**2. The effects of cholesterol challenges that result in atherogenesis on TSPO binding density in aorta and heart**

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 Macedonia), named "standard feed" hereafter.

At 16 weeks of age, animals were randomized into experimental groups: i) Two control groups (WT mice, n = 10) and (apoE KO mice, n = 10), both these control groups received standard feed for a additional period of 10 weeks; ii) Two experimental groups receiving the same feed for the same 10 weeks but supplemented with 1% cholesterol (1% WT mice, n = 10) and (1% apoE KO mice, n= 10); and iii) Two experimental groups received the same feed for the same 10 weeks but supplemented with 3% cholesterol (3% WT mice, n = 10) and (3% apoE KO mice, n = 10). After these 10 weeks, animals were sacrificed by cardiac puncture, under ketamine/xylazine anaesthesia, followed by the appropriated storage until application or procedures required for assays of TSPO binding characteristics, ROS parameters, and histopathology, as described in detail previously (Dimitrova-Shumkovska et al., 2010 a, b, c, 2012). Tissue homogenates of aorta and heart were prepared for our various assays. For TSPO binding assays, tissue homogenates were prepared in 50 mM PBS on ice with a Kinematika Polytron (Luzerne, Switzerland), as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c). To prepare homogenates for assays of oxidative stress parameters, we used an Ultrasonic Homogenizer (Cole-Parmer Instrument Co., Chicago, IL) as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c). For advanced oxidation protein products (AOPPs, Witko-Sarsat et al., 1996), tissue homogenates were prepared in 50 mM PBS at + 4 ºC, as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c). For the other assays of oxidative stress (see below), tissue homogenates were prepared in 1.12 % KCl at + 4 ºC, as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c). These later parameters of oxidative injury included: lipid peroxidation products [TBARs] (Draper and Hadley, 1990); protein carbonylation, PC (Shacter, 2000); superoxide dismutase activity (SOD assay kit, RA20408, Fluka, Biochemika, Steinheim, Germany), glutathione (GSH assay kit CS0260, Sigma-Aldrich, Steinheim, Germany), glutathione reductase (GSSG-Red), GRSA 114K4000, Sigma-Aldrich, Steinheim, Germany], Finally, aortas were prepared for anatomical observation and histopathology as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c).

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 99

(Davignon et al., 1999; Johnson et al., 2005). ApoE KO mice can also develop interplaque hemorrhage and features of plaque instability that are accelerated by feeding westernized diet (Rosenfeld et al., 2000). "Western type diets for mice" typically utilize just one

**Figure 1.** Representative cross-sections of mice aortas. A) No atherosclerotic lesions were found in

characterized by a thin fibrous tissue cap (elbow black arrow), particularly ssuperficial accumulation of foam cells (green arrow) without a necrotic core and encapsulated by collagen rich fibrous tissue in apoE KO mice given standard feed; C) accelerated atherosclerosis and deposition of cholesterol crystals (black arrow) in the endothelium of the aorta wall in 1% apoE KO; D) advanced lesions are developed in 3% apoE mice. Initial xanthoma formation, cartilage tissue (asterix) and calcified nodules (yellow arrow) with an underlying fibrocalcific plaque with minimal or absence of necrosis occur (H&E

wild-type mice regardless of the diet; B) atherosclerotic plaque (outlined)

staining, microscopic magnification applied x 100).

ingredient (milk fat or lard) as the primary source of energy from fat.

