**3. Discussion**

There is strong evidence that accumulation of plasma derived lipoproteins in the arterial wall launches specific cell reactions that account for atherosclerosis process: enhanced NO production, amplification of the inflammatory response, apoptosis, endothelial function impairment, enhanced smooth muscle cell migration and proliferation, and macrophage foam cell formation (Steinberg et al., 2002; Whitman, 2004; Zhao et al., 2008; Singh et al., 2009). Mice lacking apoE have a substantial delay in the metabolism of lipoproteins, particularly VLDL, even fed with a regular standard chow feed (Hoen et al., 2003; Kato et al., 2009). Lesions in apoE-deficient mouse have many features in common with human atherosclerosis, even that the progression can be advantageous in many experimental situations (Dansky et al, 1999). At 26 weeks, atherosclerotic lesions are in the early stages of development, characterized by lipoprotein accumulation, leukocyte gathering, and foam cell formation. This model develops atherosclerotic lesions which progress to occlusion of coronary artery by 8th to 11 months after regular feeding (Piedrahita et al., 1992; Whitman, 2004). Aged (42-54 weeks) apoE KO mice develop intraplaque hemorrhage and plaque instability features, accelerated by feeding westernized diets (Seo et al., 2005; Singh et al., 2009). We found, similar to previous observations, advanced fibrous plaque development accompanying prolonged cholesterol feeding (Figure 1C) in apoE mice but not in WT mice. Another study by Molnar et al. (2005) showed that although high fat feeding induced endothelial cell dysfunction in WT mice, it did not enhance neointimal formation in WT mice. Also in WT rats, a high fat, high cholesterol diet does not appear to lead to atherosclerosis, although modest morphological alterations in the aortic wall could be observed (Dimitrova-Shumkovska et al., 2010a)

104 Lipid Metabolism

Discussion.

0.05, \*\* = p < 0.01, \*\*\* = p < 0.001 vs. control.

**3. Discussion** 

As the effects on TSPO binding density in heart and aorta due to intake of cholesterol supplemented diet take place primarily in the WT groups, and especially not in the 3% cholesterol diet fed apoE KO mice, these data suggest that decreases of TSPO binding density in heart and aorta may serve to counteract processes typically leading to cardiovascular damage, including atherosclerosis, as explained in more detail in the

**Bb - wild type mice**

**Apo E KO mice**

**Apo E - Control Apo E - 1% Chol. Apo E - 3 % Chol.**

 **Bb - Control Bb - 1% Chol. Bb - 3 % Chol.**

**Tissue B max** (fmol/mg) **Kd** (nmol) **n B max** (fmol/mg) **Kd** (nmol) **n B max** (fmol/mg) **Kd** (nmol) **n**

**Heart 1740 ± 180 0.65 ± 0.1 5 1005 ± 240\*\* 1.12 ± 0.3 6 1006 ± 140 \*\* 1.32 ± 0.5 5**

**Aorta 29 000 ± 9700 2.50 ± 1.2 9 14 900 ± 3370\*\*\* 2.31 ± 0.8 6 12 200 ± 2920\*\* 2.62 ± 0.5 5**

**Table 4.** Average Bmax values **fmoles / mg** protein and Kd values (nM) of [3H]PK 11195 binding to TSPO in aorta and heart homogenates of WT (Bb-Control) and apoE KO mice, fed with standard feed, and feed supplemented with 1% and 3% cholesterol (Chol). One-way analysis of variance ANOVA was used, with Mann-Whitney as the post-hoc, non-parametric test. Data are expressed as mean ± SD; \* = p <

