< 0.05, respectively) (1-vessel disease: 28.0 ± 21.1 ng/ml, 2-vessel disease: 39.0 ± 25.6 ng/ml, 3-vessel disease: 41.3 ± 23.2 ng/ml). Comparisons of s-7KCHO between subjects with stable angina pectoris and those with AMI are shown in Fig. 3. s-7KCHO were significantly higher in subjects with AMI than in those with stable angina pectoris even though the number of affected vessels were similar in the two groups (p < 0.01) (Stable angina pectoris: 26.1 ± 15.2

Fig. 3. Comparisons of 7-ketocholesterol concentrations between subjects with stable angina

Table 2. Correlation between 7-keto cholesterol concentration and serum parameters.

Correlation between serum parameters and s-7KCHO in CAD subjects were shown in Table 2. There were no significant correlations between TC or LDL cholesterol concentrations and s-7KCHO. Conversely, s-7KCHO had significant correlations with HDL-cholesterol and hs-CRP concentrations. Other coronary risk factors such as age, sex, hypertension, diabetes mellitus,

ng/ml, AMI: 39.1 ± 27.7 ng/ml).

Data are expressed mean ± SD

Stable AP (n=69): stable angina pectoris AMI (n=68): acute myocardial infarction

pectoris and acute myocardial infarction.

\*p<0.01 vs stable AP

Data are expressed mean ± SD \*p<0.01 vs NCA

Fig. 1. Comparisons of 7-ketocholesterol concentrations between subjects with normal coronary artery and coronary artery disease

Fig. 2. Relation of 7-ketocholesterol concentrations and number of affected vessels.

Comparisons of s-7KCHO between NCA and CAD subjects are shown in Fig. 1. s-7KCHO were significantly higher in subjects with CAD than in those with NCA (p < 0.01) (NCA: 19.0 ± 11.3 ng/ml, CAD: 32.4 ± 23.1 ng/ml). We also examined s-7KCHO in subjects without taking statins. s-7KCHO were significantly higher in subjects with CAD than in those with NCA (p < 0.01) (NCA: 17.7 ± 9.6 ng/ml, CAD: 35.4 ± 25.4 ng/ml). The relationship between s-7KCHO and the number of affected vessels is shown in Fig. 2. s-7KCHO were significantly higher in subjects with 2 or 3 vessel disease than those with single vessel disease (p < 0.05, p < 0.05, respectively) (1-vessel disease: 28.0 ± 21.1 ng/ml, 2-vessel disease: 39.0 ± 25.6 ng/ml, 3-vessel disease: 41.3 ± 23.2 ng/ml). Comparisons of s-7KCHO between subjects with stable angina pectoris and those with AMI are shown in Fig. 3. s-7KCHO were significantly higher in subjects with AMI than in those with stable angina pectoris even though the number of affected vessels were similar in the two groups (p < 0.01) (Stable angina pectoris: 26.1 ± 15.2 ng/ml, AMI: 39.1 ± 27.7 ng/ml).

Data are expressed mean ± SD \*p<0.01 vs stable AP Stable AP (n=69): stable angina pectoris AMI (n=68): acute myocardial infarction

114 Coronary Artery Diseases

Fig. 1. Comparisons of 7-ketocholesterol concentrations between subjects with normal

Fig. 2. Relation of 7-ketocholesterol concentrations and number of affected vessels.

Comparisons of s-7KCHO between NCA and CAD subjects are shown in Fig. 1. s-7KCHO were significantly higher in subjects with CAD than in those with NCA (p < 0.01) (NCA: 19.0 ± 11.3 ng/ml, CAD: 32.4 ± 23.1 ng/ml). We also examined s-7KCHO in subjects without taking statins. s-7KCHO were significantly higher in subjects with CAD than in those with NCA (p < 0.01) (NCA: 17.7 ± 9.6 ng/ml, CAD: 35.4 ± 25.4 ng/ml). The relationship between s-7KCHO and the number of affected vessels is shown in Fig. 2. s-7KCHO were significantly higher in subjects with 2 or 3 vessel disease than those with single vessel disease (p < 0.05, p

Data are expressed mean ± SD \*p<0.01 vs NCA

coronary artery and coronary artery disease

Data are expressed mean ± SD

1VD (n=85): 1-vessel disease 2VD (n=36): 2-vessel disease 3VD (n=18): 3-vessel disease

\*p<0.05 vs 1VD

Fig. 3. Comparisons of 7-ketocholesterol concentrations between subjects with stable angina pectoris and acute myocardial infarction.

Table 2. Correlation between 7-keto cholesterol concentration and serum parameters.

Correlation between serum parameters and s-7KCHO in CAD subjects were shown in Table 2. There were no significant correlations between TC or LDL cholesterol concentrations and s-7KCHO. Conversely, s-7KCHO had significant correlations with HDL-cholesterol and hs-CRP concentrations. Other coronary risk factors such as age, sex, hypertension, diabetes mellitus,

Relatioship Between Serum 7-Ketocholesterol Concentrations and Coronary Artery Disease 117

diagnosed by coronary angiography. Furthermore, it has been reported that almost 70% of culprit lesion on acute myocardial infarction, now called acute coronary syndrome, which is highly correlated with mortality, occurs in less than 50% of angiographic stenosis (Falk E et al., 1995). Therefore, it is difficult to predict the occurrence of acute coronary syndrome and it is important that we consider the presence of coronary atherosclerosis; especially presence of vulnerable plaque, before angiographic stenosis. IVUS studies have clarified the future of plaque on acute coronary syndrome; plaque volume is large and shows abnormal plaque quality (Yamagishi M et al., 2000; Namiki N et al., 1999; Kotani J et al., 2003) such as eccentricity, lipid pool and mild calcified lesions. Therefore, next, we examined relation of s-

66 subjects with normal coronary artery diagnosed by coronary angiography were enrolled. Proxymal range of left anterior descending coronary artery was observed by intravascular ultrasound using auto-pullback methods and examined relation of s-7KCHO and coronary

Coronary angiography was performed the transfemoral approach using a standard technique. Two experienced angiographers reviewed all coronary angiograms and confirmed that there were no stenotic lesion, and consequently performed IVUS study. To avoid spasm, 1 to 2 mg of nitroglycerin was administrated before insertion of the 0.014 inch coronary guidewire and IVUS catheter through a coronary guiding catheter. After the IVUS catheter was inserted more than 20 mm beyond proximal of left descending coronary artery (LAD), a motorized auto pullback was performed at 1 mm/second velocity and the percent plaque volume was calculated for 20 mm length of LAD proximal side as plaque quantity. We also evaluated plaque quality, such as eccentricity, calcification and lipid pool into plaque. Eccentricity was identified when the minimum dimension of plaque thickness/dimension of the other side plaque thickness at max plaque area was less than 0.5. Calcification was defined when a high echo area with back reflection was detected and lipid pool was defined as when echo lucent zone was detected

There were significantly positive correlation between s-7-KCHO and %Plaque volume (r=0.37, p<0.001, Fig. 4.); furthermore, multiple regression analysis showed that high s-7- KCHO were selected as independent variables for %Plaque volume (p<0.05). On the other hand, subjects with high s-7-KCHO (≧20ng/ml, n=33) were detected high incidence of abnormal plaque quality such as eccentric plaque (76%), mild calcified lesion (64%) and lipid pool (30%) (Table 5). Fig. 5. shows the relationship of high s-7-KCHO and degree of calcification. Subjects with high s-7-KCHO had mild degree and plural number of

7KCHO and coronary plaque in angiographic normal stage using IVUS.

**4. IVUS study**

plaque.

**4.1 Study population**

**4.2 Methods of IVUS study**

during the observation period.

calcification into plaque.

**4.3 Results**

obesity, smoking, and family history of CAD were not related to s-7KCHO (data not shown). We also examined relationship between statin use and s-7KCHO. Subjects with statin use showed higher s-7KCHO than those without statin use, although the difference was not significant (p=0.09) (without statin use: 28.9 ± 19.9 ng/ml, statin use: 35.4 ± 25.7 ng/ml).

Multivariate analysis revealed that s-7KCHO were selected as an independent variable for CAD (p < 0.01, Table 3). In CAD subjects, the presence of acute myocardial infarction, number of affected vessels, and high sensitive C-reactive protein concentrations strongly correlated with s-7KCHO (p < 0.01, < 0.05, < 0.05, respectively, Table 4).

OR: odds ratio, CI: confidence interval, (n=182)

Table 3. Results of multivariate logistic regression analysis for coronary artery disease.

R2=0.27, F value=3.8, p<0.0001, (n=139)

Table 4. Results of multiple regression analysis for 7-ketocholesterol concentrations in subjects with coronary artery disease.

Our angiographic study indicated that high s-7KCHO were closely associated with acute myocardial infarction. On the other hand, recent clinical studies using IVUS have clarified that coronary artery plaque already exists in subjects with normal coronary artery, which is diagnosed by coronary angiography. Furthermore, it has been reported that almost 70% of culprit lesion on acute myocardial infarction, now called acute coronary syndrome, which is highly correlated with mortality, occurs in less than 50% of angiographic stenosis (Falk E et al., 1995). Therefore, it is difficult to predict the occurrence of acute coronary syndrome and it is important that we consider the presence of coronary atherosclerosis; especially presence of vulnerable plaque, before angiographic stenosis. IVUS studies have clarified the future of plaque on acute coronary syndrome; plaque volume is large and shows abnormal plaque quality (Yamagishi M et al., 2000; Namiki N et al., 1999; Kotani J et al., 2003) such as eccentricity, lipid pool and mild calcified lesions. Therefore, next, we examined relation of s-7KCHO and coronary plaque in angiographic normal stage using IVUS.

## **4. IVUS study**

116 Coronary Artery Diseases

obesity, smoking, and family history of CAD were not related to s-7KCHO (data not shown). We also examined relationship between statin use and s-7KCHO. Subjects with statin use showed higher s-7KCHO than those without statin use, although the difference was not significant (p=0.09) (without statin use: 28.9 ± 19.9 ng/ml, statin use: 35.4 ± 25.7 ng/ml).

Multivariate analysis revealed that s-7KCHO were selected as an independent variable for CAD (p < 0.01, Table 3). In CAD subjects, the presence of acute myocardial infarction, number of affected vessels, and high sensitive C-reactive protein concentrations strongly

Table 3. Results of multivariate logistic regression analysis for coronary artery disease.

Table 4. Results of multiple regression analysis for 7-ketocholesterol concentrations in

Our angiographic study indicated that high s-7KCHO were closely associated with acute myocardial infarction. On the other hand, recent clinical studies using IVUS have clarified that coronary artery plaque already exists in subjects with normal coronary artery, which is

correlated with s-7KCHO (p < 0.01, < 0.05, < 0.05, respectively, Table 4).

OR: odds ratio, CI: confidence interval, (n=182)

R2=0.27, F value=3.8, p<0.0001, (n=139)

subjects with coronary artery disease.

## **4.1 Study population**

66 subjects with normal coronary artery diagnosed by coronary angiography were enrolled. Proxymal range of left anterior descending coronary artery was observed by intravascular ultrasound using auto-pullback methods and examined relation of s-7KCHO and coronary plaque.

## **4.2 Methods of IVUS study**

Coronary angiography was performed the transfemoral approach using a standard technique. Two experienced angiographers reviewed all coronary angiograms and confirmed that there were no stenotic lesion, and consequently performed IVUS study. To avoid spasm, 1 to 2 mg of nitroglycerin was administrated before insertion of the 0.014 inch coronary guidewire and IVUS catheter through a coronary guiding catheter. After the IVUS catheter was inserted more than 20 mm beyond proximal of left descending coronary artery (LAD), a motorized auto pullback was performed at 1 mm/second velocity and the percent plaque volume was calculated for 20 mm length of LAD proximal side as plaque quantity. We also evaluated plaque quality, such as eccentricity, calcification and lipid pool into plaque. Eccentricity was identified when the minimum dimension of plaque thickness/dimension of the other side plaque thickness at max plaque area was less than 0.5. Calcification was defined when a high echo area with back reflection was detected and lipid pool was defined as when echo lucent zone was detected during the observation period.

### **4.3 Results**

There were significantly positive correlation between s-7-KCHO and %Plaque volume (r=0.37, p<0.001, Fig. 4.); furthermore, multiple regression analysis showed that high s-7- KCHO were selected as independent variables for %Plaque volume (p<0.05). On the other hand, subjects with high s-7-KCHO (≧20ng/ml, n=33) were detected high incidence of abnormal plaque quality such as eccentric plaque (76%), mild calcified lesion (64%) and lipid pool (30%) (Table 5). Fig. 5. shows the relationship of high s-7-KCHO and degree of calcification. Subjects with high s-7-KCHO had mild degree and plural number of calcification into plaque.

Relatioship Between Serum 7-Ketocholesterol Concentrations and Coronary Artery Disease 119

Fig. 5. Feature of calcification in subjects with high 7-ketocholesterol concentrations.

vulnerable coronary plaque which is not detectable by coronary angiography.

In angiographic study, s-7KCHO were significantly higher in subjects with CAD than in those with NCA; furthermore, multiple regression analysis revealed that s-7KCHO were selected as independent variable for the presence of CAD as a subordinate factor. The presence of AMI, number of affected vessels, and hs-CRP concentrations strongly correlated with s-7KCHO in CAD subjects. Furthermore, IVUS study indicated that s-7KCHO reflected

7KCHO is excessive in advanced atherosclerotic plaques (Brown AJ & Jessup W., 1998), and contributes to the development of atherosclerosis (Smith LL et al., 1996). Furthermore, 7KCHO causes apoptosis (Miyashita Y et al., 1997) and inhibits migration of smooth muscle cells (Oyama T et a.l, 2001). These findings suggest that accumulation of 7KCHO in atherosclerotic lesions may decrease the amount of cells and render atherosclerotic plaques unstable. Thus, 7KCHO is important not only in the progression of coronary atherosclerosis but is also the cause of plaque rupture which is a major factor of AMI. In the present study, s-7KCHO reflected the severity of coronary atherosclerosis, estimated by coronary angiography; furthermore, s-7KCHO strongly correlated with presence of AMI and vulnerable coronary plaque. Therefore, s-7KCHO may reflect 7KCHO in the coronary artery plaque and is expected to be a predictor of AMI occurrence which is strongly associated

Hypercholesterolemia is established as one of the most important coronary risk factors; however, the average serum TC or LDL-C concentration in subjects with CAD often appears to be within the normal range. The present study also indicated that serum TC or LDL-C concentrations were not higher in CAD subjects than in NCA subjects. Furthermore, there is no relationship between serum TC or LDL-C concentrations and s-7KCHO. Another study also indicated that blood 7KCHO had no relationship with blood cholesterol concentrations in CAD subjects (Tomasik A et al., 2000). Therefore, these results suggest that s-7KCHO is a different marker from blood cholesterol concentrations even though 7KCHO were oxidation

**5. Discussion**

with mortality.

