**Part 3**

**Oxidative Stress in Atherosclerosis** 

410 Atherogenesis

Zeno, S., Zaaroor, M., Leschiner, S., Veenman, L., Gavish, M. (2009). CoCl(2) induces

Zeno, S., Veenman, L., Katz, Y., Bode, J., Gavish, M., Zaaroor, M. (2011). The 18 kDa

*Biochem.* 48, 4652-61.

apoptosis via the 18kDa translocator protein in U118MG human glioblastoma cells.

mitochondrial Translocator Protein (TSPO) prevents accumulation of protoporphyrin IX. A TSPO knockdown study. *Curr Molecular Medicine.* In press.

**19** 

*Israel* 

**Are CVD Patients Under Oxidative Stress?** 

*Dept. of Physiology and Pharmacology, Sackler Medical School, Tel Aviv University* 

Oxidative stress has long been associated with cardiovascular disease (CVD) (Abuja & Albertini, 2001; Halliwell & Gutteridge, 1990; Parthasarathy et al., 2001; Steinbrecher et al., 1984). It was even assumed that the prevalence of CVD alone indicates the prevalence of oxidative stress (Witztum, 1994). Moreover, cross-sectional studies indicated that supplementation of low molecular weight antioxidants is associated with a relatively low incidence of CVD (Jha et al., 1995). By contrast, in most of the interventional studies the antioxidant supplementation did not prevent the progression of CVD nor did it improve any of the many clinical endpoints (Shekelle et al., 2004; Vivekananthan et al., 2003; Miller 2005; Bjelakovic, 2007; Dotan, 2009a; Dotan, 2009b). Based on these findings, Witztum (Witztum, 1998) and Morrow (Morrow, 2003) hypothesized that only individuals under oxidative stress may benefit from antioxidant supplementation. This, of course, implies that only people under oxidative stress should be treated with antioxidants. This, in turn, means that a criterion must be established for the ill-defined, intuitively understood term

This issue is of special importance in light of our previous study that demonstrated that no single index can be used as a universal criterion, indicating that there are several types of oxidative stress (Dotan et al., 2004). Hence, the question remains which criterion (or criteria) can be used to identify who is likely to benefit from antioxidant supplementation. The answer to this question can, theoretically, be based either on a criterion for the relevant type of oxidative stress (e.g. lipid peroxidation, as assessed by the concentrations of MDA or isoprostanes) or/and on diagnosis of specific diseases for which there is sufficient evidence

In the current study, we present the results of a meta-analysis of case-control studies used to assess the association between CVD and oxidative stress, as evaluated on the basis of different criteria. Unlike previous meta-analyses, we analyzed the association between CVD and criteria for each of the types of OS. We hope that eventually this analysis will enable us to define threshold values of relevant indices of the relevant type of oxidative stress for

This work was performed according to the guidelines outlined by the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group (Stroup et al., 2000). A detailed

**1. Introduction** 

"oxidative stress".

**2. Methods** 

for benefit of antioxidant supplementation.

treatment with antioxidants (Deeks, 2001; Deeks & Altman, 2004).

Yedidya Dotan, Dov Lichtenberg and Ilya Pinchuk

### **Are CVD Patients Under Oxidative Stress?**

Yedidya Dotan, Dov Lichtenberg and Ilya Pinchuk

*Dept. of Physiology and Pharmacology, Sackler Medical School, Tel Aviv University Israel* 

#### **1. Introduction**

Oxidative stress has long been associated with cardiovascular disease (CVD) (Abuja & Albertini, 2001; Halliwell & Gutteridge, 1990; Parthasarathy et al., 2001; Steinbrecher et al., 1984). It was even assumed that the prevalence of CVD alone indicates the prevalence of oxidative stress (Witztum, 1994). Moreover, cross-sectional studies indicated that supplementation of low molecular weight antioxidants is associated with a relatively low incidence of CVD (Jha et al., 1995). By contrast, in most of the interventional studies the antioxidant supplementation did not prevent the progression of CVD nor did it improve any of the many clinical endpoints (Shekelle et al., 2004; Vivekananthan et al., 2003; Miller 2005; Bjelakovic, 2007; Dotan, 2009a; Dotan, 2009b). Based on these findings, Witztum (Witztum, 1998) and Morrow (Morrow, 2003) hypothesized that only individuals under oxidative stress may benefit from antioxidant supplementation. This, of course, implies that only people under oxidative stress should be treated with antioxidants. This, in turn, means that a criterion must be established for the ill-defined, intuitively understood term "oxidative stress".

