 11195 in

Previous studies have shown that TSPO as well as apoE can be associated with processes such as: cholesterol metabolism, oxidative stress, apoptosis, glial activation, inflammation, and immune responses. As a ligand for cell-surface lipoprotein receptors, apoE can prevent atherosclerosis by clearing cholesterol-rich lipoproteins from plasma (Mahley and Huang, 1999). The TSPO protein has also been shown to be present in the plasma membrane of red blood cells, as well as in the plasma membrane of neutrofils, where it was shown to stimulate NADPH-oxidase activation of these cells. The plasma membrane forms of TSPO may be involved in heme metabolism, calcium channel modulation, cell growth, and immunomodulation. Furthermore, nucleus expulsion in mature erythrocytes is inhibited by excess cellular cholesterol (Fan et al., 2009). However, the involvement of the TSPO in this process has not been investigated. A recent study in cell culture showed that TSPO is important for the regulation of mitochondrial protoporphyrin IX and heme levels (Zeno et al., 2012). Thus, the TSPO appears to take part in various stages of red blood cell formation. Furthermore, TSPO takes part in the regulation of gene expression for proteins involved in adhesion, which potentially may play a role in platelet aggregation (Bode et al., 2012; Veenman et al., 2012). ApoE has also been found to be involved in platelet aggregation, while TSPO platelet levels have been found to be increased with various neurological disorders, in particular stress related disorders (Veenman and Gavish, 2000, 2006, 2012). It has been suggested that platelet aggregation may be affected by nitric oxide (NO) generation via apoE, while other studies suggest that NO requires the TSPO to induce collapse of the mitochondrial membrane potential (ΔΨm), mitochondrial reactive oxygen species (ROS) generation and cell death (Shargorodsky et al., 2012). Thus, the TSPO may present one pathway whereby NO does affect platelet aggregation. Furthermore, various alteration in TSPO density in the heart as a response to stress have been reported (Gavish et al., 1992; Veenman and Gavish, 2006), suggesting one aspect of involvement of TSPO in

(Onyimba et al., 2011; Bird et al., 2010; Dimitrova-Shumkovska et al., 2010a,b,c, 2012).

heart appeared not to be affected by the HFHC diet in this rat model.

Various studies have shown the presence of TSPO in all cell types of the immune system, thus proposed functional roles of the TSPO included modulation of stress-induced immunosuppression and immune cell activity (Lenfant et al., 1985; Ruff et al., 1985; Bessier et al., 1992; Marchetti et al., 1996; Bono et al., 1999; Veenman & Gavish, 2006). TSPO are present in platelets, lymphocytes, and mononuclear cells, and are also found in the endothelium, the striated cardiac muscle, the vascular smooth muscles, and the mast cells of the cardiovascular system (Veenman & Gavish, 2006). TSPO in the cardiovascular system appears to play roles in several aspects of the immune response, such as phagocytosis and the secretion of interleukin-2, interleukin-3, and immunoglobulin A (Veenman & Gavish, 2006). Mast cells are considered to be important for immune response to pathogens (Marshall, 2004) and they have also been implicated in the regulation of thrombosis and inflammation and cardiovascular disease processes such as atherosclerosis as well as in neoplastic conditions (Wojta et al., 2003). Studies have shown that benzodiazepines' inhibition of serotonin release in mast cells could reduce blood brain barrier permeability, influence pain levels, and decrease vascular smooth muscle contractions (Veenman and Gavish, 2006). Benzodiazepines have been found to bind to specific receptors constituted by the TSPO on macrophages and to modulate *in vitro* their metabolic oxidative responsiveness (Lenfant et al., 1985). TSPO in the cardiovascular system also has been associated with the development of atherosclerosis (Camici et al., 2012). It for example has been suggested that reductions in TSPO levels may act as a protective mechanisms against the development of oxidative stress in aorta and liver (Dimitrova-Shumkovska et al., 2010a, b, c; 2012).

#### 96 Lipid Metabolism

Anti-inflammatory properties of TSPO ligands have been demonstrated in various tissues. TSPO ligands have been shown to reduce inflammation in animal models of rheumatoid arthritis (Waterfield et al., 1999), carrageenan-induced pleurisy (Torres et al., 2000), and pulmonary inflammation (Bribes et al., 2003). Taupin et al. (1993) have also demonstrated *in vivo* that the synthetic TSPO ligand Ro5-4864 increases brain IL-1, IL-6 and TNF-α production after brain trauma. These cytokines are known to play a role in the inflammatory reaction to brain injury (Heumann et al., 1987). Interestingly, one study showed that PK 11195, but not Ro5-4864, could exert anti-inflammatory actions on mononuclear phagocytes, regulating the release of IL-1b (Klegeris et al., 2000). In addition, *in vivo* studies have shown that TSPO ligands can reduce the typical inflammatory response presented by reactive microglia and reactive astroglia resulting from brain trauma (Ryu et al., 2005; Veiga et al., 2005).