Fig. 4. Correlation between 7-keto cholesterol concentration and %Plaque volume.

Table 5. 7-ketocholesterol concentrations and abnormality of plaque quality.

Fig. 5. Feature of calcification in subjects with high 7-ketocholesterol concentrations.

## **5. Discussion**

118 Coronary Artery Diseases

Fig. 4. Correlation between 7-keto cholesterol concentration and %Plaque volume.

Table 5. 7-ketocholesterol concentrations and abnormality of plaque quality.

In angiographic study, s-7KCHO were significantly higher in subjects with CAD than in those with NCA; furthermore, multiple regression analysis revealed that s-7KCHO were selected as independent variable for the presence of CAD as a subordinate factor. The presence of AMI, number of affected vessels, and hs-CRP concentrations strongly correlated with s-7KCHO in CAD subjects. Furthermore, IVUS study indicated that s-7KCHO reflected vulnerable coronary plaque which is not detectable by coronary angiography.

7KCHO is excessive in advanced atherosclerotic plaques (Brown AJ & Jessup W., 1998), and contributes to the development of atherosclerosis (Smith LL et al., 1996). Furthermore, 7KCHO causes apoptosis (Miyashita Y et al., 1997) and inhibits migration of smooth muscle cells (Oyama T et a.l, 2001). These findings suggest that accumulation of 7KCHO in atherosclerotic lesions may decrease the amount of cells and render atherosclerotic plaques unstable. Thus, 7KCHO is important not only in the progression of coronary atherosclerosis but is also the cause of plaque rupture which is a major factor of AMI. In the present study, s-7KCHO reflected the severity of coronary atherosclerosis, estimated by coronary angiography; furthermore, s-7KCHO strongly correlated with presence of AMI and vulnerable coronary plaque. Therefore, s-7KCHO may reflect 7KCHO in the coronary artery plaque and is expected to be a predictor of AMI occurrence which is strongly associated with mortality.

Hypercholesterolemia is established as one of the most important coronary risk factors; however, the average serum TC or LDL-C concentration in subjects with CAD often appears to be within the normal range. The present study also indicated that serum TC or LDL-C concentrations were not higher in CAD subjects than in NCA subjects. Furthermore, there is no relationship between serum TC or LDL-C concentrations and s-7KCHO. Another study also indicated that blood 7KCHO had no relationship with blood cholesterol concentrations in CAD subjects (Tomasik A et al., 2000). Therefore, these results suggest that s-7KCHO is a different marker from blood cholesterol concentrations even though 7KCHO were oxidation

Relatioship Between Serum 7-Ketocholesterol Concentrations and Coronary Artery Disease 121

of atheromatous plaques (Moreau M et al., 1999). Thus, 7KCHO and inflammation in coronary atherosclerosis are closely associated with each other and may promote

In IVUS study, calcification was frequently detected in subjects with high s-7KCHO

Calcification into plaque is vulnerable and has been controversial. However, some clinical studies have shown that coronary calcification, which is detected by computed tomography is a powerful predictor for cardiac events (Kondos GT et al., 2003; Greenland P et al., 2004). Raggi et al (Raggi P et al., 2003) reported that coronary calcification identified by electronbeam computed tomography was highly prevalent in patients with acute myocardial infarction; thus, coronary calcification might be indicated in the presence of vulnerable plaque. Furthermore, a recent IVUS study (Ehara S et al., 2004) reported that culprit lesion on acute coronary syndrome had significantly high incidence of mild calcification than those with stable angina pectoris. Therefore, our results can be interpreted as follows: from the viewpoint of calcification, subjects with high s-7KCHO already have vulnerable plaque in

Our data which is accurately measured s-7KCHO indicate that high s-7KCHO are closely associated with not only progression of coronary atherosclerosis but also formation of vulnerable plaque. To achieve the clinical usefulness, simple and reliable methods to measure blood 7KCHO concentrations are needed and examination of many clinical studies

Azumi H, Inoue N, Ohashi Y, Terashima M, Mori T, Fujita H, Awano K, Kobayashi K,

Austin MA & Krauss RM (1986). Genetic control of low density lipoprotein subclasses.

de Graaf J, Hak-Lemmers HL, Hectors MP, Demacker PN, Hendriks JC, & Stalenhoef

Brown AJ & Jessup W (1998). Oxysterols and atherosclerosis. *Atherosclerosis,* 142: 1–28 Castelli WP, Garrison RJ, Wilson PW, Abbott RD, Kalousdian S, & Kannel WB (1986).

Framingham Study. *J Am Med Assoc,* 256: 2835–2838

Maeda K, Hata K, Shinke T, Kobayashi S, Hirata K, Kawashima S, Itabe H, Hayashi Y, Imajoh-Ohmi S, Itoh H, &Yokoyama M (2002). Superoxide generation in directional coronary atherectomy specimens of patients with angina pectoris: important role of NAD(P)H oxidase. *Arterioscler Thromb Vasc Biol,* 22:1838-

Incidence of coronary heart disease and lipoprotein cholesterol levels. The

AF (1991). Enhanced susceptibility to in vitro oxidation of the dense low density lipoprotein subfraction in healthy subjects. *Arterioscler Thromb,* 11: 298-

investigating the significance of s-7KCHO in diagnosis and treatment are required.

vulnerable plaque formation.

compared with low s-7KCHO.

early stage coronary atherosclerosis.

**6. Conclusion**

**7. References**

1844

306

*Lancet,* 2: 592-595

products of cholesterol. This allows us to predict coronary events which are not detectable by serum TC or LDL-C concentrations if we can measure s-7KCHO in the clinic. In a basic study, degenerative LDL such as small sized LDL or glycated LDL was shown to be easily oxidized (de Graaf J et al., 1991; Lyons TJ., 1992), consequently, promoting atherosclerotic lesion. Some clinical studies emphasized an increase in degenerative LDL in CAD (Austin MA & Krauss RM., 1984; Krauss RM., 1994). 7KCHO may relate to oxidation products of degenerative LDL, however, the relationship is not fully understood. Therefore, further studies are required to investigate the significance of s-7KCHO from the perspective of precise lipid profiles.

Among the serum lipid concentrations, only HDL-C had significant relationship with s-7KCHO. A number of studies reported that low HDL-C concentrations were inversely related to the risk of cardiovascular disease (Castelli WP et al., 1986; Rhoads GG et al., 1976) and the present study may indicate that HDL retards the progression of coronary atherosclerosis by decreasing 7KCHO in the coronary vessel wall or blood flow. The relationship between HDL-C concentrations and s-7KCHO in coronary atherosclerosis is not fully understood. Recently, however, Terasaka reported that HDL exerted a protective effect against apoptosis induced by 7KCHO using ATP-binding cassette transporter ABCG1+/+ mice (Terasaka N et al., 2007). The ATP-binding cassette transporter ABCG1 was recently shown to promote efflux of cholesterol from macrophage to HDL and reverse the relationship between HDL-C concentrations and s-7KCHO. This may be partly explained by the role of ATP-binding cassette transporter ABCG1.

In the present study, there was a significant correlation between s-7KCHO and hs-CRP concentrations in CAD subjects. Furthermore, these two factors had a significant association with each other after adjustment of related factors. Thus, this result suggests that s-7KCHO are closely associated with inflammation in coronary atherosclerosis. Recent clinical investigations showed that inflammation plays an important role in the progression of atherosclerosis (Ross R., 1999; Libby P et al., 2002) and epidemiological studies clarified the link between high levels of CRP concentration and cardiovascular events (Ridker PM et al., 2002; Matsumoto K et al., 2003). Paul et al. showed that CRP is present in the human arterial intima of the atherosclerotic lesion by histological examination (Paul A et al., 2004); furthermore, Kobayashi et al. reported that, by using a sample of directional coronary atherectomy, expression of CRP was colocalized with p22phox and CRP directly induced p22phox expression generating reactive oxygen species in cultured smooth muscle cells (Kobayashi S et al., 2003). Therefore, CRP may promote the production of 7-KCHO in coronary artery plaques; consequently, increasing s-7KCHO. However, some reports indicate that oxysterol influences proinflammatory properties (Wang N et al., 1996; Liu Y et al., 1997). Joffre et al. reported that 7KCHO enhanced interleukin-8 gene expression using porcine retinal pigment epithelial cells (Joffre C et al., 2007). Interleukin-8 is a proinflammatory and chemotactic cytokine which might play a crucial role in the recruitment of monocytes and T lymphocytes into the arterial subendothelial space, consequently, promoting atherosclerotic lesion (Terkeltaub R et al., 1998). Furthermore, interleukin-8 could have a potential atherogenic role by inhibiting the local tissue inhibitor of metalloproteinase-1 expression, thereby leading to an imbalance between matrix metalloproteinases and metalloproteinase-1 at focal sites of atherosclerotic plaque, and to local extracellular degradation, causing the rupture of atheromatous plaques (Moreau M et al., 1999). Thus, 7KCHO and inflammation in coronary atherosclerosis are closely associated with each other and may promote vulnerable plaque formation.

In IVUS study, calcification was frequently detected in subjects with high s-7KCHO compared with low s-7KCHO.

Calcification into plaque is vulnerable and has been controversial. However, some clinical studies have shown that coronary calcification, which is detected by computed tomography is a powerful predictor for cardiac events (Kondos GT et al., 2003; Greenland P et al., 2004). Raggi et al (Raggi P et al., 2003) reported that coronary calcification identified by electronbeam computed tomography was highly prevalent in patients with acute myocardial infarction; thus, coronary calcification might be indicated in the presence of vulnerable plaque. Furthermore, a recent IVUS study (Ehara S et al., 2004) reported that culprit lesion on acute coronary syndrome had significantly high incidence of mild calcification than those with stable angina pectoris. Therefore, our results can be interpreted as follows: from the viewpoint of calcification, subjects with high s-7KCHO already have vulnerable plaque in early stage coronary atherosclerosis.

## **6. Conclusion**

120 Coronary Artery Diseases

products of cholesterol. This allows us to predict coronary events which are not detectable by serum TC or LDL-C concentrations if we can measure s-7KCHO in the clinic. In a basic study, degenerative LDL such as small sized LDL or glycated LDL was shown to be easily oxidized (de Graaf J et al., 1991; Lyons TJ., 1992), consequently, promoting atherosclerotic lesion. Some clinical studies emphasized an increase in degenerative LDL in CAD (Austin MA & Krauss RM., 1984; Krauss RM., 1994). 7KCHO may relate to oxidation products of degenerative LDL, however, the relationship is not fully understood. Therefore, further studies are required to investigate the significance of s-7KCHO from the perspective of

Among the serum lipid concentrations, only HDL-C had significant relationship with s-7KCHO. A number of studies reported that low HDL-C concentrations were inversely related to the risk of cardiovascular disease (Castelli WP et al., 1986; Rhoads GG et al., 1976) and the present study may indicate that HDL retards the progression of coronary atherosclerosis by decreasing 7KCHO in the coronary vessel wall or blood flow. The relationship between HDL-C concentrations and s-7KCHO in coronary atherosclerosis is not fully understood. Recently, however, Terasaka reported that HDL exerted a protective effect against apoptosis induced by 7KCHO using ATP-binding cassette transporter ABCG1+/+ mice (Terasaka N et al., 2007). The ATP-binding cassette transporter ABCG1 was recently shown to promote efflux of cholesterol from macrophage to HDL and reverse the relationship between HDL-C concentrations and s-7KCHO. This may be partly explained by

In the present study, there was a significant correlation between s-7KCHO and hs-CRP concentrations in CAD subjects. Furthermore, these two factors had a significant association with each other after adjustment of related factors. Thus, this result suggests that s-7KCHO are closely associated with inflammation in coronary atherosclerosis. Recent clinical investigations showed that inflammation plays an important role in the progression of atherosclerosis (Ross R., 1999; Libby P et al., 2002) and epidemiological studies clarified the link between high levels of CRP concentration and cardiovascular events (Ridker PM et al., 2002; Matsumoto K et al., 2003). Paul et al. showed that CRP is present in the human arterial intima of the atherosclerotic lesion by histological examination (Paul A et al., 2004); furthermore, Kobayashi et al. reported that, by using a sample of directional coronary atherectomy, expression of CRP was colocalized with p22phox and CRP directly induced p22phox expression generating reactive oxygen species in cultured smooth muscle cells (Kobayashi S et al., 2003). Therefore, CRP may promote the production of 7-KCHO in coronary artery plaques; consequently, increasing s-7KCHO. However, some reports indicate that oxysterol influences proinflammatory properties (Wang N et al., 1996; Liu Y et al., 1997). Joffre et al. reported that 7KCHO enhanced interleukin-8 gene expression using porcine retinal pigment epithelial cells (Joffre C et al., 2007). Interleukin-8 is a proinflammatory and chemotactic cytokine which might play a crucial role in the recruitment of monocytes and T lymphocytes into the arterial subendothelial space, consequently, promoting atherosclerotic lesion (Terkeltaub R et al., 1998). Furthermore, interleukin-8 could have a potential atherogenic role by inhibiting the local tissue inhibitor of metalloproteinase-1 expression, thereby leading to an imbalance between matrix metalloproteinases and metalloproteinase-1 at focal sites of atherosclerotic plaque, and to local extracellular degradation, causing the rupture

precise lipid profiles.

the role of ATP-binding cassette transporter ABCG1.