This issue is of special importance in light of our previous study that demonstrated that no single index can be used as a universal criterion, indicating that there are several types of oxidative stress (Dotan et al., 2004). Hence, the question remains which criterion (or criteria) can be used to identify who is likely to benefit from antioxidant supplementation. The answer to this question can, theoretically, be based either on a criterion for the relevant type of oxidative stress (e.g. lipid peroxidation, as assessed by the concentrations of MDA or isoprostanes) or/and on diagnosis of specific diseases for which there is sufficient evidence for benefit of antioxidant supplementation.

In the current study, we present the results of a meta-analysis of case-control studies used to assess the association between CVD and oxidative stress, as evaluated on the basis of different criteria. Unlike previous meta-analyses, we analyzed the association between CVD and criteria for each of the types of OS. We hope that eventually this analysis will enable us to define threshold values of relevant indices of the relevant type of oxidative stress for treatment with antioxidants (Deeks, 2001; Deeks & Altman, 2004).

#### **2. Methods**

This work was performed according to the guidelines outlined by the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group (Stroup et al., 2000). A detailed

Are CVD Patients Under Oxidative Stress? 415

SMD, which reflects both the mean difference and the standard deviation. Our study required the use of SMD for two reasons: (i) it allows pooling of results obtained from different studies and (ii) its use compensates, at least partially, for the use of different methods, different units of measurement and inter-lab differences (Deeks et al., 2001). We implemented SMD by using "Hedges' adjusted g" because it contains a correction for small

Fig. 1. An outline of the flow of studies in the meta-analyses. We identified many

publications that contained the required sets of keywords (e.g. [CVD or CHD] and oxidative

To pool our results, we have considered two possible models, namely the "fixed effect model", which assumes that all the studies and trials are the repeating of the same experiment (Lau et al., 1998) or the "random effects model" of DerSimonian and Laird, which assumes that the clinical trials estimate a different, yet related, variables with a common distribution (Higgins et al., 2002; Lau et al., 1998). From these two models, we chose to use the random effects model, because we expected heterogeneous results from observational studies (Egger et al., 1998). In our view, the "fixed effect model" oversimplifies the problem and its accuracy has been questioned by others in the same context (Lau et al., 1998), particularly when there was a need to pool results of

In our analyses of the data, we relate not only to the mean value of the pooled variables. We also used the collected data to assess the credibility of the results as previously proposed by Smith et al. (Smith et al., 1997). Assessment of heterogeneity in a meta-analysis of observational studies is as important as the mean results (Egger et al., 1998; Egger et al., 1997a). We assessed the heterogeneity in terms of the following indices as proposed by Song

sample bias (Bennett et al., 2004).

stress).

heterogeneous populations (Lau et al., 1998).

description of the data retrieval process, the selection criteria and the analyses of the data is given in the following subsections.

#### **2.1 Retrieval and selection of studies to be included in the analysis**

First, we had to define criteria to be used in our search for clinical trials to be included in our meta-analysis (Fig. 1). The following criteria were defined for our search:


The latter demand requires further definition of the two populations that can be regarded as being "CVD patients" and their respective controls. We included in the control group only "pathology-free" individuals. To be included in the group of "CVD patients", a person had to be diagnosed with one of the following conditions: (i) either stable angina pectoris (STP) or unstable angina pectoris (UTP), diagnosed either by angiogram or by physical examination, (ii) patients suffering from any of the stages of coronary stenosis and (iii) a recorded history of CVD (e.g. myocardial infraction).

With these criteria in mind, we searched through two major databases, namely, the Medline database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed) and the Thomson Reuters ISI Web of Science. Reference mining was conducted to find an arbitrarily predefined number of studies (20) that fit all the above selection criteria. We defined a list of keywords to search and conducted a simple keyword search, similar to that described by Shekelle et al. (Shekelle et al., 2004).