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 97

Cholesterol lowering by diet is associated with a reduction in DNA damage, at least in animal models (Singh et al., 2009). In general, modification of atherosclerotic risk factors by lipid lowering therapies, cessation of smoking, weight loss, and improved glucose control reduces circulating markers of inflammation. These and other findings suggest that inflammation is a primary process for atherosclerosis (Ziccardi et al., 2002; Rodriguez-Moran et al., 2003). Although high dietary intake of the anti-oxidant vitamin E and C has been associated with reduced risk of cardiovascular disease (CVD), well powered clinical trials in atherosclerosis-related CVD have indicated that supplements with vitamin C or vitamin E alone do not provide sufficient benefit, in comparison to, for example, statins (Kunitomo et al., 2009). Furthermore, specific antioxidants scavenge or metabolize some, but not all of the relevant oxidized molecules (Stocker and Keaney, 2004). Stocker and Keaney (2005) conclude that whenever a physiological process goes unchecked in case of disease, treatment strategies cannot simply rely on scavenging ROS. Nonetheless, drugs that have been proven to alter plaque progression have also been shown to alter vascular oxidative stress. For example, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA) inhibitors (Statins) reduce NAD(P)H oxidase activation and superoxide production *in vitro*, in part because of their capability to inhibit the membrane translocation (and thus activity) of the small GTP-binding protein Rac-1, which is a regulatory component of vascular NAD(P)H oxidase activation (Costopoulos et al., 2008). In conclusion, it appears that beneficial therapeutic treatments to prevent atherosclerosis include lowering of lipid levels and also reduction of oxidative stress. However, restricting a treatment to only reduction of oxidative stress does not appear to generate sufficient beneficial effects to counteract

**2. The effects of cholesterol challenges that result in atherogenesis on** 

As apoE deficiency may increase cholesterol levels and induce NO generation, which in turn may affect TSPO function, we were interested to study whether TSPO binding characteristics may be affected in heart and aorta of apoE-knockout (B6.129P2-apoE*tm1* N11) mice, in comparison to their C57BL/6 background mice (i.e. wild type, WT). For the present study homogenates of whole heart organ and aorta segments (aortic arch and descendending aorta) were used. For this approach, it was taken into consideration that accumulation of proatherogenic lipid affects all cells types present into vascular wall, and the response of the entire tissue to the cholesterol exposure is relevant as an indication of vascular defense as a whole (Hoen et al., 2003). All procedures with the animals were in accordance with National Institutes of Health (USA) guidelines for the care and use of experimental animals (NIH publication No. 85-23, revised 1996), and the experimental protocol was reviewed and approved by the local ethics committee. The mice were housed in polycarbonate cages in a pathogen – free facility set on a 12h light-dark cycle and given *ad libitum* access to water and standard laboratory feed. Prior to the experimental procedures, the rats were fed a commercial standard pellet feed (Filpaso, 52.11, Skopje, Republic of

**TSPO binding density in aorta and heart** 

Macedonia), named "standard feed" hereafter.

atherosclerosis.

#### **1.3. Animal models and strategies for atherosclerosis study**

Atherosclerotic plaques may appear early in life and might progress into severe, symptomatic plaques many decades later, dependent on the coexistence of risk factors such as age, genetic background, gender, hypercholesterolemia, hypertension, smoking, diabetes, etc. (Ross, 1999; Whitman, 2004). Rupture of lipid-rich coronary plaques can trigger an atherothrombotic event and probably is the most important mechanism inducing acute coronary syndrome (ACS) (Vilahuer et al., 2011).

Plaque rupture presents a major factor in ischemic processes associated with atherosclerosis (Zhao et al., 2008; Cheng et al., 2009; Gaemerli et al., 2011). Plaque rupture in the human condition, including the cardiovascular processes and events leading up to it, presently is virtually inaccessible for research. Therefore, animal models have been developed to study atherosclerosis, including plaque rupture and thrombus formation, and also how to take measures to prevent these from happening. Nonetheless, more sophisticated models need to be developed and tested to be able to better mimic the human condition. This is so, as mice and rats, for example, do not develop atherosclerosis without genetic manipulation, because they have a lipid physiology that is radically different from that in humans, as most of the cholesterol is being transported in HDL-like particles (Whitman, 2004; Singh et al., 2009; Vilahur et al., 2011). Furthermore, all of the existing animal models, including biological and mechanical triggering of atherogenesis, e.g., the Watanabe heritable hyperlipidemic (WHHL) rabbit model, the apolipoprotein E (ApoE) mouse model, and the LDL-receptor mouse model) suffer the drawback of lacking an end-stage atherosclerosis that would show plaque rupture accompanied by platelet and fibrin-rich occlusive thrombus at the rupture site (Singh et al., 2009). Another restriction of current models for cardiovascular disorders is that most of the studies explore only male mice to avoid effects of estrogens to the extent of lesion development and diminishing LDL oxidation (Caligiuri et al., 1999; Yang et al., 2004). As cardiovascular disorders also occur in women, it would be valuable to also study female animal research subjects. Furthermore, it would give direction to research relating hormonal conditions to atherosclerosis.