Our data which is accurately measured s-7KCHO indicate that high s-7KCHO are closely associated with not only progression of coronary atherosclerosis but also formation of vulnerable plaque. To achieve the clinical usefulness, simple and reliable methods to measure blood 7KCHO concentrations are needed and examination of many clinical studies investigating the significance of s-7KCHO in diagnosis and treatment are required.

## **7. References**

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Kunsch C & Medford RM (1999). Oxidative stress as a regulator of gene expression in the

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Krauss RM (1994). Heterogeneity of plasma low-density lipoproteins and atherosclerosis

Kondos GT, Hoff JA, Sevrukov A, Daviglus ML, Garside DB, Devries SS, Chomka EV,& Liu

Bui AB, Pichard AD, Satler LF, Suddath WO, Waksman R, Laird JR Jr, Kent KM, &

Libby P, Ridker PM, & Maseri A (2002). Inflammation and atherosclerosis. Circulation,

Lyons TJ (1992). Lipoprotein glycation and its metabolic consequences. *Diabetes*, 41: 67-73 Liu Y, Hulten LM, & Wiklund O (1997). Macrophages isolated from human atherosclerotic

production. *Arterioscler Thromb Vasc Biol,* 17: 317–323

asymptomatic individuals. *JAMA,* 291: 210-215

vasculature. *Circ Res,* 85: 753-766

risk. *Curr Opin Lipidol,* 5: 339-349

*Circulation,* 107: 2571-2576

105:1135-1143

*Arterioscler Thromb Vasc Biol*, 23: 1398-1404

Kotani J, Mintz GS, Castagna MT, Pinnow E, Berzingi CO,

infarction. *Circulation,* 107: 2889-2893

1960

3424-3429

280

Takano T, Tsukamoto Y, Yoshiyama M, Takeuchi K, Yoshikawa J, & Becker AE (2001). Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. *Circulation,* 103: 1955-

Yamashita H, Yamagishi H, Takeuchi K, Naruko T, Haze K, Becker AE, Yoshikawa J, & Ueda M (1995). Spotty calcification typifies the culprit plaque in patients with acute myocardial infarction: an intravascular ultrasound study. *Circulation,* 110:

calcium score combined with Framingham score for risk prediction in

Creuzot-Garcher C,& Bretillon L (2007). Oxysterols induced inflammation and oxidation in primary porcine retinal pigment epithelial cells. *Curr Eye Res,* 32: 271-

Kobayashi K, Azumi H, Ejiri J, Hirata K, Kawashima S, Hayashi Y, Yokozaki H, Itoh H,& Yokoyama M (2003). Interaction of oxidative stress and inflammatory response in coronary plaque instability: important role of C-reactive protein.

K (2003) Electron-beam tomography coronary artery calcium and cardiac events: a 37-month follow-up of 5635 initially asymptomatic low- to intermediate-risk adults.

Weissman NJ (2003). Intravascular ultrasound analysis of infarct-related and noninfarct-related arteries in patients who presented with an acute myocardial

plaques produce IL-8, and oxysterols may have a regulatory function for IL-8

**8**

*Greece*

**Oxidized Low Density Lipoprotein,**

Elias Skopelitis, Dimitrios Levisianou,

*General Hospital of Nikaia and Piraeus*

Theodore Gialernios and Sofoklis Kougialis

**Statin Therapy and Carotid Stenosis**

Carotid artery stenosis due to atherosclerosis is a major complication of hyperlipidemia, diabetes mellitus and hypertension. Moreover, the extent of carotid intima media thickness

The effect of cholesterol in the process of atheromatosis is now well established. High levels of total cholesterol (TC), as well as of low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), lipoprotein a (Lp-α), and triglycerides (TG), coupled with decreased levels of high-density lipoprotein (HDL) are responsible for the creation of atheromatous plaques (Assmann & Schulte, 1992; Hokanson & Autsin, 1996; Katsouras et al., 2001). Of the above factors, LDL cholesterol, and especially the oxidized LDL (oxLDL) is considered as the most important contributor of atheromatosis

1. In the first stage, LDL cholesterol enters the vessel wall, binds to glucosaminoglucanes, which are part of the extracellular matrix of the intima. This binding is facilitated by apolipoprotein B-100 (ApoΒ–100). The accumulation of LDL in the vessel wall contributes to the formation of fatty strikes. Following adhesion to the vessel wall, LDL undergoes oxidation by free radicals produced locally, the molecule is altered and chemokines are produced by adjacent vessel wall cells, such as monocyte chemoattractant protein-1 (MCP–1), together with growth factors, which are responsible for the accumulation of monocytes and macrophages. The latter cause further oxidation of LDL, resulting in negative charge, recognition by scavenger receptors located on macrophage membrane and increased uptake of LDL inside the macrophages, as these receptors are not inhibited by increased intracellular concentration of cholesterol. The final result is an enormous accumulation of LDL in the macrophages, which are transformed to foam cells. These cells represent the first step in

is a measure of atheromatosis and therefore of cardiovascular disease (CVD).

The atheromatous process is completed in the following three stages:

the atheromatous process (Durrington & Sinderman, 2002) (figure 1).

2. During the second stage, the atheromatous plaque is formed. Foam cells produce growth factors and together with oxLDL result to the attraction of smooth muscle cells.

**1. Introduction**

(Anderson et al., 1996).

**1.1 Carotid stenosis and atheromatous process**

## **Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis**

Elias Skopelitis, Dimitrios Levisianou, Theodore Gialernios and Sofoklis Kougialis *General Hospital of Nikaia and Piraeus Greece*

## **1. Introduction**

124 Coronary Artery Diseases

Wang N, Tabas I, Winchester R, Ravalli S, Rabbani LE,& Tall A (1996). Interleukin 8 is

Yamagishi M, Terashima M, Awano K, Kijima M, Nakatani S, Daikoku S, Ito K, Yasumura

foam cells in human atheroma. *J Biol Chem,* 271: 8837-8842

coronary syndrome. *J Am Coll Cardiol,* 35: 106-111

induced by cholesterol loading of macrophages and expressed by macrophage

Y, & Miyatake K (2000). Morphology of vulnerable coronary plaque: insights from follow-up of patients examined by intravascular ultrasound before an acute

#### **1.1 Carotid stenosis and atheromatous process**

Carotid artery stenosis due to atherosclerosis is a major complication of hyperlipidemia, diabetes mellitus and hypertension. Moreover, the extent of carotid intima media thickness is a measure of atheromatosis and therefore of cardiovascular disease (CVD).

The effect of cholesterol in the process of atheromatosis is now well established. High levels of total cholesterol (TC), as well as of low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), lipoprotein a (Lp-α), and triglycerides (TG), coupled with decreased levels of high-density lipoprotein (HDL) are responsible for the creation of atheromatous plaques (Assmann & Schulte, 1992; Hokanson & Autsin, 1996; Katsouras et al., 2001). Of the above factors, LDL cholesterol, and especially the oxidized LDL (oxLDL) is considered as the most important contributor of atheromatosis (Anderson et al., 1996).

The atheromatous process is completed in the following three stages:

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 127

Avoiding the formation and the instability of the atheromatous plaque is top priority for patients at risk for cardiovascular events. Statins may contribute towards this direction

Fig. 2. Advanced atheromatous plaque causing arterial lumen occlusion of 70% (adopted

Cholesterol reservoir

Foamy cells Fibrous capsid Arterial lumen

Arterial wall

Fig. 3. The point of the atheromatous plaque, on which active enlargement occurs: formation of new foam cells and increased cholesterol uptake contribute to increased plaque instability

(Corti et al., 2002; Nissen et al., 2004)

Cholesterol reservoir

from Durrington & Sinderman, 2002).

Arterial lumen

Fibrous capsid

(adopted from Durrington & Sinderman, 2002).

The latter are then differentiated to fibroblasts and start producing collagen. This collagen covers foam cells, which either are destroyed or are forced to apoptosis. The final result is the formation of a pool of extracellular cholesterol trapped under a fibrous capsid (figure 2). The part which is close to the yet intact vessel wall is the active site of the plaque, where the foam cells are produced. As the plaque extents to the inner layers of the vessel wall, the point of foam cell formation becomes instable and may cause rapture of the plaque (Durrington & Sinderman, 2002) (figure 3).

3. In the third stage, that of the complicated lesion, the rapture of the fibrous capsid of the atheromatous plaque leads to massive evacuation of the cholesterol reservoir. The artery may occult due to the accumulation of platelets and subsequent clotting, leading to acute ischemia or infarction (figure 4). If not so, then the plaque will be further enlarged (Durrington & Sinderman, 2002).

Fig. 1. Atherogenesis. Fatty strikes are characterized by macrophages containing an excess of lipids (foam cells). Foam cells are derived by blood monocytes which are attracted to vessel intima and start phagocytosing lipoproteins, such as oxLDL. The conversion of fatty strike to atheroma depends on proliferation and differentiation of smooth muscle cells to fibroblasts. The latter produce collagen resulting in intima thickening. As the lesion extents further, foam cells are destroyed releasing large amounts of cholesterol trapped in a fibrous capsid. The active site of atheroma is the point which is adjacent to normal endothelium, where foam cells are formed (adopted with persmission from Durrington & Sinderman, 2002).

3. In the third stage, that of the complicated lesion, the rapture of the fibrous capsid of the atheromatous plaque leads to massive evacuation of the cholesterol reservoir. The artery may occult due to the accumulation of platelets and subsequent clotting, leading to acute ischemia or infarction (figure 4). If not so, then the plaque will be further

Fig. 1. Atherogenesis. Fatty strikes are characterized by macrophages containing an excess of lipids (foam cells). Foam cells are derived by blood monocytes which are attracted to vessel intima and start phagocytosing lipoproteins, such as oxLDL. The conversion of fatty strike

growth factors

scavenger receptors

Fc receptors

Smooth muscle cell proliferation

Decreased

chemotactic agents

macrophage mobility

fibroblasts. The latter produce collagen resulting in intima thickening. As the lesion extents further, foam cells are destroyed releasing large amounts of cholesterol trapped in a fibrous capsid. The active site of atheroma is the point which is adjacent to normal endothelium, where foam cells are formed (adopted with persmission from Durrington & Sinderman,

to atheroma depends on proliferation and differentiation of smooth muscle cells to

Collagen

2002).

rapture of the plaque (Durrington & Sinderman, 2002) (figure 3).

enlarged (Durrington & Sinderman, 2002).

Circulating LDL

cytotoxicity oxidation

Oxidized LDL

autoantibodies

The latter are then differentiated to fibroblasts and start producing collagen. This collagen covers foam cells, which either are destroyed or are forced to apoptosis. The final result is the formation of a pool of extracellular cholesterol trapped under a fibrous capsid (figure 2). The part which is close to the yet intact vessel wall is the active site of the plaque, where the foam cells are produced. As the plaque extents to the inner layers of the vessel wall, the point of foam cell formation becomes instable and may cause Avoiding the formation and the instability of the atheromatous plaque is top priority for patients at risk for cardiovascular events. Statins may contribute towards this direction (Corti et al., 2002; Nissen et al., 2004)

Fig. 2. Advanced atheromatous plaque causing arterial lumen occlusion of 70% (adopted from Durrington & Sinderman, 2002).

Fig. 3. The point of the atheromatous plaque, on which active enlargement occurs: formation of new foam cells and increased cholesterol uptake contribute to increased plaque instability (adopted from Durrington & Sinderman, 2002).

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 129

1984). Oxidized LDL then acts chemotactic for monocytes and smooth muscle cells through binding to scavenger receptors (D. Li et al., 2002), leading to the formation of foamy cells. Oxidized LDL is also capable to elicit endothelial dysfunction by altering the secretory activity of endothelial cells (D. Li et al., 2002), inhibiting the nitric oxide-mediated vasodilatation through reduction of the expression of endothelial nitric-oxide synthase (eNOS), inducing the expression of adhesion molecules on the endothelium thus mediating the adhesion of monocytes to intima (D. Li et al., 2002), and inducing the expression of inflammatory cytokines (Steinberg, 1997). Indeed, oxLDL is a potent inducer of inflammation (Steinberg et al., 1989), contributing to the chronic inflammatory process

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, reduce serum TC, LDL cholesterol, apolipoprotein B (apoB), and, to a lesser degree, TG and Lp-a levels. Statins also have pleiotropic effects (Bellosta et al., 2000), such as the modulation of inflammatory molecules and monocyte maturation and differentiation (Bellosta et al., 2000), the suppression of smooth muscle-cells migration and proliferation (Bellosta et al., 2000), the reduction of the monocyte adhesion to the endothelium (Weber et al., 1997), the restoration of the impaired endothelium-dependent vessel wall relaxation (Jarvisalo et al., 1999), and the modification of cell-mediated LDL oxidation (Giroux et al., 1993; Aviram et al., 1998). All of the above mechanisms contribute to the reversion of atheromatosis. Undeniably, statins reduce the incidence of coronary events and are a cornerstone in the primary and secondary prevention of CHD (D.Y. Li et al., 2001). Previous studies have detected some efficacy in reducing the circulating oxLDL levels, but whether this effect is due to the reduction of LDL or is an independent, pleiotropic phenomenon remains a matter of controversy (Kwak & Mach, 2001; Robinson et al., 2005). Furthermore, little is known about the definite clinical

The aim of the present study was to evaluate the efficacy of atorvastatin in reducing stenosis, to investigate the effect on oxLDL and to search for possible associations of oxLDL modification with changes of stenosis in patients managed conservatively and in pre-treated with percutaneous catheter interventional procedures patients with carotid atheromatosis. We hypothesise that atorvastatin therapy will confer remission of oxLDL levels in vivo and

Between January 2005 and February 2008 a total of 100 patients were randomly selected from the lipid clinic and the carotid angioplasty clinic of a large tertiary hospital in Athens for inclusion in the study. Informed consent was obtained from each patient at recruitment according to our institutional policies. Eligible were patients with carotid artery atheromatosis from various causes (not only dyslipidemia) and with a range of predisposing factors. Exclusion criteria included: acute cardiovascular disease, severe or unstable angina pectoris, clinically evident cardiac failure, severe arrhythmias, recent surgical procedures, inflammatory diseases, active liver disease or liver impairment, excessive alcohol consumption (>4 drinks /day) or history of alcohol abuse, known allergic reaction to statins, poorly controlled diabetes mellitus as defined by a haemoglobin A1c (HbA1c) level of >7 %,

this will be associated with significant reduction of carotid artery stenosis.

which results to atherosclerosis (Ross, 1999).

benefit of such oxidative marker reduction.