Data of case-control studies may be biased towards the prevailing paradigm and the findings of other recent trials (French et al., 2005; Moher & Tsertsvadze, 2006). Such bias must be considered when conducting a search for studies to be included in meta-analyses because it may cause a change in the significance of the pooled variables (French et al., 2005; Moher & Tsertsvadze, 2006). This is of special importance in the present meta-analysis because the prevailing paradigm changed from viewing "oxidative stress" as a cause of atherosclerosis (Witztum, 1994) to viewing it as being merely a result of questionable significance (Witztum & Steinberg, 2001). In addition, we had to assess the completeness of the retrieved data and its validity with respect to the complete body of published data (Bennett et al., 2004). Towards this end, we have devised an algorithm similar to the "capture-recapture technique" proposed by Spoor et al. (Spoor et al., 1996). Spoor et al. used different methods of data retrieval to assess their results. We used, for the same purpose, different time frames. Briefly, to achieve as random as possible subset of studies (Furukawa et al., 2002), we searched the Medline database to identify studies published between 1.1.1990 and 1.8.2003 and used studies dating from 1.8.2003 until 1.2.2006 to reassert our findings, as proposed by Moher & Tsertsvadze (Moher & Tsertsvadze, 2006). By that, we have minimized the bias towards recent publications.

#### **2.2 Data analysis**

The difference between individuals with prevalent CVD and their controls, as observed in each of the selected studies, was expressed in terms of two factors: (i) the standard mean difference (SMD), which is a composite index, and (ii) the 95% confidence interval of the

description of the data retrieval process, the selection criteria and the analyses of the data is

First, we had to define criteria to be used in our search for clinical trials to be included in our

ii. Methods: for a clinical study to be included in our meta-analysis it had to use only generally accepted methods for the evaluation of oxidative stress (Dotan et al., 2004). iii. Size: to be included in our meta-analysis, the study had to involve at least 20 CVD

The latter demand requires further definition of the two populations that can be regarded as being "CVD patients" and their respective controls. We included in the control group only "pathology-free" individuals. To be included in the group of "CVD patients", a person had to be diagnosed with one of the following conditions: (i) either stable angina pectoris (STP) or unstable angina pectoris (UTP), diagnosed either by angiogram or by physical examination, (ii) patients suffering from any of the stages of coronary stenosis and (iii) a

With these criteria in mind, we searched through two major databases, namely, the Medline database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=pubmed) and the Thomson Reuters ISI Web of Science. Reference mining was conducted to find an arbitrarily predefined number of studies (20) that fit all the above selection criteria. We defined a list of keywords to search and conducted a simple keyword search, similar to that

Data of case-control studies may be biased towards the prevailing paradigm and the findings of other recent trials (French et al., 2005; Moher & Tsertsvadze, 2006). Such bias must be considered when conducting a search for studies to be included in meta-analyses because it may cause a change in the significance of the pooled variables (French et al., 2005; Moher & Tsertsvadze, 2006). This is of special importance in the present meta-analysis because the prevailing paradigm changed from viewing "oxidative stress" as a cause of atherosclerosis (Witztum, 1994) to viewing it as being merely a result of questionable significance (Witztum & Steinberg, 2001). In addition, we had to assess the completeness of the retrieved data and its validity with respect to the complete body of published data (Bennett et al., 2004). Towards this end, we have devised an algorithm similar to the "capture-recapture technique" proposed by Spoor et al. (Spoor et al., 1996). Spoor et al. used different methods of data retrieval to assess their results. We used, for the same purpose, different time frames. Briefly, to achieve as random as possible subset of studies (Furukawa et al., 2002), we searched the Medline database to identify studies published between 1.1.1990 and 1.8.2003 and used studies dating from 1.8.2003 until 1.2.2006 to reassert our findings, as proposed by Moher & Tsertsvadze (Moher & Tsertsvadze, 2006). By that, we

The difference between individuals with prevalent CVD and their controls, as observed in each of the selected studies, was expressed in terms of two factors: (i) the standard mean difference (SMD), which is a composite index, and (ii) the 95% confidence interval of the

patients and at least 20 "CVD-free" individuals, serving as a control group.

**2.1 Retrieval and selection of studies to be included in the analysis** 

meta-analysis (Fig. 1). The following criteria were defined for our search: i. Design: only case-control or nested case control studies were included.

recorded history of CVD (e.g. myocardial infraction).

described by Shekelle et al. (Shekelle et al., 2004).

have minimized the bias towards recent publications.

**2.2 Data analysis** 

given in the following subsections.

SMD, which reflects both the mean difference and the standard deviation. Our study required the use of SMD for two reasons: (i) it allows pooling of results obtained from different studies and (ii) its use compensates, at least partially, for the use of different methods, different units of measurement and inter-lab differences (Deeks et al., 2001). We implemented SMD by using "Hedges' adjusted g" because it contains a correction for small sample bias (Bennett et al., 2004).