Effects of cholesterol supplements to the apoE KO mice on plaque formation in the aorta are shown in Figure 1. No atherosclerotic formation was found in WT mice regardless of diet (Figure 1A). Control aortas of apoE KO mice having access to standard feed are characterized by the presence of thin fibrous tissue caps i.e. encapsulations of collagen rich fibrous tissue without a necrotic core that showed only superficial accumulation of foam cells (Figure 1B). Cholesterol diet accelerated atherosclerosis in apoE KO mice, increasing the total surface area of plaque formation significantly over the intimal area (Figure 1C) compared to apoE mice receiving standard feed. In 1% cholesterol fed apoE KO mice, expansion of the necrotic core presenting an important pathogenic process contributing to plaque vulnerability was observed in comparison to standard fed apoE mice (Figure 1C). After administration of 3% cholesterol diet to apoE KO mice even more advanced lesions have developed. Initial xanthoma formation, cartilage tissue, and calcified nodules with an underlying fibrocalcific plaque with minimal or absence of necrosis occurred (Figure 1D). Furthermore, plaques become more progressive and lesions show luminal stenosis with pathologic intimal thickening. These observations are in line with other research data, where plague rupture was seen in apoE KO mice especially when exposed to western type diet (Davignon et al., 1999; Johnson et al., 2005). ApoE KO mice can also develop interplaque hemorrhage and features of plaque instability that are accelerated by feeding westernized diet (Rosenfeld et al., 2000). "Western type diets for mice" typically utilize just one ingredient (milk fat or lard) as the primary source of energy from fat.

98 Lipid Metabolism

At 16 weeks of age, animals were randomized into experimental groups: i) Two control groups (WT mice, n = 10) and (apoE KO mice, n = 10), both these control groups received standard feed for a additional period of 10 weeks; ii) Two experimental groups receiving the same feed for the same 10 weeks but supplemented with 1% cholesterol (1% WT mice, n = 10) and (1% apoE KO mice, n= 10); and iii) Two experimental groups received the same feed for the same 10 weeks but supplemented with 3% cholesterol (3% WT mice, n = 10) and (3% apoE KO mice, n = 10). After these 10 weeks, animals were sacrificed by cardiac puncture, under ketamine/xylazine anaesthesia, followed by the appropriated storage until application or procedures required for assays of TSPO binding characteristics, ROS parameters, and histopathology, as described in detail previously (Dimitrova-Shumkovska et al., 2010 a, b, c, 2012). Tissue homogenates of aorta and heart were prepared for our various assays. For TSPO binding assays, tissue homogenates were prepared in 50 mM PBS on ice with a Kinematika Polytron (Luzerne, Switzerland), as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c). To prepare homogenates for assays of oxidative stress parameters, we used an Ultrasonic Homogenizer (Cole-Parmer Instrument Co., Chicago, IL) as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c). For advanced oxidation protein products (AOPPs, Witko-Sarsat et al., 1996), tissue homogenates were prepared in 50 mM PBS at + 4 ºC, as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c). For the other assays of oxidative stress (see below), tissue homogenates were prepared in 1.12 % KCl at + 4 ºC, as described previously (Dimitrova-Shumkovska et al., 2010 a, b, c). These later parameters of oxidative injury included: lipid peroxidation products [TBARs] (Draper and Hadley, 1990); protein carbonylation, PC (Shacter, 2000); superoxide dismutase activity (SOD assay kit, RA20408, Fluka, Biochemika, Steinheim, Germany), glutathione (GSH assay kit CS0260, Sigma-Aldrich, Steinheim, Germany), glutathione reductase (GSSG-Red), GRSA 114K4000, Sigma-Aldrich, Steinheim, Germany], Finally, aortas were prepared for anatomical observation and histopathology as described