**2. Patients and methods**

**1.3 Statins**

Fig. 4. A raptured ahteromatous plaque, in which the cholesterol reservoir has evacuated itself under the fibrous capsid. A clot in the endothelial surface at the site of rapture occults the lumen completely (adopted from Durrington & Sinderman, 2002).

## **1.2 Oxidized LDL**

Oxidized LDL cholesterol in humans is found mainly in two types:

Oxidized LDL is produced following oxidation of LDL by free radicals and other oxidadive factors, a procedure called oxidative stress. The circulating oxLDL is the measurable fraction of oxLDL in plasma. Oxidized LDL is a key element of the pathway leading to the formation of the atheromatous plaque and has been extensively studied both as a marker of atheromatosis and as a possible target of therapeutic intervention. Circulating oxLDL is considered a risk marker for atherosclerosis (Toshima, 2000) and coronary heart disease (CHD) ( Ehara et al., 2001; Holvoet et al., 2003; Toshima et al., 2000). Increased oxLDL levels in circulation and the vessel wall are associated with endothelial dysfunction (Penny et al., 2001) in such patients (Ehara et al., 2001; Holvoet et al., 1999; Nishi et al., 2002), contributing to atheromatous plaque instability (Ehara et al., 2001).

Oxidative modification of LDL leads to rapid focal accumulation in macrophages (Witztum & Steinberg, 1991), which is the first step of the atheromatous process. The increased retention time of LDL in the intima offers enhanced probability to be oxidized by free radicals produced by endothelium, smooth muscle cells or macrophages (Steinbrecher et al., 1984). Oxidized LDL then acts chemotactic for monocytes and smooth muscle cells through binding to scavenger receptors (D. Li et al., 2002), leading to the formation of foamy cells. Oxidized LDL is also capable to elicit endothelial dysfunction by altering the secretory activity of endothelial cells (D. Li et al., 2002), inhibiting the nitric oxide-mediated vasodilatation through reduction of the expression of endothelial nitric-oxide synthase (eNOS), inducing the expression of adhesion molecules on the endothelium thus mediating the adhesion of monocytes to intima (D. Li et al., 2002), and inducing the expression of inflammatory cytokines (Steinberg, 1997). Indeed, oxLDL is a potent inducer of inflammation (Steinberg et al., 1989), contributing to the chronic inflammatory process which results to atherosclerosis (Ross, 1999).

## **1.3 Statins**

128 Coronary Artery Diseases

Fibrous capsid

Empty space (the former cholesterol reservoir)

Fig. 4. A raptured ahteromatous plaque, in which the cholesterol reservoir has evacuated itself under the fibrous capsid. A clot in the endothelial surface at the site of rapture occults

Oxidized LDL is produced following oxidation of LDL by free radicals and other oxidadive factors, a procedure called oxidative stress. The circulating oxLDL is the measurable fraction of oxLDL in plasma. Oxidized LDL is a key element of the pathway leading to the formation of the atheromatous plaque and has been extensively studied both as a marker of atheromatosis and as a possible target of therapeutic intervention. Circulating oxLDL is considered a risk marker for atherosclerosis (Toshima, 2000) and coronary heart disease (CHD) ( Ehara et al., 2001; Holvoet et al., 2003; Toshima et al., 2000). Increased oxLDL levels in circulation and the vessel wall are associated with endothelial dysfunction (Penny et al., 2001) in such patients (Ehara et al., 2001; Holvoet et al., 1999; Nishi et al., 2002), contributing

Oxidative modification of LDL leads to rapid focal accumulation in macrophages (Witztum & Steinberg, 1991), which is the first step of the atheromatous process. The increased retention time of LDL in the intima offers enhanced probability to be oxidized by free radicals produced by endothelium, smooth muscle cells or macrophages (Steinbrecher et al.,

the lumen completely (adopted from Durrington & Sinderman, 2002).

Oxidized LDL cholesterol in humans is found mainly in two types:

a. conjugated form, attached to the atheromatous plaque and

to atheromatous plaque instability (Ehara et al., 2001).

**1.2 Oxidized LDL**

Site of rapture

Clot occulting the arterial lumen

b. circulating form found in serum.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, reduce serum TC, LDL cholesterol, apolipoprotein B (apoB), and, to a lesser degree, TG and Lp-a levels. Statins also have pleiotropic effects (Bellosta et al., 2000), such as the modulation of inflammatory molecules and monocyte maturation and differentiation (Bellosta et al., 2000), the suppression of smooth muscle-cells migration and proliferation (Bellosta et al., 2000), the reduction of the monocyte adhesion to the endothelium (Weber et al., 1997), the restoration of the impaired endothelium-dependent vessel wall relaxation (Jarvisalo et al., 1999), and the modification of cell-mediated LDL oxidation (Giroux et al., 1993; Aviram et al., 1998). All of the above mechanisms contribute to the reversion of atheromatosis. Undeniably, statins reduce the incidence of coronary events and are a cornerstone in the primary and secondary prevention of CHD (D.Y. Li et al., 2001). Previous studies have detected some efficacy in reducing the circulating oxLDL levels, but whether this effect is due to the reduction of LDL or is an independent, pleiotropic phenomenon remains a matter of controversy (Kwak & Mach, 2001; Robinson et al., 2005). Furthermore, little is known about the definite clinical benefit of such oxidative marker reduction.

The aim of the present study was to evaluate the efficacy of atorvastatin in reducing stenosis, to investigate the effect on oxLDL and to search for possible associations of oxLDL modification with changes of stenosis in patients managed conservatively and in pre-treated with percutaneous catheter interventional procedures patients with carotid atheromatosis. We hypothesise that atorvastatin therapy will confer remission of oxLDL levels in vivo and this will be associated with significant reduction of carotid artery stenosis.

## **2. Patients and methods**

Between January 2005 and February 2008 a total of 100 patients were randomly selected from the lipid clinic and the carotid angioplasty clinic of a large tertiary hospital in Athens for inclusion in the study. Informed consent was obtained from each patient at recruitment according to our institutional policies. Eligible were patients with carotid artery atheromatosis from various causes (not only dyslipidemia) and with a range of predisposing factors. Exclusion criteria included: acute cardiovascular disease, severe or unstable angina pectoris, clinically evident cardiac failure, severe arrhythmias, recent surgical procedures, inflammatory diseases, active liver disease or liver impairment, excessive alcohol consumption (>4 drinks /day) or history of alcohol abuse, known allergic reaction to statins, poorly controlled diabetes mellitus as defined by a haemoglobin A1c (HbA1c) level of >7 %,

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 131

al., 2002). For the measurement of circulating oxLDL, a commercially available kit (Mercodia, Uppsala, Sweden), based on a double antibody (4E6 and mouse monoclonal antiapoB) (Holvoet et al., 1996) capture ELISA test, was used. This method primarily detects malondialdehyde LDL (MDA-LDL). The normative range (reference range) in our lab was 31-61 mU/l. Apolipoprotein A, B and Lp-a were measured using immuno-nephelometry

The evaluation of stenosis was conducted by Triplex ultrasonography using an Apogee 800 plus scanner with a 7.5 MHz transducer (ATL Inc., Bothell WA, USA) at baseline and 12 months. The stenosis was calculated in three sections in each common and internal carotid artery, and the final measure was the mean value of the three. The value of stenosis in the most occluded vessel was used in the statistical analysis. Specifically, the internal carotid artery (ICA) and common carotid artery (CCA) bilaterally were evaluated for each patient using coloured and grey Doppler ultrasonography. An effort was made to completely visualize the vessels. Additionally, the pulse wave was estimated with Doppler phasmatometry as well as the blood flow velocity of the two vessels. Results were recorded in a validated form. Stenosis was defined as the presence of visual plaque in coloured or grey Doppler. The degree of stenosis was calculated by measuring the decrease of the lumen diameter and the maximum systolic blood flow velocity. In difficult cases, other parameters were taken into account, such as ICA/CCA max blood flow velocity ratio and the ICA enddiastolic velocity. A degree of stenosis >70% was considered as sever and angioplasty was advised. A degree of stenosis between 60 – 70% was defined as high, between 50 – 60% as moderate and <50% as mild. High, moderate and mild stenoses were treated conservatively. The intima media thickness (IMT) and plaque morphology were not studied due to specific

Continuous variables were presented as mean values ± standard deviation, while qualitative variables were presented as absolute and relative frequencies. Normality tests were applied using the Kolmogorov-Smirnov criterion as well as Shapiro-Wilk test. Univariate analysis was initially applied to test the associations of oxLDL with carotid stenosis for each patient group as well as to identify first order correlations with various clinical parameters. Correlations between skewed continuous or discrete variables were evaluated using Spearman's p-coefficient, whereas correlations of normally distributed variables were evaluated by calculating the Pearson's r-coefficient. Comparisons between normally distributed, continuous variables and categorical variables were made using the Student ttest. Analysis of categorical data was carried out with the [chi]2 test or Fischer's exact test

The association of oxLDL with carotid stenosis was also tested through multiple Cox proportional hazard model. The results obtained were presented as Hazard Ratios (HR) and the 95% Confidence Intervals (CI). A backward elimination procedure was applied to all multivariate models (using P<5% as the threshold for removing a variable from the models). All models were adjusted for age, gender, SBP and TC. Kaplan-Meier curves concerning stenosis over the study period were plotted and Log rank test was performed. All reported P-values were based on two-sided tests and compared to a significance level of 5%. STATA

8.0 software (Stata Corporation, 2003, Texas, USA) was used for the analysis.

with rabbit antisera (Dade Behring, Newark, USA) in a Dade Behring analyser.

lab requirements, not readily available in our institution.

**2.1 Statistical analysis**

when appropriate.

uncontrolled hypertension indicated by systolic blood pressure (SBP) >140 mmHg and/or diastolic pressure >85 mmHg, history of deep vein thrombosis, bleeding tendency, serum triglycerides >350 mg/dl, evidence of thyroid dysfunction, use of systemic steroids or other anabolics, pernicious anaemia, impaired vitamin B12 or folate acid levels, abnormal serum urate at baseline, serum creatinine phosphokinase elevation of >1.5 fold at baseline, pregnancy or lactation, and end-stage renal disease or dialysis.

Patients were allocated into two groups according to the degree of carotid artery stenosis: those with arterial lumen occlusion of ≥70% in at least one common or internal carotid vessel consisted group A; those with stenosis <70% comprised group B. Patients in both groups were naive to statin therapy or if otherwise, a 6-month washout period was allowed before enrolment in the study. Group A underwent percutaneous transluminal carotid angioplasty with stenting by the same interventional cardiologist, prior to the initiation of statin therapy. Those patients were additionally administered clopidrogrel and salicylate. Both groups had to follow an American Heart Association step II diet and were encouraged to exercise.

According to the study protocol, all patients were placed on atorvastatin once daily at bedtime in individualised doses, tittered to achieve and maintain serum LDL cholesterol levels of <100 mg/dl (and ideally <70 mg/dl, if hypertension, renal impairment, smoking, hyperlipidemia, symptomatic peripheral arterial obstructive disease, or diabetes mellitus were present). Patients were prescribed statins even in the absence of hyperlipidemia, as the aim of the study was to investigate the effect of statin on oxLDL and carotid stenosis in a common atherogenic patient population. The most common doses used to achieve the above levels of LDL ranged between 10 to 40 mg, while seldom it was required to administer higher doses such as 60 mg (median atorvastatin dose for the total population = 20 mg, range 10 – 60 mg). The use of other drugs known to act synergistically with statins causing rhabdomyolysis was prohibited during the study. Adverse events were assessed in every visit in a non-specific manner: every newly reported symptom was documented as possible adverse reaction due to statin therapy and subsequently evaluated by an expert in clinical biochemistry. Adherence to the medication regimen was assessed indirectly by the low LDL levels compared with baseline.

Medical anamnesis, anthropometrics, smoking habits, blood pressure, and laboratory investigations comprising of complete blood count, fasting glucose, HbA1c, liver and kidney biochemistry, detailed lipid profile (TC, LDL cholesterol, HDL cholesterol, serum TG, apoB, and apolipoprotein A), urate, B12 and folate, thyroid function tests, homocysteine, Lp-a, and oxLDL were obtained at baseline and during follow-up visits, which were arranged at baseline, one, three, and six months; the final assessment was carried out in 12 months. Blood samples were collected after an at least 12-hour fast and a light, low-fat meal the night before sample collection was advised. Venous blood samples were collected in standard biochemistry vacutainer tubes. For the analysis of homocysteine and whole blood count, ethylenediaminetetraacetic acid (EDTA) vacutainer was used. Serum for biochemistry analysis was obtained by centrifugation (4000 g) at 4°C for 7 min and was immediately tested.

Lipid profiles (TC, HDL, TG) were determined using commercially available enzymatic colourimetric methods (Dade Behring, Newark, USA) with a Dade Behring analyser. LDL was calculated with the use of Friedewald's formula as all had TG <350 mg/dl (Puccetti et al., 2002). For the measurement of circulating oxLDL, a commercially available kit (Mercodia, Uppsala, Sweden), based on a double antibody (4E6 and mouse monoclonal antiapoB) (Holvoet et al., 1996) capture ELISA test, was used. This method primarily detects malondialdehyde LDL (MDA-LDL). The normative range (reference range) in our lab was 31-61 mU/l. Apolipoprotein A, B and Lp-a were measured using immuno-nephelometry with rabbit antisera (Dade Behring, Newark, USA) in a Dade Behring analyser.