**Aorta 24 500 ± 4100 1.9 ± 1.0 9 16 670 ± 3800 \*\* 1.48 ± 0.5 7 20 800 ± 6850 2.68 ± 1.44 7**

**Tissue B max** (fmol/mg) **Kd** (nmol) **n B max** (fmol/mg) **Kd** (nmol) **n B max** (fmol/mg) **Kd** (nmol) **n**

**Heart 1590 ± 390 0.92 ± 0.4 5 1260 ± 370 1.57 ± 0.8 7 2 580 ± 1890 1.62 ± 0.9 7**

There is strong evidence that accumulation of plasma derived lipoproteins in the arterial wall launches specific cell reactions that account for atherosclerosis process: enhanced NO production, amplification of the inflammatory response, apoptosis, endothelial function impairment, enhanced smooth muscle cell migration and proliferation, and macrophage foam cell formation (Steinberg et al., 2002; Whitman, 2004; Zhao et al., 2008; Singh et al., 2009). Mice lacking apoE have a substantial delay in the metabolism of lipoproteins, particularly VLDL, even fed with a regular standard chow feed (Hoen et al., 2003; Kato et al., 2009). Lesions in apoE-deficient mouse have many features in common with human atherosclerosis, even that the progression can be advantageous in many experimental situations (Dansky et al, 1999). At 26 weeks, atherosclerotic lesions are in the early stages of development, characterized by lipoprotein accumulation, leukocyte gathering, and foam cell formation. This model develops atherosclerotic lesions which progress to occlusion of We also checked in blood plasma of apoE KO and WT mice the levels of total cholesterol, including triglycerides, high-density lipoprotein and low-density lipoprotein, since it can increase the risk of heart disease and atherosclerosis (Steinberg, 2002; Stocker and Keany, 2004, 2005). Mice naturally have high levels of HDL and low levels of LDL, lacking the cholesterol ester transfer protein, an enzyme responsible for trafficking cholesterol from HDL to VLDL and LDL. As reported also by others previously, we found clear cut differences in abundance of cholesterol related particles between apoE KO mice and WT mice (Table 1), (Hoen et al., 2003; Kato et al., 2009). In particular, each group of apoE KO mice had five times more plasma cholesterol than their WT counterparts. The apoE KO mice also always had higher TAG levels. HDL levels in apoE KO mice supplied with standard feed and 1% cholesterol supplemented diet was also twice as high than in WT mice. Interestingly, 3% cholesterol supplemented diet resulted in a reversal, meaning that HDL levels (i.e. "good" HDL-lipoproteins) in WT mice became twice as high as in apoE KO mice (Table 1). The generally low LDL cholesterol levels in WT mice even with cholesterol supplemented diet may be due to the capability of WT mice to efficiently suppress the percentage of dietary cholesterol absorption by increasing the excretion of gallbladder biliary cholesterol concentration (Sehayek et al., 2000).

We used this model, of apoE KO mice fed with cholesterol supplemented diet that shows well developed atherosclerosis, to assess oxidative stress in the aorta in correlation with TSPO binding density and atherosclerosis. For this purpose, homogenates of the aorta were used for ROS analysis and antioxidant enzymes activities. As accumulation of proatherogenic lipid affects all cell types present within the vascular wall, the response of the entire tissue vs. isolated cells to the hyperlipidemic conditions is relevant as an indication of vascular defense as a whole. The increase in plasma cholesterol levels was paralleled by changes in oxidative stress parameters in WT mice and ApoE KO mice, as discussed in detail below.

An indicator of cellular defence capacity against oxidative stress is the presence of reduced GSH, which we determined in the aorta homogenates after application of feed with cholesterol supplements. As seen in table 3, a reduction of GSH content in was evident compared to the corresponding controls, when 3% cholesterol diet was administered to WT as well as apoE mice. This shows that cholesterol diet regime indeed constitutes an elevated risk factor for ROS formation, due to a reduction in GSH levels in this model. It has been

#### 106 Lipid Metabolism

reported that ROS induce vascular cells to express cell adhesion molecules that trigger adhesion of leukocytes to the endothelium, which is part of the initiation atherosclerosis (Yang et al., 2009). Interestingly, it was also found that TSPO expression correlates positively with expression of adhesion molecules (Bode et al., 2012; Veenman et al., 2012). This may suggest that the reduction in TSPO levels seen in this study may counteract adhesion of leukocytes to the endothelium, and thereby prevent initiation atherosclerosis in particular in WT mice.

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 107

discussed previously that the reduced TSPO levels accompanying atherogenic challenges may be a compensatory mechanism to counteract oxidative stress in the aorta and liver (Dimitrova-Shumkovska et al., 2010 a, b, c). This would be in effect similar to increased levels of SOD observed, which also counteract oxidative stress (see above). Our present study suggests that reduced TSPO binding density as observed in WT mice subjected to cholesterol supplemented diet may counteract oxidative stress as one mechanism to attenuate the development of atherosclerosis. As TSPO binding density is not affected in apoE mice subjected to cholesterol supplemented diet mentioned TSPO dependent mechanism is not available for apoE KO mice to counteract development of atherosclerosis. Presently, it is not known which components of the vascular wall, i.e. mast cells, smooth muscular, or dermal vascular endothelial cells, would be important for the potential correlation between TSPO expression, oxidative stress, and atherosclerosis (Stoebner et al., 1999; 2001; Morgan et al., 2004; Veenman and Gavish, 2006; Dimitrova-Shumkovska et al.,