Effects of cholesterol supplements to the apoE KO mice on plaque formation in the aorta are shown in Figure 1. No atherosclerotic formation was found in WT mice regardless of diet (Figure 1A). Control aortas of apoE KO mice having access to standard feed are characterized by the presence of thin fibrous tissue caps i.e. encapsulations of collagen rich fibrous tissue without a necrotic core that showed only superficial accumulation of foam cells (Figure 1B). Cholesterol diet accelerated atherosclerosis in apoE KO mice, increasing the total surface area of plaque formation significantly over the intimal area (Figure 1C) compared to apoE mice receiving standard feed. In 1% cholesterol fed apoE KO mice, expansion of the necrotic core presenting an important pathogenic process contributing to plaque vulnerability was observed in comparison to standard fed apoE mice (Figure 1C). After administration of 3% cholesterol diet to apoE KO mice even more advanced lesions have developed. Initial xanthoma formation, cartilage tissue, and calcified nodules with an underlying fibrocalcific plaque with minimal or absence of necrosis occurred (Figure 1D). Furthermore, plaques become more progressive and lesions show luminal stenosis with pathologic intimal thickening. These observations are in line with other research data, where plague rupture was seen in apoE KO mice especially when exposed to western type diet

previously (Dimitrova-Shumkovska et al., 2010 a, b, c).

**Figure 1.** Representative cross-sections of mice aortas. A) No atherosclerotic lesions were found in wild-type mice regardless of the diet; B) atherosclerotic plaque (outlined) characterized by a thin fibrous tissue cap (elbow black arrow), particularly ssuperficial accumulation of foam cells (green arrow) without a necrotic core and encapsulated by collagen rich fibrous tissue in apoE KO mice given standard feed; C) accelerated atherosclerosis and deposition of cholesterol crystals (black arrow) in the endothelium of the aorta wall in 1% apoE KO; D) advanced lesions are developed in 3% apoE mice. Initial xanthoma formation, cartilage tissue (asterix) and calcified nodules (yellow arrow) with an underlying fibrocalcific plaque with minimal or absence of necrosis occur (H&E staining, microscopic magnification applied x 100).

#### 100 Lipid Metabolism


The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 101

**Table 2.** Effects of cholesterol (Chol) supplemented diet for 10 weeks on aorta oxidative stress

**Variables / Aorta WT Control 1% Chol 3% Chol**

 **TBARs nmol/mg** 0.16 ± 0.04 (n=8) 0.17 ± 0.06 (n=7) **0.32 ± 0.08\*\*** (n=8)

 **AOPP nmol/mg** 37.1 ± 11.3 (n=7) 44.1 ± 19.3 (n=8) **86.8 ± 21.4\*\* (n=8)**

 **PC pmol/mg** 45.7 ± 11.0 (n=7) 55.2 ± 22.8 (n=8) 55.3 ± 11.8 (n=8)

**Variables / Aorta Apo E Control 1% Chol 3% Chol**

 **TBARs nmol/mg** 0.24 ± 0.07 (n=8) 0.29 ± 0.11 (n=8) **0.54 ± 0.25\*\*\* (n=10)**

 **AOPP nmol/mg** 22.0 ± 17.1 (n=8) 27.9 ± 7.1 (n=8) **60.5 ± 30.6\*\*\* (n=10)**

 **PC pmol/mg** 46.6 ± 20.3 (n=8) 45.3 ± 11.3 (n=8) 52.1 ± 10.6 (n=12)

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

0.05) and in WT mice (by 47% p < 0.05).

parameters in apoE KO mice and their WT counterparts. 1-way ANOVA followed by application of the Tukey test to assess the significance of specific intergroup differences. Data are expressed as mean ± SD;

The capacity of glutathione as an electron donor to regenerate the most important antioxidants (vitamin E, glutathione peroxidase (GPx), lipid hydroperoxides), is linked with the redox state of the glutathione disulfide – glutathione couple GSSG/2GSH (Schafer and Buettner, 2001). This in turn, has a high impact on the overall redox environment in the cell. Concerning antioxidant activities in aorta tissue due to 3% cholesterol supplemented feed, significantly reduced activity of superoxide dismutase (SOD) was measured in 3% apoE KO mice compared to standard feed mice (- 41%**, Table 3**). The results also suggest a significant reverse interaction between glutathione level (GSH) and glutathione peroxidase (GPx) activity in aorta tissue. In particular, the analyzed results indicated that the glutathione content in aorta of 3% apoE animals was significantly decreased (-32%), with simultaneous slight, but significant enhancement achieved in activity of glutathione peroxidase (+10%), as compared to standard feed control (p < 0.05). In parallel, glutathione content in aorta was also significantly reduced in 3% WT mice for 70% (p < 0.01), without affecting GPx levels. Feeding the mice diet supplemented with 1% cholesterol, resulted in significantly reduced activity in SOD in apoE KO mice (by 33% p <