The evaluation of stenosis was conducted by Triplex ultrasonography using an Apogee 800 plus scanner with a 7.5 MHz transducer (ATL Inc., Bothell WA, USA) at baseline and 12 months. The stenosis was calculated in three sections in each common and internal carotid artery, and the final measure was the mean value of the three. The value of stenosis in the most occluded vessel was used in the statistical analysis. Specifically, the internal carotid artery (ICA) and common carotid artery (CCA) bilaterally were evaluated for each patient using coloured and grey Doppler ultrasonography. An effort was made to completely visualize the vessels. Additionally, the pulse wave was estimated with Doppler phasmatometry as well as the blood flow velocity of the two vessels. Results were recorded in a validated form. Stenosis was defined as the presence of visual plaque in coloured or grey Doppler. The degree of stenosis was calculated by measuring the decrease of the lumen diameter and the maximum systolic blood flow velocity. In difficult cases, other parameters were taken into account, such as ICA/CCA max blood flow velocity ratio and the ICA enddiastolic velocity. A degree of stenosis >70% was considered as sever and angioplasty was advised. A degree of stenosis between 60 – 70% was defined as high, between 50 – 60% as moderate and <50% as mild. High, moderate and mild stenoses were treated conservatively. The intima media thickness (IMT) and plaque morphology were not studied due to specific lab requirements, not readily available in our institution.

### **2.1 Statistical analysis**

130 Coronary Artery Diseases

uncontrolled hypertension indicated by systolic blood pressure (SBP) >140 mmHg and/or diastolic pressure >85 mmHg, history of deep vein thrombosis, bleeding tendency, serum triglycerides >350 mg/dl, evidence of thyroid dysfunction, use of systemic steroids or other anabolics, pernicious anaemia, impaired vitamin B12 or folate acid levels, abnormal serum urate at baseline, serum creatinine phosphokinase elevation of >1.5 fold at baseline,

Patients were allocated into two groups according to the degree of carotid artery stenosis: those with arterial lumen occlusion of ≥70% in at least one common or internal carotid vessel consisted group A; those with stenosis <70% comprised group B. Patients in both groups were naive to statin therapy or if otherwise, a 6-month washout period was allowed before enrolment in the study. Group A underwent percutaneous transluminal carotid angioplasty with stenting by the same interventional cardiologist, prior to the initiation of statin therapy. Those patients were additionally administered clopidrogrel and salicylate. Both groups had to follow an American Heart Association step II diet and were encouraged

According to the study protocol, all patients were placed on atorvastatin once daily at bedtime in individualised doses, tittered to achieve and maintain serum LDL cholesterol levels of <100 mg/dl (and ideally <70 mg/dl, if hypertension, renal impairment, smoking, hyperlipidemia, symptomatic peripheral arterial obstructive disease, or diabetes mellitus were present). Patients were prescribed statins even in the absence of hyperlipidemia, as the aim of the study was to investigate the effect of statin on oxLDL and carotid stenosis in a common atherogenic patient population. The most common doses used to achieve the above levels of LDL ranged between 10 to 40 mg, while seldom it was required to administer higher doses such as 60 mg (median atorvastatin dose for the total population = 20 mg, range 10 – 60 mg). The use of other drugs known to act synergistically with statins causing rhabdomyolysis was prohibited during the study. Adverse events were assessed in every visit in a non-specific manner: every newly reported symptom was documented as possible adverse reaction due to statin therapy and subsequently evaluated by an expert in clinical biochemistry. Adherence to the medication regimen was assessed indirectly by the low LDL

Medical anamnesis, anthropometrics, smoking habits, blood pressure, and laboratory investigations comprising of complete blood count, fasting glucose, HbA1c, liver and kidney biochemistry, detailed lipid profile (TC, LDL cholesterol, HDL cholesterol, serum TG, apoB, and apolipoprotein A), urate, B12 and folate, thyroid function tests, homocysteine, Lp-a, and oxLDL were obtained at baseline and during follow-up visits, which were arranged at baseline, one, three, and six months; the final assessment was carried out in 12 months. Blood samples were collected after an at least 12-hour fast and a light, low-fat meal the night before sample collection was advised. Venous blood samples were collected in standard biochemistry vacutainer tubes. For the analysis of homocysteine and whole blood count, ethylenediaminetetraacetic acid (EDTA) vacutainer was used. Serum for biochemistry analysis was obtained by centrifugation (4000 g) at 4°C for 7 min and was immediately

Lipid profiles (TC, HDL, TG) were determined using commercially available enzymatic colourimetric methods (Dade Behring, Newark, USA) with a Dade Behring analyser. LDL was calculated with the use of Friedewald's formula as all had TG <350 mg/dl (Puccetti et

pregnancy or lactation, and end-stage renal disease or dialysis.

to exercise.

tested.

levels compared with baseline.

Continuous variables were presented as mean values ± standard deviation, while qualitative variables were presented as absolute and relative frequencies. Normality tests were applied using the Kolmogorov-Smirnov criterion as well as Shapiro-Wilk test. Univariate analysis was initially applied to test the associations of oxLDL with carotid stenosis for each patient group as well as to identify first order correlations with various clinical parameters. Correlations between skewed continuous or discrete variables were evaluated using Spearman's p-coefficient, whereas correlations of normally distributed variables were evaluated by calculating the Pearson's r-coefficient. Comparisons between normally distributed, continuous variables and categorical variables were made using the Student ttest. Analysis of categorical data was carried out with the [chi]2 test or Fischer's exact test when appropriate.

The association of oxLDL with carotid stenosis was also tested through multiple Cox proportional hazard model. The results obtained were presented as Hazard Ratios (HR) and the 95% Confidence Intervals (CI). A backward elimination procedure was applied to all multivariate models (using P<5% as the threshold for removing a variable from the models). All models were adjusted for age, gender, SBP and TC. Kaplan-Meier curves concerning stenosis over the study period were plotted and Log rank test was performed. All reported P-values were based on two-sided tests and compared to a significance level of 5%. STATA 8.0 software (Stata Corporation, 2003, Texas, USA) was used for the analysis.

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 133

A significant correlation between LDL and oxLDL levels was detected (Pearson's correlation coefficient r=0.7, p<0.01) (figure 6). Similar correlation was found between oxLDL and apoB

Between smokers mean oxLDL was reduced from 60.68±24.09 at baseline to 45.84±24.89 mU/l at the end of study period (difference 14.84 mU/l, p = 0.0036). Similarly, between non-smokers it was reduced from 69.33±25.11 to 40.36±5.6 mU/l (difference 28.97 mU/l, p<0.001). Nonsmokers had approximately double decline of oxLDL levels compared to smokers. Carotid artery stenosis was reduced between smokers from 29.68±25.59% at baseline to 23.06±21.71% at 12 months (p = 0.002). Non-smokers also presented significant reduction of stenosis during the study period (24.67 ± 26.22% vs 20 ± 21.45%, p = 0.004). Non-smokers and smokers had

Investigations Total Group A Group B

232.23 ± 47.8 153.36±17.2 <0.0001

151.5±41.7 79.75±12.7 <0.0001

145.59 ± 73.1 111±53.1 0.0002

64.67±24.8 43.38±18.9 <0.0001

51.97±12.7 51.32±15.5 0.74

13.99±4.8 11.89±3.5 0.0006

156.57±26.7 160.35±25.3

129.95±31.3 77.1±11.8 <0.0001

25.08±23.8 27.72±29.1 0.48

0.3

LDL: low density lipoprotein, HDL: high density lipoprotein, TC: total cholesterol, TG:triglycerides,

Table 2. Comparison of mean ± standard deviation and respective P-values of measured laboratory investigations at baseline and 12 months, in the total population, and the two

235.24±49.1 154.24±16.9 <0.0001

154.16±42.8 79.54±13.2 <0.0001

146.04±73.2 112.1±54.7 0.01

65.8±25.3 42.16±17.6 <0.0001

52.12±12.1 52.22±16.3 0.97

13.5±4.6 11.88±3.8 0.057

156.46±27.3 162.02±23.7 0.28

131.84±31.4 77.58±13.1 <0.0001

25.67±24.1 29.42±29.8 0.49

229.22±46.7 152.48±17.7 <0.0001

148.84±40.9 79.96±12.3 <0.0001

145.14±73.7 109.9±51.96 0.0069

63.53±24.6 44.65±26.1 0.0007

51.82±13.4 50.42±14.8 0.62

14.48±5.1 11.9±3.4 0.0036

156.68±26.4 158.68±27.1

128.05±31.4 76.62±10.47 <0.0001

24.47±23.8 26.01±28.7 0.77

0.7

levels (r=0.65, p<0.001), while no significant correlation was shown with Lp-a.

similar decline of carotid stenosis in 12 months (6.61% vs 4.67%, table 3).

Lp-a (mg/dl) baseline Lp-a (mg/dl) 12 months

## **3. Results**

## **3.1 Patients' characteristics**

A total of 612 patients were evaluated, of which 123 fulfilled the eligibility criteria; finally, 100 had complete data to enter the analysis, 76 males and 24 females, median age 68 years (range 45-81). Diabetes mellitus was recorded in 26 of the 100 patients and hypertension in 66. Twenty patients had metabolic syndrome according to the national cholesterol education programme-adult treatment panel III (NCEP-ATP III) criteria (National Cholesterol Education Program (NCEP), 2001). Active smoking (defined as current of discontinued as far back as 5 years) was reported by 58 patients. Mean atorvastatin dose at baseline was 24.31±11.49 mg for group A and 20.62±10.39 mg for group B (p=0.1). By the end of the study period, the respective mean values were significantly increased to 30.45±16.27 mg for group A (p=0.044) and 28.75±17.57 mg for group B (p=0.007).

Each of the study group (A and B) comprised 50 patients. The two groups were comparable with regard of their baseline characteristics (table 1).

## **3.2 Lipid profile and oxidzed LDL**

Mean serum TC, LDL-cholesterol, TG, Lp-a, homocysteine, and oxLDL were significantly reduced at 12 months compared to baseline (table 2). Specifically, mean oxLDL dropped from 62.26±22.03 to 44.49±21.75 mU/l (p<0.001). A marked decrease was noticed during the first 6 months and a plateau thereafter (figure 5).

Pts: patients, DM: diabetes mellitus, HTN: arterial hypertension, MS: metabolic syndrome, SD: standard deviation, LDL: low density lipoprotein, HDL: high density lipoprotein, CAD: coronary artery disease, TC: total cholesterol, TG: triglycerides, oxLDL: oxidized LDL

Table 1. Study population baseline characteristics.

A total of 612 patients were evaluated, of which 123 fulfilled the eligibility criteria; finally, 100 had complete data to enter the analysis, 76 males and 24 females, median age 68 years (range 45-81). Diabetes mellitus was recorded in 26 of the 100 patients and hypertension in 66. Twenty patients had metabolic syndrome according to the national cholesterol education programme-adult treatment panel III (NCEP-ATP III) criteria (National Cholesterol Education Program (NCEP), 2001). Active smoking (defined as current of discontinued as far back as 5 years) was reported by 58 patients. Mean atorvastatin dose at baseline was 24.31±11.49 mg for group A and 20.62±10.39 mg for group B (p=0.1). By the end of the study period, the respective mean values were significantly increased to 30.45±16.27 mg for group

Each of the study group (A and B) comprised 50 patients. The two groups were comparable

Mean serum TC, LDL-cholesterol, TG, Lp-a, homocysteine, and oxLDL were significantly reduced at 12 months compared to baseline (table 2). Specifically, mean oxLDL dropped from 62.26±22.03 to 44.49±21.75 mU/l (p<0.001). A marked decrease was noticed during the

Characteristic Total Group A Group B P value

Pts: patients, DM: diabetes mellitus, HTN: arterial hypertension, MS: metabolic syndrome, SD: standard deviation, LDL: low density lipoprotein, HDL: high density lipoprotein, CAD: coronary artery disease,

Mean age in years ± SD 67.57±7.15 68.46±5.71 66.68±8.31 0.21 Number of pts with DM (percentage) 37 (37%) 18 (36%) 19 (38%) 0.83 Number of pts with HTN (percentage) 67 (67%) 36 (72%) 31 (62%) 0.29 Number of smokers (percentage) 54 (54%) 29 (54%) 25 (46%) 0.33 Number of pts with CAD (percentage) 51 (51%) 24 (47%) 27 (53%) 0.55 Mean ± SD TC (mg/dl) 232.23±47.8 235.24±49.2 229.22±46.7 0.53 Mean ± SD LDL cholesterol (mg/dl) 151.27±41.7 154.16±42.8 148.84±40.9 0.52 Mean ± SD HDL cholesterol (mg/dl) 51.97±12.7 52.12±12.1 51.82±13.4 0.9 Mean ± SD TG (mg/dl) 145.59±73.1 146.04±73.2 145.14±73.7 0.95 Mean ± SD oxLDL (mU/l) 64.66±24.8 65.8±25.3 63.53±24.5 0.65 Mean ± SD homocysteine (mU/l) 13.99±4.8 13.5±4.6 14.47±5.1 0.32

Males / females 72 / 28 36 / 14 36 / 14

**3. Results**

**3.1 Patients' characteristics**

A (p=0.044) and 28.75±17.57 mg for group B (p=0.007).

with regard of their baseline characteristics (table 1).

first 6 months and a plateau thereafter (figure 5).

TC: total cholesterol, TG: triglycerides, oxLDL: oxidized LDL Table 1. Study population baseline characteristics.

**3.2 Lipid profile and oxidzed LDL**

A significant correlation between LDL and oxLDL levels was detected (Pearson's correlation coefficient r=0.7, p<0.01) (figure 6). Similar correlation was found between oxLDL and apoB levels (r=0.65, p<0.001), while no significant correlation was shown with Lp-a.

Between smokers mean oxLDL was reduced from 60.68±24.09 at baseline to 45.84±24.89 mU/l at the end of study period (difference 14.84 mU/l, p = 0.0036). Similarly, between non-smokers it was reduced from 69.33±25.11 to 40.36±5.6 mU/l (difference 28.97 mU/l, p<0.001). Nonsmokers had approximately double decline of oxLDL levels compared to smokers. Carotid artery stenosis was reduced between smokers from 29.68±25.59% at baseline to 23.06±21.71% at 12 months (p = 0.002). Non-smokers also presented significant reduction of stenosis during the study period (24.67 ± 26.22% vs 20 ± 21.45%, p = 0.004). Non-smokers and smokers had similar decline of carotid stenosis in 12 months (6.61% vs 4.67%, table 3).

LDL: low density lipoprotein, HDL: high density lipoprotein, TC: total cholesterol, TG:triglycerides, oxLDL: oxidized LDL, Lp-a: lipoprotein a

Table 2. Comparison of mean ± standard deviation and respective P-values of measured laboratory investigations at baseline and 12 months, in the total population, and the two groups.