It can be assumed from the present study, that oxidative stress parameters do not absolutely correlate with the development of atherosclerotic lesions (because supplementation with 1% of cholesterol to the diet does not affect oxidative stress), but the absolute levels of cholesterol do correlate with atherosclerotic development. Nonetheless, enhancement of cholesterol percentage from 1% to 3% in the diet resulted in significant increases in ROS parameters of WT and apoE KO mice in comparison to their control groups, and also provoked advanced lesion formation in aortic intimae in apoE KO mice fed a 3% cholesterol supplemented diet (but not in WT mice). TSPO binding density is reduced due to cholesterol intake in particular in WT mice and such changes in TSPO binding density in WT mice are in negative correlation with oxidative stress measured in heart and aorta. We believe the reductions in TSPO binding density in WT mice are compensatory for oxidative stress and atherosclerotic development. Thus, the lack of a significant decrease in TSPO binding density in the aorta of 3% cholesterol fed apoE KO mice may actually correlate with the enhanced atherosclerosis in this model. The capability of apoE KO mice fed with 1% cholesterol to reduce TSPO binding density in the aorta may present a rudimentary antiatherosclerosis protective capacity. In conclusion, this study is in accord with previous studies suggesting that reductions in arterial TSPO binding density are part of a mechanism counteracting the development of atherosclerosis. A question is how the presence of apoE, in combination with enhanced dietary cholesterol levels, can result in suppression of TSPO binding density. It is also important to find out how in a mechanistic sense a reduction in TSPO levels can contribute to self protection against the development of atherosclerosis.

**Explanation of abbreviations and symbols:** ACS, acute coronary syndrome; ANOVA, analysis of variance; (AOPPs), advanced oxidation protein products; ApoE-/- KO, apolipoprotein E knockout mice; cAMP, adenosine 3,5-cyclic monophosphate; CBR, centraltype benzodiazepine receptor; DBI, Diazepam Binding Inhibitor; CAM, cell adhesion molecule; CVD, cardiovascular disease; HDL, high-density lipoprotein; HFHC- high fat high cholesterol diet; HMGCoA, 3-hydroxy-3-methylglutaryl coenzyme A reductase; H2O2, hydrogen peroxide; Hb, hemoglobin; IL-1, interleukin-1 (IL-2, etc.); kDa, kilodalton; Kd,

2010 a, b, c).

In accord with the observations of Hoen et al. (2003) that the mRNA levels of many antioxidant enzymes in apoE KO mice are higher (1.5 -5 fold) in the age of 6-15 weeks, compared to aged-matched wild type mice, we also saw that SOD activity were higher in aorta homogenates of apoE mice than those in age-matched WT mice (Table 3). Their hypothesis is that the aorta compensates for the oxidative stress induced by atherogenic stimuli, by stimulating the expression of antioxidant enzymes, thereby delaying the process of atheroma plaque formation. The latter was supported by Yang et al. (2004, 2009) providing evidence that over expression of catalase and superoxide dismutase delayed the development of atherosclerosis in apoE KO mice.

To determine the potential involvement of the TSPO in effects of apoE dysregulation, we studied TSPO binding density in heart and aorta of apoE KO mice (B6.129P2-apoE*tm1* N11) versus their wild type (WT) background mice, with and without inclusion of 1% and 3% cholesterol to the diet. TSPO has been detected in heart of normal mice before, and we found comparable levels in our control animals (Hashimoto et al., 1989; Weizman et al., 1992; Fares et al., 1990; Katz et al., 1994; Dumont et al., 1999). To our knowledge the present study is the first study regarding TSPO binding density in the aorta of mice, which are quite high (even comparable to TSPO levels in adrenal of rats (Gavish and Fares, 1985; Gavish et al., 1999). We found that enhanced cholesterol levels in the diet can result in reduced TSPO binding density in the aorta and heart of WT mice, as well as in the aorta of apoE mice (Table 4). The present study indicates that there is negative correlation between ROS parameters in heart tissue and TSPO binding density in cholesterol fed WT mice. Namely, in the heart of WT mice, the "steady state" levels of lipid peroxides (TBARs) showed a 2.5 fold enhancement after 3% cholesterol supplemented diet vs. a 1/3 fold enhancement in the group with 1% cholesterol supplemented diet. Regarding oxidized proteins in the heart tissues of WT mice fed with cholesterol supplements, AOPP and proteins carbonyls showed increases of 40% and 35%, respectively, regardless of the cholesterol percentage (data not shown). Such a relation between ROS parameters and TSPO binding density is not apparent in apoE mice, since in apoE mice little effect is seen on TSPO binding density.