Fig. 1. An outline of the flow of studies in the meta-analyses. We identified many publications that contained the required sets of keywords (e.g. [CVD or CHD] and oxidative stress).

To pool our results, we have considered two possible models, namely the "fixed effect model", which assumes that all the studies and trials are the repeating of the same experiment (Lau et al., 1998) or the "random effects model" of DerSimonian and Laird, which assumes that the clinical trials estimate a different, yet related, variables with a common distribution (Higgins et al., 2002; Lau et al., 1998). From these two models, we chose to use the random effects model, because we expected heterogeneous results from observational studies (Egger et al., 1998). In our view, the "fixed effect model" oversimplifies the problem and its accuracy has been questioned by others in the same context (Lau et al., 1998), particularly when there was a need to pool results of heterogeneous populations (Lau et al., 1998).

In our analyses of the data, we relate not only to the mean value of the pooled variables. We also used the collected data to assess the credibility of the results as previously proposed by Smith et al. (Smith et al., 1997). Assessment of heterogeneity in a meta-analysis of observational studies is as important as the mean results (Egger et al., 1998; Egger et al., 1997a). We assessed the heterogeneity in terms of the following indices as proposed by Song

Are CVD Patients Under Oxidative Stress? 417

McMurray et al., 1992; Schisterman et al., 2002; Singh et al., 1995; Tamer et al., 2002; Turgan

Fig. 2. Indices of oxidative stress in cardiovascular disease. We present both graphically and numerically the pooled standardized mean difference (SMD) and the 95% confidence interval. Also given is the heterogeneity, as defined by χ²/df + 1, of the studies regarding each index. Note that MDA is the only accepted index of oxidative stress that shows a mean

Given the prevailing paradigm regarding the association between atherosclerosis and lipid peroxidation (Witztum & Steinberg, 2001), it is not surprising that MDA was the most frequently used index utilized to assess oxidative stress (13/20 studies). Furthermore, only MDA exhibited a strong effect size, given by a SMD of 1.60 (0.75 to 2.45). Using the "Trim and Fill" method (Duval & Tweedie, 2000) revealed that at least three studies were missing from the funnel plot. Adjusting for the latter finding resulted in a much lower SMD of 0.71 (0.37 to 1.05). The "Egger method" (Egger et al., 1997b) identified an intercept that was significant and positive, supporting the conclusion that the previously observed differences

We performed a mixed stepwise regression for MDA concentrations, factoring in age, gender, smoking habits, severity of CVD, prevalence of hypertension and diabetes mellitus as well as the use of NSAIDs. This analysis indicated that the inclusion of smokers in both CVD patients and control groups (estimate1 = -1.3, p =0.04), results in an underestimation of the association between CVD and oxidative stress. By contrast, the inclusion of patients with both acute (severe CVD) and chronic (mild CVD) coronary syndromes (estimate =3.1,

We stratified the results from those studies that assessed MDA concentrations into three groups: (i) patients with unstable angina pectoris (UAP), (ii) patients with stable angina

<sup>1</sup>**Estimate:** A numerical value obtained from a statistical sample and assigned to a population

p=0.01), results in an overestimation of the latter associations.

et al., 1999; Weinbrenner et al., 2003).

difference greater the 1SMD.

were overestimated.

parameter.

(Song et al., 2001): (i) χ² (Deeks et al., 2001), (ii) *i*² (used by the RevMan 4.2.8 software) and (iii) log(χ²/DF+1), where DF is the number of degrees of freedom (Deeks et al., 2001), for comparing the degree of heterogeneity between heterogeneous results.

To detect variables that affect our models, we used stratification, clustering of associated variables and meta-regression models (Smith et al., 1997; Song et al., 2001). Yet, unlike in the case of clinical trials, we analyzed either the complete trial or a specific subgroup within the trial (Lau et al., 1998). We also analyzed the correlations between indices of oxidative stress and various stages of CVD, to determine causality (Egger et al., 1998; Mulrow, 1994; Smith et al., 1997). To assess the effects of exposure variables on the outcome, we used stratification for common risk-related variables such as age (in subgroups), gender and history of smoking (Smith et al., 1997; Sterne, et al., 2001). We also used stratification for variables specific to the pathology (e.g. stable and unstable angina pectoris in CVD), as proposed by Smith et al (Smith et al., 1997). We used meta-regression (logistic and multiple linear) to model the increase in continuous and ordinal SMD, as well as the annual event rates (Lau et al., 1998). We used the observed SMD as our dependent variable and the covariates of interest (independent variables) to assess the sources of heterogeneity (Lau et al., 1998). A mixed stepwise regression was used to detect trends and factors affecting SMD of MDA concentration in CVD (Lau et al., 1998).