To determine TSPO binding characteristics in this paradigm we applied binding assays with the TSPO specific ligand [3H] PK 11195. The present study sought to determine whether cholesterol supplementation affects TSPO binding characteristics in aorta and heart of apoE KO mice in association with parameters for oxidative stress. Binding assays of the heart and


**Table 1.** Effects of cholesterol (Chol) supplemented diet for 10 weeks, on lipoprotein levels in apoE KO mice and their WT counterparts. Unpaired Student t*-test* was performed. Data are expressed as mean ± SD; \* = p < 0.05, \*\* = p < 0.01, \*\*\* = p < 0.001.

Changes in the serum levels of total cholesterol, triglycerides and HDL-cholesterol in each group are shown in **Table 1.** Corroborating previous studies (Davignon et al., 1999; Seo et al., 2005; Zhao et al., 2008) at 16 weeks of age, even before application of the cholesterol enriched diets, apoE KO mice, already displayed approximately 5 times higher levels of total cholesterol in comparison with WT mice. At this time point, no significant differences in triglycerides (TAG) levels were observed between WT mice and apoE KO mice. However, 3% diet regimes, caused significant increases in total cholesterol level in apoE KO mice (by 44%, p < 0.001), compared to standard feed. The enhanced total cholesterol levels, included an almost 90% representation of non HDL – cholesterol (calculated from Friedewald formula; Friedewald et al., 1972). In contrast, 3% WT mice, showed significantly higher cholesterol levels (by 62%, p < 0.01), including an almost 70% representation of HDLlipoproteins. Supplement of 3% cholesterol also provoked significantly higher triglycerides levels: by 35 % (p < 0.01) in apoE mice and by 36% (p < 0.01) in WT mice. Supplement of 1% cholesterol, resulted in slight increases in total cholesterol in apoE mice (by 20%, p < 0.05), but did not significantly affect the triglycerides levels. The same type of diet did not affect lipoprotein levels in WT mice.

In the aorta, 3% cholesterol diet supplement, caused significant increases in "steady-state" levels of lipid peroxides (TBARs) and oxidized proteins in WT as well as apoE KO mice (**Table 2**). In detail, regarding lipid peroxidation, TBARs production was significantly increased by 2 fold in WT and apoE KO mice subjected to 3% cholesterol supplemented diet (+100%, p < 0.01 for WT mice, and +125%, p < 0.001 for ApoE KO mice). In parallel, protein oxidation products levels (AOPP) were also significantly higher (+135%, p < 0.01 in 3% WT mice and +177%, p < 0.001, in 3% apoE KO mice). Protein carbonyls (PC) showed a slight but non-significant increase in 3% cholesterol fed WT and apoE KO mice, compared to their controls. In contrast to the 3% diet regime, 1% cholesterol supplemented diet did not affect ROS parameters in aortic tissue in both WT and apoE KO mice.

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 101


100 Lipid Metabolism

SD; \* = p < 0.05, \*\* = p < 0.01, \*\*\* = p < 0.001.

lipoprotein levels in WT mice.