Also in a previous study, enhanced plasma lipid levels due to HFHC diet supplied to rats, enhanced oxidative stress parameters and decreased indicators for antioxidant activity in the aorta, which were associated with reduced TSPO density in this organ (Dimitrova-Shumkovska et al., 2010a). Notably, wild type rats are not prone to develop atherosclerosis even when subjected to HFHC diet (Dimitrova-Shumkovska et al., 2010a). We have shown that reduction of TSPO expression by genetic manipulation in vitro in cell culture reduces mitochondrial ROS generation (Veenman et al., 2008, 2012; Zeno et al., 2009). We have discussed previously that the reduced TSPO levels accompanying atherogenic challenges may be a compensatory mechanism to counteract oxidative stress in the aorta and liver (Dimitrova-Shumkovska et al., 2010 a, b, c). This would be in effect similar to increased levels of SOD observed, which also counteract oxidative stress (see above). Our present study suggests that reduced TSPO binding density as observed in WT mice subjected to cholesterol supplemented diet may counteract oxidative stress as one mechanism to attenuate the development of atherosclerosis. As TSPO binding density is not affected in apoE mice subjected to cholesterol supplemented diet mentioned TSPO dependent mechanism is not available for apoE KO mice to counteract development of atherosclerosis. Presently, it is not known which components of the vascular wall, i.e. mast cells, smooth muscular, or dermal vascular endothelial cells, would be important for the potential correlation between TSPO expression, oxidative stress, and atherosclerosis (Stoebner et al., 1999; 2001; Morgan et al., 2004; Veenman and Gavish, 2006; Dimitrova-Shumkovska et al., 2010 a, b, c).

106 Lipid Metabolism

WT mice.

development of atherosclerosis in apoE KO mice.

since in apoE mice little effect is seen on TSPO binding density.

reported that ROS induce vascular cells to express cell adhesion molecules that trigger adhesion of leukocytes to the endothelium, which is part of the initiation atherosclerosis (Yang et al., 2009). Interestingly, it was also found that TSPO expression correlates positively with expression of adhesion molecules (Bode et al., 2012; Veenman et al., 2012). This may suggest that the reduction in TSPO levels seen in this study may counteract adhesion of leukocytes to the endothelium, and thereby prevent initiation atherosclerosis in particular in

In accord with the observations of Hoen et al. (2003) that the mRNA levels of many antioxidant enzymes in apoE KO mice are higher (1.5 -5 fold) in the age of 6-15 weeks, compared to aged-matched wild type mice, we also saw that SOD activity were higher in aorta homogenates of apoE mice than those in age-matched WT mice (Table 3). Their hypothesis is that the aorta compensates for the oxidative stress induced by atherogenic stimuli, by stimulating the expression of antioxidant enzymes, thereby delaying the process of atheroma plaque formation. The latter was supported by Yang et al. (2004, 2009) providing evidence that over expression of catalase and superoxide dismutase delayed the

To determine the potential involvement of the TSPO in effects of apoE dysregulation, we studied TSPO binding density in heart and aorta of apoE KO mice (B6.129P2-apoE*tm1* N11) versus their wild type (WT) background mice, with and without inclusion of 1% and 3% cholesterol to the diet. TSPO has been detected in heart of normal mice before, and we found comparable levels in our control animals (Hashimoto et al., 1989; Weizman et al., 1992; Fares et al., 1990; Katz et al., 1994; Dumont et al., 1999). To our knowledge the present study is the first study regarding TSPO binding density in the aorta of mice, which are quite high (even comparable to TSPO levels in adrenal of rats (Gavish and Fares, 1985; Gavish et al., 1999). We found that enhanced cholesterol levels in the diet can result in reduced TSPO binding density in the aorta and heart of WT mice, as well as in the aorta of apoE mice (Table 4). The present study indicates that there is negative correlation between ROS parameters in heart tissue and TSPO binding density in cholesterol fed WT mice. Namely, in the heart of WT mice, the "steady state" levels of lipid peroxides (TBARs) showed a 2.5 fold enhancement after 3% cholesterol supplemented diet vs. a 1/3 fold enhancement in the group with 1% cholesterol supplemented diet. Regarding oxidized proteins in the heart tissues of WT mice fed with cholesterol supplements, AOPP and proteins carbonyls showed increases of 40% and 35%, respectively, regardless of the cholesterol percentage (data not shown). Such a relation between ROS parameters and TSPO binding density is not apparent in apoE mice,