To detect (and assess) the degree of bias in our meta-analyses, we have used both the "Trim and Fill method" (Duval & Tweedie , 2000) and a simple funnel plots (Sutton et al., 2000). A funnel plot is a regression of each trial's effect size against a measure of its size (e.g. 1/standard error) (Sutton et al., 2000). An asymmetry in a forest plot is attributed to the high probability that smaller studies with less statistical power are not published. Asymmetric publications were "trimmed" and then the number of studies missing was calculated. We used a simulation to fill the missing studies as described by Duval & Tweedie (Duval & Tweedie, 2000), thus verifying our initial assumption. We used additional methods to assess the relations between study size and results, mainly because publication-bias is not the only reason for asymmetry in funnel plots, and because this method may be subjective and have a relatively high false-positive rate of detecting bias (Sterne et al., 2001). We compensated for the subjective nature of graphical assessment by using the rank correlation method (Thornton and Lee, 2000) and an adjustment of the "Egger's method" (Egger et al., 1997b). Rank correlation is the statistical analogue of the funnel plot, namely a regression of the effect size (SMD) against both sizes of study and 1/SE (Thornton and Lee, 2000). We used an adjustment of the "Egger's method" to detect publication bias and its direction. We defined the standard normal deviate (SND) as the mean difference, divided by its standard error regressed against the estimate's precision (Bennett et al., 2004). We defined the threshold pvalue for the intercept at a value of 0.1 to detect possible bias (Bennett et al., 2004).

#### **3. Results**

As depicted in Fig. 1, we reviewed 172 studies, of which 20 were selected for analysis. These chosen 20 studies compared 1068 CVD patients to 2128 matched controls, using 15 common indices to assess oxidative stress. A summary of the results is given in Fig. 2 (Akkus et al., 1996; Cavalca et al., 2001; Chiu et al., 1994; Cipollone et al., 2000; Clejan et al., 2002; Delanty et al., 1997; Durak et al., 2001; Ferns et al., 2000; Gackowski et al., 2001; Haidari et al., 2001; Halevy et al., 1997; Karmansky et al., 1996; Kesavulu et al., 2001; Kostner et al., 1997;

416 Atherogenesis

(Song et al., 2001): (i) χ² (Deeks et al., 2001), (ii) *i*² (used by the RevMan 4.2.8 software) and (iii) log(χ²/DF+1), where DF is the number of degrees of freedom (Deeks et al., 2001), for

To detect variables that affect our models, we used stratification, clustering of associated variables and meta-regression models (Smith et al., 1997; Song et al., 2001). Yet, unlike in the case of clinical trials, we analyzed either the complete trial or a specific subgroup within the trial (Lau et al., 1998). We also analyzed the correlations between indices of oxidative stress and various stages of CVD, to determine causality (Egger et al., 1998; Mulrow, 1994; Smith et al., 1997). To assess the effects of exposure variables on the outcome, we used stratification for common risk-related variables such as age (in subgroups), gender and history of smoking (Smith et al., 1997; Sterne, et al., 2001). We also used stratification for variables specific to the pathology (e.g. stable and unstable angina pectoris in CVD), as proposed by Smith et al (Smith et al., 1997). We used meta-regression (logistic and multiple linear) to model the increase in continuous and ordinal SMD, as well as the annual event rates (Lau et al., 1998). We used the observed SMD as our dependent variable and the covariates of interest (independent variables) to assess the sources of heterogeneity (Lau et al., 1998). A mixed stepwise regression was used to detect trends and factors affecting SMD of MDA