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 135

In further analysis, the group of smokers was subdivided to mild (≤5 cigarettes/day), moderate (5 – 15 cigarettes/day) and heavy (≥15 cigarettes/day) smokers. The statistical significant reduction of oxLDL levels and degree of carotid stenosis was apparent in the subgroup of mild smokers (oxLDL at baseline 48.24±8.74 mU/l vs 41.54±9 mU/l at 12 months, p = 0.027 and stenosis at baseline 27.63±25.68% vs 23.42±21.74% at 12 months, p = 0.009), while it was not apparent in the subgroups of moderate and heavy smokers (oxLDL at baseline 86.82±37.7 mU/l vs 42.92±10.77 mU/l at 12 months, p = 0.077 and stenosis at baseline 34±31.9% vs 22±24.9% at 12 months, p = 0.186, for moderate smokers; respective values for oxLDL were 66.29±15.88 mU/l vs 34.81±5.48 mU/l, p = 0.06 and for stenosis 32.14±24.13% vs 22.86±22.8%, p = 0.174, for heavy smokers). The above described effect of

Smokers P value Non Smokers P value

0.0036 69.33±25.11

0.002 24.67±26.22

0.021 Pearson's

Stenosis >60<70% Stenosis <60% P value

40.36±5.6 28.97

20±21.45 4.67

r = 0.198

0.001

0.004

0.03

smoking was taken into consideration during Cox-regression analysis.

60.68±24.09 45.48±24.89 14.84

29.68±25.59 23.06±21.71 6.61

Pearson's r = 0.412

during the 1 year follow-up period, between smokers and non-smokers.

Table 3. Comparison of mean oxidized LDL values and degree of carotid stenosis change

Within group B, the subgroup of patients with high degree of stenosis (>60%) had oxLDL 63.47±19.18 mU/l at baseline, while those with moderate and mild degree of stenosis (<60%) had 40.32±20.72 mU/l (p<0.001). Corresponding values at 12-months were 33.18±17.78 mU/l and 38.81±29.02 mU/l, representing a marked decline for patients with >60% initial stenosis and a far less decline for patients with <60% initial stenosis; yet those differences

Mean oxLDL 63.47±19.18 mU/l 40.32±20.72 mU/l < 0.001

Mean oxLDL 33.18±17.78 mU/l 38.81±29.02 mU/l NS

Table 4. Comparison of mean oxidized LDL levels at baseline and 12 months within patients

LDL: low density lipoprotein cholesterol, oxLDL: oxidized LDL, NS: non significant.

of group Β (n = 50), according to degree of stenosis at enrollment.

OxLDL (mU/l) baseline 12 months difference

> Stenosis (%) baseline 12 months difference

Correlation of oxLDL change with stenosis change in 12 months

Baseline

12 months

were not statistically significant (table 4).

oxLDL: oxidized LDL.

TC: total cholesterol, LDL: low density lipoprotein cholesterol, oxLDL: oxidized LDL, TG: triglycerides, 0=baseline, 1=one month, 2=three months, 3=six months, 4=twelve months.

Fig. 5. Time curve of change of total cholesterol, LDL cholesterol, triglycerides and oxidized LDL levels during the observation period.

LDL: low density lipoprotein cholesterol, oxLDL: oxidized LDL, CI: confidence intervals.

Fig. 6. Correlation of low density lipoprotein with oxidized LDL levels at baseline (Pearson's correlation coefficient r=0.7, p<0.001).

In further analysis, the group of smokers was subdivided to mild (≤5 cigarettes/day), moderate (5 – 15 cigarettes/day) and heavy (≥15 cigarettes/day) smokers. The statistical significant reduction of oxLDL levels and degree of carotid stenosis was apparent in the subgroup of mild smokers (oxLDL at baseline 48.24±8.74 mU/l vs 41.54±9 mU/l at 12 months, p = 0.027 and stenosis at baseline 27.63±25.68% vs 23.42±21.74% at 12 months, p = 0.009), while it was not apparent in the subgroups of moderate and heavy smokers (oxLDL at baseline 86.82±37.7 mU/l vs 42.92±10.77 mU/l at 12 months, p = 0.077 and stenosis at baseline 34±31.9% vs 22±24.9% at 12 months, p = 0.186, for moderate smokers; respective values for oxLDL were 66.29±15.88 mU/l vs 34.81±5.48 mU/l, p = 0.06 and for stenosis 32.14±24.13% vs 22.86±22.8%, p = 0.174, for heavy smokers). The above described effect of smoking was taken into consideration during Cox-regression analysis.

oxLDL: oxidized LDL.

134 Coronary Artery Diseases

Lipids alteration during observation period

0 1 2 3 4 Time periods of observation

TC: total cholesterol, LDL: low density lipoprotein cholesterol, oxLDL: oxidized LDL, TG: triglycerides,

Fig. 5. Time curve of change of total cholesterol, LDL cholesterol, triglycerides and oxidized

Correlation LDL with Ox LDL

LDL: low density lipoprotein cholesterol, oxLDL: oxidized LDL, CI: confidence intervals.

Fitted values

Fig. 6. Correlation of low density lipoprotein with oxidized LDL levels at baseline (Pearson's

50 100 150 200 250 LDL

Ox LDL 95% CI

0=baseline, 1=one month, 2=three months, 3=six months, 4=twelve months.

LDL levels during the observation period.

correlation coefficient r=0.7, p<0.001).

TC LDL oxLDL TG

50

0

p<0.001

50

100

150

Ox LDL %

100

150

mgr/dl

200

250

> Table 3. Comparison of mean oxidized LDL values and degree of carotid stenosis change during the 1 year follow-up period, between smokers and non-smokers.

Within group B, the subgroup of patients with high degree of stenosis (>60%) had oxLDL 63.47±19.18 mU/l at baseline, while those with moderate and mild degree of stenosis (<60%) had 40.32±20.72 mU/l (p<0.001). Corresponding values at 12-months were 33.18±17.78 mU/l and 38.81±29.02 mU/l, representing a marked decline for patients with >60% initial stenosis and a far less decline for patients with <60% initial stenosis; yet those differences were not statistically significant (table 4).

LDL: low density lipoprotein cholesterol, oxLDL: oxidized LDL, NS: non significant.

Table 4. Comparison of mean oxidized LDL levels at baseline and 12 months within patients of group Β (n = 50), according to degree of stenosis at enrollment.

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 137

This study demonstrates that atorvastatin administered in indivudualised doses, tittered to maintain serum LDL cholesterol levels <100 mg/dl, significantly decreased lipid profile and oxLDL, reduced carotid artery stenosis in patients managed conservatively and prevented restenosis in patients with prior angioplasty. Oxidized LDL in this study correlated positively with the degree of carotid artery stenosis; it was also shown by multivariate analysis that oxLDL represented an independent risk factor for restenosis. To our knowledge this is the first prospective study with a long observation period of 12 months to report such a clear, significant reduction of oxLDL levels following atorvastatin therapy for carotid atheromatosis of various causes and to report an association of the degree of oxLDL reduction with remission of carotid stenosis. It is also of major importance that this robust, long-standing decline of oxLDL was achieved with doses of atorvastatin used in everyday clinical practice. Interestingly, this beneficial effect was completed in the first six months,

while practically no further reduction was noticed past this time point.

Kaplan Meier Curves for Carotid Stenosis

oxLDL=0: low quartile of oxLDL levels, oxLDL=1: high quartile of oxLDL levels, 0=baseline, 1=one

% <sup>0</sup> <sup>1</sup> <sup>2</sup> <sup>3</sup> <sup>4</sup> <sup>5</sup> Time of observation (in five periods)

oxLDL = 0 oxLDL = 1

Logrank test p<0.001

Fig. 7. Kaplan Meier survival analysis for the estimation of the risk ratio for restenosis according to the levels of oxidized LDL (oxLDL). With red line those with oxLDL levels in the highest quartile of the values. With blue line those with oxLDL levels in the lowest

The mechanism by which statins modulate oxLDL levels has been controversial in the literature. Moreover, the association of oxLDL level modification with improvement of carotid atheromatosis and clinical outcome is not unequivocally established by large, double-blinded, randomised trials. Under this perspective, the present observational study provides reasonable evidence that reducing oxLDL may independently improve carotid

quartile of the measurements (risk ratio 1.025, logrank test p<0.001).

month, 2=three months, 3=six months, 4=twelve months.

stenosis.

0.00

0.25

0.50

0.75

1.00 C a R O T I D S T E N O S I S

**4. Discussion**

## **3.3 Anthropometrics**

Body mass index (BMI), weight, waist circumference and waist:hip ratio did not change significantly during the study period.

## **3.4 Carotid stenosis**

Patients in group A had null stenosis at recruitment due to prior angioplasty with stenting. At the end of the 12-month statin therapy, no case of clinically important restenosis (>70%) was reported in this group (as restenosis was defined any increase of the carotid lumen diameter >5%). Patients in group B had mean percentage of stenosis at baseline 47.6±13.2%, which was significantly reduced following 12-month statin therapy (37.7±15.7%, p<0.001) (table 5).

Table 5. Change of the percentage of carotid artery stenosis between baseline and 12months for patients in group B.

## **3.5 Association of stenosis with oxidized LDL**

Group B patients in the highest quartile of oxLDL values had a 12-month risk ratio for restenosis of 1.025, 95%CI=1.006-1.044, p=0.0083 (figure 7). After adjusting for gender, age, smoking, SBP, TC, and LDL levels, these patients demonstrated a HR for restenosis of 4.319 compared with those in the lowest quartile (p<0.001, figure 7). This means that an increase of oxLDL by one unit increases the degree of carotid stenosis by 2.5%, for patients in group B. A weak but significant correlation was detected between oxLDL levels and the degree of carotid artery stenosis (r=0.17, p=0.018). Similar correlation was found between LDL cholesterol levels and carotid stenosis (r=0.18, p=0.0085). The strength of Pearson's correlation of mean oxLDL change with degree of carotid stenosis change during the 12-month period was greater for smokers compared to non-smokers (table 3).

## **3.6 The effect of LDL levels**

Patients in group B who achieved LDL levels <70 mg/dl during the observation period had a greater (28.08±28% vs 22.31±22.7%, difference 5.77%, p = 0.06) reduction of carotid stenosis compared to those with LDL levels between 70 and 100 mg/dl (26.98±25.3% vs 21.35±21.3%, difference 5.63%, p < 0.001), but this difference was not statistically significant. Thus, in conservatively treated group B, further reduction of LDL than the limit of 100 mg/dl was not associated with additional improvement of stenosis.

## **4. Discussion**

136 Coronary Artery Diseases

Body mass index (BMI), weight, waist circumference and waist:hip ratio did not change

Patients in group A had null stenosis at recruitment due to prior angioplasty with stenting. At the end of the 12-month statin therapy, no case of clinically important restenosis (>70%) was reported in this group (as restenosis was defined any increase of the carotid lumen diameter >5%). Patients in group B had mean percentage of stenosis at baseline 47.6±13.2%, which was significantly reduced following 12-month statin therapy (37.7±15.7%, p<0.001)

deviation 47.6 ± 13.2 37.7 ± 15.7 0.001

Table 5. Change of the percentage of carotid artery stenosis between baseline and 12months

Group B patients in the highest quartile of oxLDL values had a 12-month risk ratio for restenosis of 1.025, 95%CI=1.006-1.044, p=0.0083 (figure 7). After adjusting for gender, age, smoking, SBP, TC, and LDL levels, these patients demonstrated a HR for restenosis of 4.319 compared with those in the lowest quartile (p<0.001, figure 7). This means that an increase of oxLDL by one unit increases the degree of carotid stenosis by 2.5%, for patients in group B. A weak but significant correlation was detected between oxLDL levels and the degree of carotid artery stenosis (r=0.17, p=0.018). Similar correlation was found between LDL cholesterol levels and carotid stenosis (r=0.18, p=0.0085). The strength of Pearson's correlation of mean oxLDL change with degree of carotid stenosis change during the 12-month period was greater for smokers compared to non-smokers

Patients in group B who achieved LDL levels <70 mg/dl during the observation period had a greater (28.08±28% vs 22.31±22.7%, difference 5.77%, p = 0.06) reduction of carotid stenosis compared to those with LDL levels between 70 and 100 mg/dl (26.98±25.3% vs 21.35±21.3%, difference 5.63%, p < 0.001), but this difference was not statistically significant. Thus, in conservatively treated group B, further reduction of LDL than the limit of 100 mg/dl was

Baseline 12 months P value

**3.3 Anthropometrics**

**3.4 Carotid stenosis**

(table 5).

(table 3).

**3.6 The effect of LDL levels**

significantly during the study period.

Mean (%) carotid stenosis ± standard

**3.5 Association of stenosis with oxidized LDL**

not associated with additional improvement of stenosis.

for patients in group B.

This study demonstrates that atorvastatin administered in indivudualised doses, tittered to maintain serum LDL cholesterol levels <100 mg/dl, significantly decreased lipid profile and oxLDL, reduced carotid artery stenosis in patients managed conservatively and prevented restenosis in patients with prior angioplasty. Oxidized LDL in this study correlated positively with the degree of carotid artery stenosis; it was also shown by multivariate analysis that oxLDL represented an independent risk factor for restenosis. To our knowledge this is the first prospective study with a long observation period of 12 months to report such a clear, significant reduction of oxLDL levels following atorvastatin therapy for carotid atheromatosis of various causes and to report an association of the degree of oxLDL reduction with remission of carotid stenosis. It is also of major importance that this robust, long-standing decline of oxLDL was achieved with doses of atorvastatin used in everyday clinical practice. Interestingly, this beneficial effect was completed in the first six months, while practically no further reduction was noticed past this time point.

oxLDL=0: low quartile of oxLDL levels, oxLDL=1: high quartile of oxLDL levels, 0=baseline, 1=one month, 2=three months, 3=six months, 4=twelve months.

Fig. 7. Kaplan Meier survival analysis for the estimation of the risk ratio for restenosis according to the levels of oxidized LDL (oxLDL). With red line those with oxLDL levels in the highest quartile of the values. With blue line those with oxLDL levels in the lowest quartile of the measurements (risk ratio 1.025, logrank test p<0.001).