Cholesterol lowering by diet is associated with a reduction in DNA damage, at least in animal models (Singh et al., 2009). In general, modification of atherosclerotic risk factors by lipid lowering therapies, cessation of smoking, weight loss, and improved glucose control reduces circulating markers of inflammation. These and other findings suggest that inflammation is a primary process for atherosclerosis (Ziccardi et al., 2002; Rodriguez-Moran et al., 2003). Although high dietary intake of the anti-oxidant vitamin E and C has been associated with reduced risk of cardiovascular disease (CVD), well powered clinical trials in atherosclerosis-related CVD have indicated that supplements with vitamin C or vitamin E alone do not provide sufficient benefit, in comparison to, for example, statins (Kunitomo et al., 2009). Furthermore, specific antioxidants scavenge or metabolize some, but not all of the relevant oxidized molecules (Stocker and Keaney, 2004). Stocker and Keaney (2005) conclude that whenever a physiological process goes unchecked in case of disease, treatment strategies cannot simply rely on scavenging ROS. Nonetheless, drugs that have been proven to alter plaque progression have also been shown to alter vascular oxidative stress. For example, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA) inhibitors (Statins) reduce NAD(P)H oxidase activation and superoxide production *in vitro*, in part because of their capability to inhibit the membrane translocation (and thus activity) of the small GTP-binding protein Rac-1, which is a regulatory component of vascular NAD(P)H oxidase activation (Costopoulos et al., 2008). In conclusion, it appears that beneficial therapeutic treatments to prevent atherosclerosis include lowering of lipid levels and also reduction of oxidative stress. However, restricting a treatment to only reduction of oxidative stress does not appear to generate sufficient beneficial effects to counteract atherosclerosis.

96 Lipid Metabolism

2005).

Anti-inflammatory properties of TSPO ligands have been demonstrated in various tissues. TSPO ligands have been shown to reduce inflammation in animal models of rheumatoid arthritis (Waterfield et al., 1999), carrageenan-induced pleurisy (Torres et al., 2000), and pulmonary inflammation (Bribes et al., 2003). Taupin et al. (1993) have also demonstrated *in vivo* that the synthetic TSPO ligand Ro5-4864 increases brain IL-1, IL-6 and TNF-α production after brain trauma. These cytokines are known to play a role in the inflammatory reaction to brain injury (Heumann et al., 1987). Interestingly, one study showed that PK 11195, but not Ro5-4864, could exert anti-inflammatory actions on mononuclear phagocytes, regulating the release of IL-1b (Klegeris et al., 2000). In addition, *in vivo* studies have shown that TSPO ligands can reduce the typical inflammatory response presented by reactive microglia and reactive astroglia resulting from brain trauma (Ryu et al., 2005; Veiga et al.,

Atherosclerotic plaques may appear early in life and might progress into severe, symptomatic plaques many decades later, dependent on the coexistence of risk factors such as age, genetic background, gender, hypercholesterolemia, hypertension, smoking, diabetes, etc. (Ross, 1999; Whitman, 2004). Rupture of lipid-rich coronary plaques can trigger an atherothrombotic event and probably is the most important mechanism inducing acute

Plaque rupture presents a major factor in ischemic processes associated with atherosclerosis (Zhao et al., 2008; Cheng et al., 2009; Gaemerli et al., 2011). Plaque rupture in the human condition, including the cardiovascular processes and events leading up to it, presently is virtually inaccessible for research. Therefore, animal models have been developed to study atherosclerosis, including plaque rupture and thrombus formation, and also how to take measures to prevent these from happening. Nonetheless, more sophisticated models need to be developed and tested to be able to better mimic the human condition. This is so, as mice and rats, for example, do not develop atherosclerosis without genetic manipulation, because they have a lipid physiology that is radically different from that in humans, as most of the cholesterol is being transported in HDL-like particles (Whitman, 2004; Singh et al., 2009; Vilahur et al., 2011). Furthermore, all of the existing animal models, including biological and mechanical triggering of atherogenesis, e.g., the Watanabe heritable hyperlipidemic (WHHL) rabbit model, the apolipoprotein E (ApoE) mouse model, and the LDL-receptor mouse model) suffer the drawback of lacking an end-stage atherosclerosis that would show plaque rupture accompanied by platelet and fibrin-rich occlusive thrombus at the rupture site (Singh et al., 2009). Another restriction of current models for cardiovascular disorders is that most of the studies explore only male mice to avoid effects of estrogens to the extent of lesion development and diminishing LDL oxidation (Caligiuri et al., 1999; Yang et al., 2004). As cardiovascular disorders also occur in women, it would be valuable to also study female animal research subjects. Furthermore, it would give direction to research relating hormonal

**1.3. Animal models and strategies for atherosclerosis study** 

coronary syndrome (ACS) (Vilahuer et al., 2011).

conditions to atherosclerosis.