To detect (and assess) the degree of bias in our meta-analyses, we have used both the "Trim and Fill method" (Duval & Tweedie , 2000) and a simple funnel plots (Sutton et al., 2000). A funnel plot is a regression of each trial's effect size against a measure of its size (e.g. 1/standard error) (Sutton et al., 2000). An asymmetry in a forest plot is attributed to the high probability that smaller studies with less statistical power are not published. Asymmetric publications were "trimmed" and then the number of studies missing was calculated. We used a simulation to fill the missing studies as described by Duval & Tweedie (Duval & Tweedie, 2000), thus verifying our initial assumption. We used additional methods to assess the relations between study size and results, mainly because publication-bias is not the only reason for asymmetry in funnel plots, and because this method may be subjective and have a relatively high false-positive rate of detecting bias (Sterne et al., 2001). We compensated for the subjective nature of graphical assessment by using the rank correlation method (Thornton and Lee, 2000) and an adjustment of the "Egger's method" (Egger et al., 1997b). Rank correlation is the statistical analogue of the funnel plot, namely a regression of the effect size (SMD) against both sizes of study and 1/SE (Thornton and Lee, 2000). We used an adjustment of the "Egger's method" to detect publication bias and its direction. We defined the standard normal deviate (SND) as the mean difference, divided by its standard error regressed against the estimate's precision (Bennett et al., 2004). We defined the threshold p-

value for the intercept at a value of 0.1 to detect possible bias (Bennett et al., 2004).

As depicted in Fig. 1, we reviewed 172 studies, of which 20 were selected for analysis. These chosen 20 studies compared 1068 CVD patients to 2128 matched controls, using 15 common indices to assess oxidative stress. A summary of the results is given in Fig. 2 (Akkus et al., 1996; Cavalca et al., 2001; Chiu et al., 1994; Cipollone et al., 2000; Clejan et al., 2002; Delanty et al., 1997; Durak et al., 2001; Ferns et al., 2000; Gackowski et al., 2001; Haidari et al., 2001; Halevy et al., 1997; Karmansky et al., 1996; Kesavulu et al., 2001; Kostner et al., 1997;

comparing the degree of heterogeneity between heterogeneous results.

concentration in CVD (Lau et al., 1998).

**3. Results** 


McMurray et al., 1992; Schisterman et al., 2002; Singh et al., 1995; Tamer et al., 2002; Turgan et al., 1999; Weinbrenner et al., 2003).

Fig. 2. Indices of oxidative stress in cardiovascular disease. We present both graphically and numerically the pooled standardized mean difference (SMD) and the 95% confidence interval. Also given is the heterogeneity, as defined by χ²/df + 1, of the studies regarding each index. Note that MDA is the only accepted index of oxidative stress that shows a mean difference greater the 1SMD.

Given the prevailing paradigm regarding the association between atherosclerosis and lipid peroxidation (Witztum & Steinberg, 2001), it is not surprising that MDA was the most frequently used index utilized to assess oxidative stress (13/20 studies). Furthermore, only MDA exhibited a strong effect size, given by a SMD of 1.60 (0.75 to 2.45). Using the "Trim and Fill" method (Duval & Tweedie, 2000) revealed that at least three studies were missing from the funnel plot. Adjusting for the latter finding resulted in a much lower SMD of 0.71 (0.37 to 1.05). The "Egger method" (Egger et al., 1997b) identified an intercept that was significant and positive, supporting the conclusion that the previously observed differences were overestimated.

We performed a mixed stepwise regression for MDA concentrations, factoring in age, gender, smoking habits, severity of CVD, prevalence of hypertension and diabetes mellitus as well as the use of NSAIDs. This analysis indicated that the inclusion of smokers in both CVD patients and control groups (estimate1 = -1.3, p =0.04), results in an underestimation of the association between CVD and oxidative stress. By contrast, the inclusion of patients with both acute (severe CVD) and chronic (mild CVD) coronary syndromes (estimate =3.1, p=0.01), results in an overestimation of the latter associations.

We stratified the results from those studies that assessed MDA concentrations into three groups: (i) patients with unstable angina pectoris (UAP), (ii) patients with stable angina

<sup>1</sup>**Estimate:** A numerical value obtained from a statistical sample and assigned to a population parameter.