The mechanism by which statins modulate oxLDL levels has been controversial in the literature. Moreover, the association of oxLDL level modification with improvement of carotid atheromatosis and clinical outcome is not unequivocally established by large, double-blinded, randomised trials. Under this perspective, the present observational study provides reasonable evidence that reducing oxLDL may independently improve carotid stenosis.

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 139

monocyte maturation and differentiation, and the modification of production of

Atorvastatin suppresses cellular uptake of oxLDL from differentiating monocytes by reducing the expression of LOX-1 and scavenger receptors (Fuhrman et al., 2002) and accelerates the LDL-receptor-mediated removal of the non oxidized LDL particles (Vasankari et al., 2005). Hydroxymetabolites of atorvastatin protect the LDL against oxidation (van Tits et al., 2006). The antioxidant potency of atorvastatin metabolites has been confirmed by the reduction of IgG antibodies against LDL, a marker well-associated with CHD (Aviram et al., 1998). It has even been reported that these active atorvastatin metabolites may have greater anti-atherosclerotic effects than other statin molecules (Mason

In acute coronary syndromes, atorvastatin therapy was linked to modulation of short- and long-term immune response towards LDL due to inhibition of lipoprotein-associated phospholipase A2 (Lp-LPA2) enzyme (Papathanasiou et al., 2008). The apparent benefit from statin therapy after acute coronary events may also be attributed to the stabilization of the plaque and removal of oxLDL from the vessel wall (Tsimikas et al., 2004). Increased mobilization of oxidized phospholipids from the vessel wall, transient binding with apoB-100 particles and clearance from the circulation may be the possible underlying mechanism. Under this perspective the increase in oxLDL:apoB ratio detected with atorvastatin therapy might represent a marker of oxLDL efflux from the vessel wall. Removal of oxLDL contributes to improved endothelial function as oxLDL is highly immunogenic and vasoconstrictive. In our study there was no significant change in oxLDL:apoB ratio. Atorvastatin also inhibits the oxLDL-mediated LOX-1 expression by endothelial cells, the uptake of oxLDL in endothelium and the oxLDL-mediated reduction of protein kinase B (PKB) phosphorylation (Li et al., 2001). The activation of PKB is critical for the expression of eNOS, which promotes vessel relaxation. However, a meta-analysis provided no clear

evidence that statin therapy have a favourable effect on oxLDL (Balk et al., 2003).

accelerated decline of oxLDL levels followed by a milder reduction rate thereafter.

In STAT trial (Mulder et al., 2007) the antibodies against oxLDL were equally decreased with both aggressive and conventional lipid-lowering therapy. This indicates that the statin-related reduction of oxLDL is not a dose-dependent phenomenon, a finding which is in agreement with our results. It might therefore represent a pleiotropic effect, independent -at least partially- from the hypo-cholesterolemic action. A study by Orem et al. (2002) detected a significant decrease of autoantibodies against oxLDL with low doses of atorvastatin (10 mg), similar to doses used in our study. In statin exposed patients, intensification of the regimen offers no additional benefit and only those with LDL >125 mg/dl benefited from a more aggressive statin therapy (Mulder et al., 2007). Statins have a dose-related response with regard to clinical outcome, but this dose-related response has not been confirmed with regard to oxidative stress (Ky et al., 2008). This might alternatively be explained by the hypothesis that statins achieve their uttermost benefit on oxLDL within a certain time point (Mulder et al., 2007), after which further continuation of treatment serves only the purpose of maintenance. Atorvastatin has been shown to reduce small dense LDL subfractions, remnant-like particles cholesterol and oxLDL, and improve endothelial function, after just few weeks of therapy (Miyagishima et al., 2007; Sakabe et al., 2003). Such time-related effect has not been fully elucidated, but may possibly account for our finding that in the first six months there was an

inflammatory cytokines (Rothe et al., 1999).

et al., 2004).

Carotid IMT is a validated measure of carotid atherosclerosis. It is well established that carotid atherosclerosis, serves as an independent surrogate marker for CHD (Vasankari et al., 2001) and CVD (van Tits et al., 2006). Nevertheless, in the present study it was preferred to estimate the degree of carotid stenosis with a more direct approach, because this is more readily available in most hospital settings and because there is an obvious relation with clinical symptoms and signs. Besides, it represents a reliable method with sufficient reproducibility and it is practically the method of choice when evaluated patients candidate for endarterectomy or angioplasty. Evaluating carotid stenosis in turn, is an established method for estimating coronary risk (Vasankari et al., 2001) and cardiovascular risk (van Tits et al., 2006). Other parameters of vessel wall function, such as IMT and plaque morphology, even if clearly associated with cardiovascular risk in the literature, require well equipped laboratory and are not readily available in our hospital. Future research on the field should, ideally, comprise such measurements.

Oxidized LDL has long been recognized as a risk factor for carotid atherosclerosis in asymptomatic men (Liu et al., 2004) and has also been linked with CVD (Robbesyn et al., 2004). Oxidized LDL levels (Papathanasiou et al., 2008), autoantibodies against epitopes of oxLDL (Papathanasiou et al., 2008) and oxLDL:LDL ratio (Vasankari et al., 2001) are independently associated with increased risk for coronary atheromatosis and ischemic heart disease. Increased levels of oxLDL (Ehara et al., 2001) and MDA-LDL (Holvoet et al., 1999) in such cases are related to plaque instability. On the other hand, it has been reported that oxLDL is weakly associated with carotid IMT, but not with carotid plaque occurrence (Hulthe & Fagerberg, 2002). Oxidized LDL impairs endothelium relaxation (Harrison et al., 1987) by inhibition of the expression of eNOS and of the transport pathways of nitric oxide (NO) from the endothelial cell, reduces the responsiveness of smooth muscle cell to NO (Keaney et al., 1996), inhibits the NO-mediated vasodilation (Harrison et al., 1987; Simon et al., 1990; Steinberg, 1997), induces the expression of adhesion molecules (Frostegard et al., 1990), acts directly chemotactic to circulating monocytes (Steinberg, 1997), stimulates endothelial cells to produce MCP-1 (Cushing et al., 1990), facilitates monocyte adhesion to intima (Mehta et al., 1995), exhibits cytotoxic properties against endothelial cells (Steinberg, 1997), and induces the expression of inflammatory molecules (Steinberg, 1997). All of the above contribute directly to dysfunction of the endothelium (Witztum & Steinberg, 1991) and foam cell formation, which is the first step in the development of fatty streaks (Ross, 1999), the first visible step of atherosclerosis. These effects are mediated by preferential binding of oxLDL with type A scavenger receptors (SRA, SRA-II and CD36) on subendothelial resident macrophages and smooth muscle cells (Li et al., 1995) and lectin-like oxLDL receptor-1 (LOX-1) on endothelial cells (Sawamura et al., 1997) rather than the typical LDL receptor, resulting in an unrestricted uptake of cholesterol.

Statins reduce the incidence of cardiovascular events, an effect attributable to their hypocholesterolemic properties (Archbold & Timmis, 1999). However, the extent of clinical benefit and accumulating laboratory evidence suggest additional mechanisms of action, the so-called pleiotropic effects (Bellosta et al., 2000). The most important among such effects are the suppression of smooth muscle cell migration and proliferation (Bellosta et al., 1998), the reduction of monocyte adhesion to the vascular endothelium (Weber et al., 1997), the improvement of endothelial function (Jarvisalo et al., 1999), the inhibition of cell-mediated LDL oxidation (Aviram et al., 1998; Giroux et al., 1993), the immuno-modulation of

Carotid IMT is a validated measure of carotid atherosclerosis. It is well established that carotid atherosclerosis, serves as an independent surrogate marker for CHD (Vasankari et al., 2001) and CVD (van Tits et al., 2006). Nevertheless, in the present study it was preferred to estimate the degree of carotid stenosis with a more direct approach, because this is more readily available in most hospital settings and because there is an obvious relation with clinical symptoms and signs. Besides, it represents a reliable method with sufficient reproducibility and it is practically the method of choice when evaluated patients candidate for endarterectomy or angioplasty. Evaluating carotid stenosis in turn, is an established method for estimating coronary risk (Vasankari et al., 2001) and cardiovascular risk (van Tits et al., 2006). Other parameters of vessel wall function, such as IMT and plaque morphology, even if clearly associated with cardiovascular risk in the literature, require well equipped laboratory and are not readily available in our hospital. Future research on the

Oxidized LDL has long been recognized as a risk factor for carotid atherosclerosis in asymptomatic men (Liu et al., 2004) and has also been linked with CVD (Robbesyn et al., 2004). Oxidized LDL levels (Papathanasiou et al., 2008), autoantibodies against epitopes of oxLDL (Papathanasiou et al., 2008) and oxLDL:LDL ratio (Vasankari et al., 2001) are independently associated with increased risk for coronary atheromatosis and ischemic heart disease. Increased levels of oxLDL (Ehara et al., 2001) and MDA-LDL (Holvoet et al., 1999) in such cases are related to plaque instability. On the other hand, it has been reported that oxLDL is weakly associated with carotid IMT, but not with carotid plaque occurrence (Hulthe & Fagerberg, 2002). Oxidized LDL impairs endothelium relaxation (Harrison et al., 1987) by inhibition of the expression of eNOS and of the transport pathways of nitric oxide (NO) from the endothelial cell, reduces the responsiveness of smooth muscle cell to NO (Keaney et al., 1996), inhibits the NO-mediated vasodilation (Harrison et al., 1987; Simon et al., 1990; Steinberg, 1997), induces the expression of adhesion molecules (Frostegard et al., 1990), acts directly chemotactic to circulating monocytes (Steinberg, 1997), stimulates endothelial cells to produce MCP-1 (Cushing et al., 1990), facilitates monocyte adhesion to intima (Mehta et al., 1995), exhibits cytotoxic properties against endothelial cells (Steinberg, 1997), and induces the expression of inflammatory molecules (Steinberg, 1997). All of the above contribute directly to dysfunction of the endothelium (Witztum & Steinberg, 1991) and foam cell formation, which is the first step in the development of fatty streaks (Ross, 1999), the first visible step of atherosclerosis. These effects are mediated by preferential binding of oxLDL with type A scavenger receptors (SRA, SRA-II and CD36) on subendothelial resident macrophages and smooth muscle cells (Li et al., 1995) and lectin-like oxLDL receptor-1 (LOX-1) on endothelial cells (Sawamura et al., 1997) rather than the

typical LDL receptor, resulting in an unrestricted uptake of cholesterol.

Statins reduce the incidence of cardiovascular events, an effect attributable to their hypocholesterolemic properties (Archbold & Timmis, 1999). However, the extent of clinical benefit and accumulating laboratory evidence suggest additional mechanisms of action, the so-called pleiotropic effects (Bellosta et al., 2000). The most important among such effects are the suppression of smooth muscle cell migration and proliferation (Bellosta et al., 1998), the reduction of monocyte adhesion to the vascular endothelium (Weber et al., 1997), the improvement of endothelial function (Jarvisalo et al., 1999), the inhibition of cell-mediated LDL oxidation (Aviram et al., 1998; Giroux et al., 1993), the immuno-modulation of

field should, ideally, comprise such measurements.

monocyte maturation and differentiation, and the modification of production of inflammatory cytokines (Rothe et al., 1999).

Atorvastatin suppresses cellular uptake of oxLDL from differentiating monocytes by reducing the expression of LOX-1 and scavenger receptors (Fuhrman et al., 2002) and accelerates the LDL-receptor-mediated removal of the non oxidized LDL particles (Vasankari et al., 2005). Hydroxymetabolites of atorvastatin protect the LDL against oxidation (van Tits et al., 2006). The antioxidant potency of atorvastatin metabolites has been confirmed by the reduction of IgG antibodies against LDL, a marker well-associated with CHD (Aviram et al., 1998). It has even been reported that these active atorvastatin metabolites may have greater anti-atherosclerotic effects than other statin molecules (Mason et al., 2004).

In acute coronary syndromes, atorvastatin therapy was linked to modulation of short- and long-term immune response towards LDL due to inhibition of lipoprotein-associated phospholipase A2 (Lp-LPA2) enzyme (Papathanasiou et al., 2008). The apparent benefit from statin therapy after acute coronary events may also be attributed to the stabilization of the plaque and removal of oxLDL from the vessel wall (Tsimikas et al., 2004). Increased mobilization of oxidized phospholipids from the vessel wall, transient binding with apoB-100 particles and clearance from the circulation may be the possible underlying mechanism. Under this perspective the increase in oxLDL:apoB ratio detected with atorvastatin therapy might represent a marker of oxLDL efflux from the vessel wall. Removal of oxLDL contributes to improved endothelial function as oxLDL is highly immunogenic and vasoconstrictive. In our study there was no significant change in oxLDL:apoB ratio. Atorvastatin also inhibits the oxLDL-mediated LOX-1 expression by endothelial cells, the uptake of oxLDL in endothelium and the oxLDL-mediated reduction of protein kinase B (PKB) phosphorylation (Li et al., 2001). The activation of PKB is critical for the expression of eNOS, which promotes vessel relaxation. However, a meta-analysis provided no clear evidence that statin therapy have a favourable effect on oxLDL (Balk et al., 2003).

In STAT trial (Mulder et al., 2007) the antibodies against oxLDL were equally decreased with both aggressive and conventional lipid-lowering therapy. This indicates that the statin-related reduction of oxLDL is not a dose-dependent phenomenon, a finding which is in agreement with our results. It might therefore represent a pleiotropic effect, independent -at least partially- from the hypo-cholesterolemic action. A study by Orem et al. (2002) detected a significant decrease of autoantibodies against oxLDL with low doses of atorvastatin (10 mg), similar to doses used in our study. In statin exposed patients, intensification of the regimen offers no additional benefit and only those with LDL >125 mg/dl benefited from a more aggressive statin therapy (Mulder et al., 2007). Statins have a dose-related response with regard to clinical outcome, but this dose-related response has not been confirmed with regard to oxidative stress (Ky et al., 2008). This might alternatively be explained by the hypothesis that statins achieve their uttermost benefit on oxLDL within a certain time point (Mulder et al., 2007), after which further continuation of treatment serves only the purpose of maintenance. Atorvastatin has been shown to reduce small dense LDL subfractions, remnant-like particles cholesterol and oxLDL, and improve endothelial function, after just few weeks of therapy (Miyagishima et al., 2007; Sakabe et al., 2003). Such time-related effect has not been fully elucidated, but may possibly account for our finding that in the first six months there was an accelerated decline of oxLDL levels followed by a milder reduction rate thereafter.