**Table 1.** Effects of cholesterol (Chol) supplemented diet for 10 weeks, on lipoprotein levels in apoE KO mice and their WT counterparts. Unpaired Student t*-test* was performed. Data are expressed as mean ±

**Apo E (-/-) <sup>16</sup>** 383.7 ± 47.3 **457.23 ± 62\* 555.4 ± 83.3\*\*\*** 117.7± 24.5 105.6 ± 11.4 **159.4 ± 59.0\*\*** 67.0 ± 37.5 66.6 ± 16.3 **32.9 ± 11.4\*\***

**wk** C **1% 3%** C **1% 3%** C **1% 3%**

 **Plasma lipoprotein levels mg/dL Strain Chol TAG HDL**

 **Plasma lipoprotein levels mg/dL Strain Chol TAG HDL**

**wk** C **1% 3%** C **1% 3%** C **1% 3%**

 **WT 16** 67.7 ± 23.3 88.07 ± 28.0 **110.0 ± 27.0\*\*** 71.0 ± 18.4 71.5 ± 5.1 **97.5 ± 16.2\*\*** 33.2 ± 5.1 33.6 ± 10.8 **74.7 ± 10.8\*\*\***

Changes in the serum levels of total cholesterol, triglycerides and HDL-cholesterol in each group are shown in **Table 1.** Corroborating previous studies (Davignon et al., 1999; Seo et al., 2005; Zhao et al., 2008) at 16 weeks of age, even before application of the cholesterol enriched diets, apoE KO mice, already displayed approximately 5 times higher levels of total cholesterol in comparison with WT mice. At this time point, no significant differences in triglycerides (TAG) levels were observed between WT mice and apoE KO mice. However, 3% diet regimes, caused significant increases in total cholesterol level in apoE KO mice (by 44%, p < 0.001), compared to standard feed. The enhanced total cholesterol levels, included an almost 90% representation of non HDL – cholesterol (calculated from Friedewald formula; Friedewald et al., 1972). In contrast, 3% WT mice, showed significantly higher cholesterol levels (by 62%, p < 0.01), including an almost 70% representation of HDLlipoproteins. Supplement of 3% cholesterol also provoked significantly higher triglycerides levels: by 35 % (p < 0.01) in apoE mice and by 36% (p < 0.01) in WT mice. Supplement of 1% cholesterol, resulted in slight increases in total cholesterol in apoE mice (by 20%, p < 0.05), but did not significantly affect the triglycerides levels. The same type of diet did not affect

In the aorta, 3% cholesterol diet supplement, caused significant increases in "steady-state" levels of lipid peroxides (TBARs) and oxidized proteins in WT as well as apoE KO mice (**Table 2**). In detail, regarding lipid peroxidation, TBARs production was significantly increased by 2 fold in WT and apoE KO mice subjected to 3% cholesterol supplemented diet (+100%, p < 0.01 for WT mice, and +125%, p < 0.001 for ApoE KO mice). In parallel, protein oxidation products levels (AOPP) were also significantly higher (+135%, p < 0.01 in 3% WT mice and +177%, p < 0.001, in 3% apoE KO mice). Protein carbonyls (PC) showed a slight but non-significant increase in 3% cholesterol fed WT and apoE KO mice, compared to their controls. In contrast to the 3% diet regime, 1% cholesterol supplemented diet did not affect

ROS parameters in aortic tissue in both WT and apoE KO mice.


**Table 2.** Effects of cholesterol (Chol) supplemented diet for 10 weeks on aorta oxidative stress parameters in apoE KO mice and their WT counterparts. 1-way ANOVA followed by application of the Tukey test to assess the significance of specific intergroup differences. Data are expressed as mean ± SD; \* = p < 0.05, \*\* = p < 0.01, \*\*\* = p < 0.001.