Also in a previous study, enhanced plasma lipid levels due to HFHC diet supplied to rats, enhanced oxidative stress parameters and decreased indicators for antioxidant activity in the aorta, which were associated with reduced TSPO density in this organ (Dimitrova-Shumkovska et al., 2010a). Notably, wild type rats are not prone to develop atherosclerosis even when subjected to HFHC diet (Dimitrova-Shumkovska et al., 2010a). We have shown that reduction of TSPO expression by genetic manipulation in vitro in cell culture reduces mitochondrial ROS generation (Veenman et al., 2008, 2012; Zeno et al., 2009). We have It can be assumed from the present study, that oxidative stress parameters do not absolutely correlate with the development of atherosclerotic lesions (because supplementation with 1% of cholesterol to the diet does not affect oxidative stress), but the absolute levels of cholesterol do correlate with atherosclerotic development. Nonetheless, enhancement of cholesterol percentage from 1% to 3% in the diet resulted in significant increases in ROS parameters of WT and apoE KO mice in comparison to their control groups, and also provoked advanced lesion formation in aortic intimae in apoE KO mice fed a 3% cholesterol supplemented diet (but not in WT mice). TSPO binding density is reduced due to cholesterol intake in particular in WT mice and such changes in TSPO binding density in WT mice are in negative correlation with oxidative stress measured in heart and aorta. We believe the reductions in TSPO binding density in WT mice are compensatory for oxidative stress and atherosclerotic development. Thus, the lack of a significant decrease in TSPO binding density in the aorta of 3% cholesterol fed apoE KO mice may actually correlate with the enhanced atherosclerosis in this model. The capability of apoE KO mice fed with 1% cholesterol to reduce TSPO binding density in the aorta may present a rudimentary antiatherosclerosis protective capacity. In conclusion, this study is in accord with previous studies suggesting that reductions in arterial TSPO binding density are part of a mechanism counteracting the development of atherosclerosis. A question is how the presence of apoE, in combination with enhanced dietary cholesterol levels, can result in suppression of TSPO binding density. It is also important to find out how in a mechanistic sense a reduction in TSPO levels can contribute to self protection against the development of atherosclerosis.

**Explanation of abbreviations and symbols:** ACS, acute coronary syndrome; ANOVA, analysis of variance; (AOPPs), advanced oxidation protein products; ApoE-/- KO, apolipoprotein E knockout mice; cAMP, adenosine 3,5-cyclic monophosphate; CBR, centraltype benzodiazepine receptor; DBI, Diazepam Binding Inhibitor; CAM, cell adhesion molecule; CVD, cardiovascular disease; HDL, high-density lipoprotein; HFHC- high fat high cholesterol diet; HMGCoA, 3-hydroxy-3-methylglutaryl coenzyme A reductase; H2O2, hydrogen peroxide; Hb, hemoglobin; IL-1, interleukin-1 (IL-2, etc.); kDa, kilodalton; Kd, equilibrium dissociation constant; *K*m, equilibrium constant related to Michaelis-Menten kinetics (similarly, *K*d, *K*a, *K*eq, *K*s); LDL, low density lipoproteins; mPTP, mitochondrial permeability transition pore; MCP-1, monocyte chemoatractant proteins-1; NADP, nicotinamide adenine dinucleotide phosphate; NADH, reduced nicotinamide adenine dinucleotide; PBR, peripheral-type benzodiazepine receptor; PC protein carbonyls; PK 11195, 1-(2- chlorophenyl)-N-methyl-N-(1-methyl-prop1)-3 isoquinolinecarboxamide; ONOO- , peroxinitrite ; Ro5-4864, (4'- chlorodiazepam); ROS , reactive oxygen species; SOD, superoxide dismutase activity; TBARs, thiobarbituric acid reactive substances; TNF, tumor necrosis factor; TSPO, 18 kDa translocator protein; VCAM-1, vascular cell adhesion molecule; VSMCs, vascular smooth muscle cells.

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 109

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