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 141

scavenger receptor expression in macrophages, and increase plaque stability via reduction

The reduction of oxLDL and of carotid stenosis in our study was relevant for both smokers and non-smokers. However, subsequent subgroup analysis showed that the beneficial effect of statin use concerns mostly the subgroup of mild smokers, while no such effect was noticed for moderate and heavy smokers. How smoking may diminish the beneficial effect of statins on oxLDL and carotid stenosis is not yet clarified in the literature. A reasonable assumption might be that, since smoking increases the oxidative stress, it contributes to enhanced LDL oxidation (Van Himbergen et al., 2004). Moreover, studies in animal models, have demonstrated that smoking alters the immunologic response to oxLDL by reducing the production of antibodies against these molecules, i.e. causing a kind of immune suppression regarding the response to oxLDL. Thus, it has been shown to increase carotid IMT (Tani et

The Mercodia oxLDL detects the MDA-modified apoB (Holvoet et al., 1996). It has been proposed that oxLDL looses its predictive value for CVD when adjustment for apoB level is performed (Ky et al., 2008). In several studies though, a significant reduction of Mercodia oxLDL with atorvastatin 10 mg was still detected even after adjustment for apoB, (Holvoet et al., 2003; Ky et al., 2008; van Tits et al., 2006), while in other studies no adjustment for LDL or apoB levels was made (Ky et al., 2008; Sasaki et al., 2002). In our study the oxLDL:apoB ratio remained unchanged, but in the multivariate analysis the reduction of oxLDL was still

In patients with familial hypercholesterolemia a lack of association between oxLDL and IMT was reported at baseline, however two years therapy with atorvastatin 80 mg was associated with regression of carotid IMT (van Tits et al., 2004). The LDL subfraction profile and autoantibodies against oxLDL remained unchanged. Nevertheless, the rate of oxidation and the amount of dienes formed decreased and this was linked to lessening of atherosclerosis. In our study the reduction of carotid stenosis was associated with decreased oxLDL levels. Besides, the unchanged oxLDL autoantibodies levels do not preclude the reduction of oxLDL, as was indicated in another study involving dialysis patients, where atorvastatin therapy reduced plasma oxLDL, whereas oxLDL autoantibodies did not

Disadvantages of the study were the relatively small size, the lack of a control group comprising of patients with carotid stenosis not on statin therapy, which would be unethical, the fact that researchers were not blinded to the patients' status, the lack of randomization of the dose-schedules and the use of only one method to detect oxLDL.

This prospective, cross-sectional study with such a long observation period provided enough evidence to postulate a favourable effect of low-dose atorvastatin therapy on oxLDL, which was additionally associated with improvement of stenosis in patients with carotid atheromatosis. We thus, assume that oxLDL may represent a far more sensitive risk factor for carotid stenosis, than LDL itself or apoB. Further studying is required to confirm such findings and to establish a clear clinical and pathophsiologic link between oxLDL and

of metalloproteinases (Hogue et al., 2008).

significant after adjustment for apoB and LDL levels.

changed significantly (van den Akker et al., 2003).

al., 2004).

**5. Conclusion**

carotid stenosis.

Additional pleiotropic effects of statins have been reported in the literature and might account for the observed beneficial effects in the current study. Lysophosphatidylcholine is elevated during LDL oxidation and is responsible for some of the biological effects of oxLDL. Atorvastatin alters the ability of oxLDL to impair the endothelium relaxation, by modulating the hydrolysis of phosphatidylcholine to lysophosphatidylcholine when LDL is being oxidized (Zhu et al., 2000). Statins remove predominately "aged LDL" from plasma, which is more prone to oxidation (Orem et al., 2002), through stimulation of hepatic LDL receptor activity and inhibition of very-low density lipoprotein (VLDL) and LDL production by the liver cells (Orem et al., 2002). Statins also reduce oxygen species generation (Ky et al., 2008). Atorvastatin promotes adipocyte uptake of oxLDL in rabbits by increasing the expression of CD36 and peroxisome proliferators-activated receptor γ (PPARγ) in adipocytes (Zhao & Zhang, 2004). The increased expression of such receptors by adipocytes results to internalization of oxLDL and clearance from plasma, converting adipocytes to an oxLDL-buffering pool (Zhao & Zhang, 2004). Reduction of oxLDL in patients with CHD with atorvastatin 10 mg parallel with an increase of adiponectin, which has anti-atherogenic, anti-inflammatory and anti-diabetic properties through reduction of insulin resistance (Miyagishima et al., 2007). The CARDS study reported a significant degree of preventive activity of atorvastatin against myocardial infarction in eucholesterolemic diabetic patients, conceivably attributed to such improvement of insulin sensitivity (Miyagishima et al., 2007). Statins also diminish the expression of CD40 and CD40 ligand in vascular cells, smooth muscle cells and macrophages, which are promoted by oxLDL and are considered proatherogenic (Schonbeck et al., 2002). Other anti-inflammatory pathways include reduction of C-reactive protein (Hogue et al., 2008), chemokines, major histocompatibility complex II molecules, matrix-degrading enzymes, and procoagulant tissue factor (Schonbeck et al., 2002). Atorvastatin reverses the oxLDL-mediated inhibition of vascular endothelial growth factor-induced endothelial progenitor cell differentiation via the phosphatidylinositol 3 kinase/Akt pathway (Imanishi et al., 2003), which restores the oxLDL-related inhibition of mature endothelial cells migration (Imanishi et al., 2003). This could improve neovascularization and collateral vessel formation in response to tissue ischemia. Atorvastatin also suppresses platelet activity (Puccetti et al., 2005) by reducing the expression of CD36 and LOX-1, which are present in platelets (Puccetti et al., 2005; Sawamura et al., 1997), thus inhibiting the oxLDL-mediated platelet hyperactivity (Puccetti et al., 2005). Statins reduce the oxLDL-derived expression of adhesion molecules (E- and Pselectins, vascular cell adhesion molecule 1 [VCAM-1] and intercellular adhesion molecule 1 [ICAM-1]) in human coronary artery endothelial cells, through up-regulation of eNOS expression, which regulates the expression of adhesion molecules in endothelial cells (Li et al., 2002). Statins also diminish the oxLDL-mediated activation of nuclear factor-κΒ (NF-κB) (D. Li et al., 2002), which regulates the transcription of adhesion molecule genes (Robbesyn et al., 2004). In diabetic patients with dyslipidemia atorvastatin reduced CVD and markers of inflammation, adhesion and oxidation, such as C-reactive protein (CRP), soluble ICAM-1, soluble VCAM-1, E-selectin, matrix metalloproteinase 9, secretory phospholipase A2 (sPLA2), and oxLDL, the latter by 38,4% (Hogue et al., 2008). Moreover, the change of oxLDL levels correlated with the change of sICAM-1 and E-selectin levels, suggesting that statins could possibly counteract the oxLDL-associated increase of NF-κΒ, and therefore, the production of such cell adhesion molecules (Hogue et al., 2008). Statins also enhance scavenger receptor expression in macrophages, and increase plaque stability via reduction of metalloproteinases (Hogue et al., 2008).

The reduction of oxLDL and of carotid stenosis in our study was relevant for both smokers and non-smokers. However, subsequent subgroup analysis showed that the beneficial effect of statin use concerns mostly the subgroup of mild smokers, while no such effect was noticed for moderate and heavy smokers. How smoking may diminish the beneficial effect of statins on oxLDL and carotid stenosis is not yet clarified in the literature. A reasonable assumption might be that, since smoking increases the oxidative stress, it contributes to enhanced LDL oxidation (Van Himbergen et al., 2004). Moreover, studies in animal models, have demonstrated that smoking alters the immunologic response to oxLDL by reducing the production of antibodies against these molecules, i.e. causing a kind of immune suppression regarding the response to oxLDL. Thus, it has been shown to increase carotid IMT (Tani et al., 2004).

The Mercodia oxLDL detects the MDA-modified apoB (Holvoet et al., 1996). It has been proposed that oxLDL looses its predictive value for CVD when adjustment for apoB level is performed (Ky et al., 2008). In several studies though, a significant reduction of Mercodia oxLDL with atorvastatin 10 mg was still detected even after adjustment for apoB, (Holvoet et al., 2003; Ky et al., 2008; van Tits et al., 2006), while in other studies no adjustment for LDL or apoB levels was made (Ky et al., 2008; Sasaki et al., 2002). In our study the oxLDL:apoB ratio remained unchanged, but in the multivariate analysis the reduction of oxLDL was still significant after adjustment for apoB and LDL levels.

In patients with familial hypercholesterolemia a lack of association between oxLDL and IMT was reported at baseline, however two years therapy with atorvastatin 80 mg was associated with regression of carotid IMT (van Tits et al., 2004). The LDL subfraction profile and autoantibodies against oxLDL remained unchanged. Nevertheless, the rate of oxidation and the amount of dienes formed decreased and this was linked to lessening of atherosclerosis. In our study the reduction of carotid stenosis was associated with decreased oxLDL levels. Besides, the unchanged oxLDL autoantibodies levels do not preclude the reduction of oxLDL, as was indicated in another study involving dialysis patients, where atorvastatin therapy reduced plasma oxLDL, whereas oxLDL autoantibodies did not changed significantly (van den Akker et al., 2003).

Disadvantages of the study were the relatively small size, the lack of a control group comprising of patients with carotid stenosis not on statin therapy, which would be unethical, the fact that researchers were not blinded to the patients' status, the lack of randomization of the dose-schedules and the use of only one method to detect oxLDL.

## **5. Conclusion**

140 Coronary Artery Diseases

Additional pleiotropic effects of statins have been reported in the literature and might account for the observed beneficial effects in the current study. Lysophosphatidylcholine is elevated during LDL oxidation and is responsible for some of the biological effects of oxLDL. Atorvastatin alters the ability of oxLDL to impair the endothelium relaxation, by modulating the hydrolysis of phosphatidylcholine to lysophosphatidylcholine when LDL is being oxidized (Zhu et al., 2000). Statins remove predominately "aged LDL" from plasma, which is more prone to oxidation (Orem et al., 2002), through stimulation of hepatic LDL receptor activity and inhibition of very-low density lipoprotein (VLDL) and LDL production by the liver cells (Orem et al., 2002). Statins also reduce oxygen species generation (Ky et al., 2008). Atorvastatin promotes adipocyte uptake of oxLDL in rabbits by increasing the expression of CD36 and peroxisome proliferators-activated receptor γ (PPARγ) in adipocytes (Zhao & Zhang, 2004). The increased expression of such receptors by adipocytes results to internalization of oxLDL and clearance from plasma, converting adipocytes to an oxLDL-buffering pool (Zhao & Zhang, 2004). Reduction of oxLDL in patients with CHD with atorvastatin 10 mg parallel with an increase of adiponectin, which has anti-atherogenic, anti-inflammatory and anti-diabetic properties through reduction of insulin resistance (Miyagishima et al., 2007). The CARDS study reported a significant degree of preventive activity of atorvastatin against myocardial infarction in eucholesterolemic diabetic patients, conceivably attributed to such improvement of insulin sensitivity (Miyagishima et al., 2007). Statins also diminish the expression of CD40 and CD40 ligand in vascular cells, smooth muscle cells and macrophages, which are promoted by oxLDL and are considered proatherogenic (Schonbeck et al., 2002). Other anti-inflammatory pathways include reduction of C-reactive protein (Hogue et al., 2008), chemokines, major histocompatibility complex II molecules, matrix-degrading enzymes, and procoagulant tissue factor (Schonbeck et al., 2002). Atorvastatin reverses the oxLDL-mediated inhibition of vascular endothelial growth factor-induced endothelial progenitor cell differentiation via the phosphatidylinositol 3 kinase/Akt pathway (Imanishi et al., 2003), which restores the oxLDL-related inhibition of mature endothelial cells migration (Imanishi et al., 2003). This could improve neovascularization and collateral vessel formation in response to tissue ischemia. Atorvastatin also suppresses platelet activity (Puccetti et al., 2005) by reducing the expression of CD36 and LOX-1, which are present in platelets (Puccetti et al., 2005; Sawamura et al., 1997), thus inhibiting the oxLDL-mediated platelet hyperactivity (Puccetti et al., 2005). Statins reduce the oxLDL-derived expression of adhesion molecules (E- and Pselectins, vascular cell adhesion molecule 1 [VCAM-1] and intercellular adhesion molecule 1 [ICAM-1]) in human coronary artery endothelial cells, through up-regulation of eNOS expression, which regulates the expression of adhesion molecules in endothelial cells (Li et al., 2002). Statins also diminish the oxLDL-mediated activation of nuclear factor-κΒ (NF-κB) (D. Li et al., 2002), which regulates the transcription of adhesion molecule genes (Robbesyn et al., 2004). In diabetic patients with dyslipidemia atorvastatin reduced CVD and markers of inflammation, adhesion and oxidation, such as C-reactive protein (CRP), soluble ICAM-1, soluble VCAM-1, E-selectin, matrix metalloproteinase 9, secretory phospholipase A2 (sPLA2), and oxLDL, the latter by 38,4% (Hogue et al., 2008). Moreover, the change of oxLDL levels correlated with the change of sICAM-1 and E-selectin levels, suggesting that statins could possibly counteract the oxLDL-associated increase of NF-κΒ, and therefore, the production of such cell adhesion molecules (Hogue et al., 2008). Statins also enhance

This prospective, cross-sectional study with such a long observation period provided enough evidence to postulate a favourable effect of low-dose atorvastatin therapy on oxLDL, which was additionally associated with improvement of stenosis in patients with carotid atheromatosis. We thus, assume that oxLDL may represent a far more sensitive risk factor for carotid stenosis, than LDL itself or apoB. Further studying is required to confirm such findings and to establish a clear clinical and pathophsiologic link between oxLDL and carotid stenosis.

Oxidized Low Density Lipoprotein, Statin Therapy and Carotid Stenosis 143

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