The capacity of glutathione as an electron donor to regenerate the most important antioxidants (vitamin E, glutathione peroxidase (GPx), lipid hydroperoxides), is linked with the redox state of the glutathione disulfide – glutathione couple GSSG/2GSH (Schafer and Buettner, 2001). This in turn, has a high impact on the overall redox environment in the cell. Concerning antioxidant activities in aorta tissue due to 3% cholesterol supplemented feed, significantly reduced activity of superoxide dismutase (SOD) was measured in 3% apoE KO mice compared to standard feed mice (- 41%**, Table 3**). The results also suggest a significant reverse interaction between glutathione level (GSH) and glutathione peroxidase (GPx) activity in aorta tissue. In particular, the analyzed results indicated that the glutathione content in aorta of 3% apoE animals was significantly decreased (-32%), with simultaneous slight, but significant enhancement achieved in activity of glutathione peroxidase (+10%), as compared to standard feed control (p < 0.05). In parallel, glutathione content in aorta was also significantly reduced in 3% WT mice for 70% (p < 0.01), without affecting GPx levels. Feeding the mice diet supplemented with 1% cholesterol, resulted in significantly reduced activity in SOD in apoE KO mice (by 33% p < 0.05) and in WT mice (by 47% p < 0.05).

To determine TSPO binding characteristics in this paradigm we applied binding assays with the TSPO specific ligand [3H] PK 11195. The present study sought to determine whether cholesterol supplementation affects TSPO binding characteristics in aorta and heart of apoE KO mice in association with parameters for oxidative stress. Binding assays of the heart and


The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 103

**Figure 2.** Representative examples of saturation curves (**A, C, E, G)** and their Scatchard plots (**B, D, F, H**) of [3H]PK 11195 binding to membrane homogenates of aorta**,** respectively of WT mice (**A , B, C, D)**  and apoE KO mice (**E, F, G, H).** Abbreviations: apoE KO = apolipoprotein deficient mice; WT- wild type

mice; B: bound; B/F: bound over free.

**Table 3.** Effects of cholesterol (Chol) supplemented diet for 10 weeks on aorta antioxidant parameters in apoE KO mice and their WT counterparts. Unpaired Student t*-test* was performed. Data are expressed as mean ± SD; \* = p < 0.05, \*\* = p < 0.01.

aorta with the TSPO specific ligand [3H]PK 11195 were done to determine potential effects of cholesterol supplementation on TSPO binding characteristics, according to methods described previously (Dimitrova-Shumkovska et al., 2010 a,b,c). For representative examples, see **Figure 2.** In heart , only in WT mice significant decreases in the Bmax of TSPO (- 42%, p < 0.001) was determined with [3H]PK 11195 binding as a consequence of both cholesterol 1% and 3% supplemented diets, compared to control standard fed WT mice. Regarding the apoE KO mice, cholesterol supplemented diet did not induce differences in the TSPO binding characteristics in the heart **(Table 4)**. Regarding heart tissues, both in the apoE KO groups and WT groups, Kd values determined with [3H] PK 11195 binding were in the nM range (0.6 – 1.6 nM) showing no significant differences between experimental and control groups.

Regarding the aorta, feeding the mice with standard feed was not accompanied by significant differences in the TSPO binding characteristics of the aorta of apoE KO mice versus WT mice (**Table 4**). Interestingly, these mouse aortas showed very TSPO binding levels, comparable to those observed in the adrenal of rats (Gavish et al., 1999). To date, the adrenal of rats is the tissue with one of highest demonstrated Bmax for TSPO ligand binding (Gavish et al., 1999). The 1% cholesterol supplemented diet significantly reduced TSPO binding capacity in aorta in both WT and apoE KO mice. In particular, reductions by 49% in WT mice and by 32% in apoE KO mice (p < 0.001 and p < 0.01, respectively) compared to their standard feed controls were observed (**Table 4**). The 3% cholesterol diet also provoked a reduction in TSPO binding density by 58% in the aorta (p < 0.01), but only in WT mice. In the aortas of both groups, apoE KO mice and WT mice, Kd values determined with [3H] PK 11195 binding were in the nM range (1.5 – 2.6 nM), showing no significant differences between the groups.

102 Lipid Metabolism

**Table 3.** Effects of cholesterol (Chol) supplemented diet for 10 weeks on aorta antioxidant parameters in apoE KO mice and their WT counterparts. Unpaired Student t*-test* was performed. Data are

**GPx mU/mg** 0.242 ± 0.02 (n=9) **0.256 ± 0.01\*(n=7) 0.266 ±0.03\* (n=7)**

**Variables / Aorta WT Control 1% Chol 3% Chol**

**SOD U/mg** 4.76 ± 1.5 (n=9) **2.5 ± 0.9\* (n=7) 1.45 ± 0.6\*\* (n=7)**

**GSH nmol/mg** 7.8 ± 4.2 (n=9) 8.3 ± 3.7 (n=9) **3.7 ± 0.8\*\* (n=9)**

**GPx mU/mg** 0.261 ± 0.01 (n=8) 0.273 ± 0.01 (n=8) 0.257 ± 0.03 (n=8)

**Variables / Aorta Apo E Control 1% Chol 3% Chol**

**SOD U/mg** 6.87 ± 1.6 (n=7) **4.6 ± 1.1\* (n=6) 4.05 ± 0.88\* (n=8)**

**GSH nmol/mg** 6.7 ± 2.9 (n=9) 7.4 ± 2.7 (n=9) **4.5 ± 2.0\* (n=9)**

aorta with the TSPO specific ligand [3H]PK 11195 were done to determine potential effects of cholesterol supplementation on TSPO binding characteristics, according to methods described previously (Dimitrova-Shumkovska et al., 2010 a,b,c). For representative examples, see **Figure 2.** In heart , only in WT mice significant decreases in the Bmax of TSPO (- 42%, p < 0.001) was determined with [3H]PK 11195 binding as a consequence of both cholesterol 1% and 3% supplemented diets, compared to control standard fed WT mice. Regarding the apoE KO mice, cholesterol supplemented diet did not induce differences in the TSPO binding characteristics in the heart **(Table 4)**. Regarding heart tissues, both in the apoE KO groups and WT groups, Kd values determined with [3H] PK 11195 binding were in the nM range (0.6 – 1.6

Regarding the aorta, feeding the mice with standard feed was not accompanied by significant differences in the TSPO binding characteristics of the aorta of apoE KO mice versus WT mice (**Table 4**). Interestingly, these mouse aortas showed very TSPO binding levels, comparable to those observed in the adrenal of rats (Gavish et al., 1999). To date, the adrenal of rats is the tissue with one of highest demonstrated Bmax for TSPO ligand binding (Gavish et al., 1999). The 1% cholesterol supplemented diet significantly reduced TSPO binding capacity in aorta in both WT and apoE KO mice. In particular, reductions by 49% in WT mice and by 32% in apoE KO mice (p < 0.001 and p < 0.01, respectively) compared to their standard feed controls were observed (**Table 4**). The 3% cholesterol diet also provoked a reduction in TSPO binding density by 58% in the aorta (p < 0.01), but only in WT mice. In the aortas of both groups, apoE KO mice and WT mice, Kd values determined with [3H] PK 11195 binding were in the nM range (1.5 – 2.6 nM), showing no significant differences

nM) showing no significant differences between experimental and control groups.

expressed as mean ± SD; \* = p < 0.05, \*\* = p < 0.01.

between the groups.

**Figure 2.** Representative examples of saturation curves (**A, C, E, G)** and their Scatchard plots (**B, D, F, H**) of [3H]PK 11195 binding to membrane homogenates of aorta**,** respectively of WT mice (**A , B, C, D)**  and apoE KO mice (**E, F, G, H).** Abbreviations: apoE KO = apolipoprotein deficient mice; WT- wild type mice; B: bound; B/F: bound over free.

#### 104 Lipid Metabolism

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 Discussion.

The 18 kDa Translocator Protein and Atherosclerosis in Mice Lacking Apolipoprotein E 105

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

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

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

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

observed (Dimitrova-Shumkovska et al., 2010a)

biliary cholesterol concentration (Sehayek et al., 2000).

discussed in detail below.



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