Are CVD Patients Under Oxidative Stress? 419

Unfortunately, those studies that used either DNA damage or total antioxidant capacity (TEAC) to assess oxidative stress in patients with CVD in comparison to healthy controls

Based on many lines of indirect evidence, oxidative stress has long been associated with CVD. The following findings have been considered to lend support to the oxidative theory of CVD: (i) oxidized LDL may cause formation of foam cell in-vitro (Chisolm & Steinberg, 2000), (ii) the development of atherosclerosis is preceded by an increase of the levels of many indicators of oxidative stress in lab animals (Chisolm & Steinberg, 2000) and (iii) the incidence of CVD in individuals with low concentrations of antioxidants is relatively high (Chisolm and Steinberg, 2000). These (and other) findings and clinical trials led researchers to two assumptions: (i) oxidative stress plays a pivotal role in the formation of atherogenic plaque and (ii) individuals with prevalent CVD are likely to be under oxidative stress and therefore have high plasma

The first assumption has long been disputed and is currently under scrutiny (Williams & Fisher, 2005). The second assumption is weakened by the results of our current metaanalysis, which shows that the prevalence of CVD is only slightly associated with OS, as defined on the basis MDA concentration. By contrast, no evidence is available for association between CVD and OS, as determined on the basis of all other indices of oxidative stress. Specifically, the concentrations of almost all the micronutrients in the plasma of CVD patients are within normal ranges (Fig. 2), the activities of relevant enzymes did not differ from those observed in matched controls (Fig. 2). The same results were observed for most of the indices of lipid peroxidation. The only index that is significantly different with the prevalence of CVD is the plasma concentration of MDA. Even if we choose to ignore the reservations regarding the use of MDA as an index of lipid peroxidation (Draper et al., 1993), the possibility of publication bias should not be ignored. Hence, we think that the existing evidence for association between oxidative stress and the prevalence of CVD is quite weak. In other words, it appears that the role of oxidative stress in atherogenesis has been overestimated. In accordance with this conclusion, is the viewpoint of the recent review, regarding association of several more OS indices (circulation levels of oxidized LDL and myeloperoxidase) with CVD (Strobel et al., 2011). The authors conclude that "results of studies using Ox-LDL have been equivocal" and that "the ability of

The latter considerations accord with the current trend in cardiovascular disease research, which views atherosclerosis as mostly an inflammatory disease (Ross, 1999) and implies that oxidative stress is a result and not the cause for atherosclerosis. This trend also accords with and therefore is strengthened by two of our findings: (i) we observed that patients with UAP, a condition commonly associated with acute inflammation, are under higher "oxidative stress", as assessed by the serum concentration of MDA, than both patients with SAP and matched controls (Fig. 3) and (ii) we observed that studies including more acute conditions are under higher "oxidative stress" than matched

In our opinion, these findings are of little relevance to the possibility that many individuals

(Fig. 2) were too few and too small to enable any conclusions.

concentrations of lipid peroxidation products and loss of LMWA.

oxidative stress biomarkers to predict CVD has yet to be established".

**4. Discussion** 

controls (Fig. 4).

may benefit from antioxidant supplementation.

pectoris (SAP) and (iii) healthy controls. As seen in Fig. 3, the MDA concentrations of patients with SAP are not different from matched controls, whereas UAP patients have significantly higher MDA concentrations than both healthy controls and patients with SAP.

We also stratified the results from two types of studies, those that had acute coronary conditions (MI, UAP) and those CVD patients who had chronic coronary conditions (SAP and occlusions). As seen in Fig. 4, patients with acute coronary conditions had marked and significantly higher MDA concentrations (SMD = 2.30, 0.93 to 3.67) than matched controls. By contrast, patients with chronic coronary conditions had only slightly higher MDA concentrations (SMD = 0.60, 0.18 to 1.01) as compared with matched controls.

The concentrations of beta-carotene, as evaluated by Singh et al. (Singh et al., 1995), were significantly lower in CVD patients than in matched controls. The difference between the concentrations all other low molecular weight antioxidants (LMWA) in patients with CVD and healthy controls were not statistically significant (Fig. 2). In six of the seven studies vitamin E concentration in CVD patients and controls was similar (SMD = 0.02, -0.17 to 0.21).

Significantly lower concentrations were observed only by Singh et al. Furthermore, pooling the results from all the seven studies failed to achieve statistically significant differences (SMD = -0.19, -0.67 to 0.29), although the heterogeneity index substantially increased from 1.35 to 11.51. The differences between the results of Singh et al. and all other studies cannot be attributed either to publication bias or to small-sample bias. A viable possibility is that the difference is due to selection bias, particularly by a biased selection of controls. Indeed, the major difference between the results obtained by Singh et al. and the results observed in all the other studies assessing LMWA was that Singh et al. selected their control group to exclude most, if not all of the CVD-related risk factors (smoking, DM and glucose intolerance), whereas the CVD patients were not devoid of these risk factors.


Fig. 3. A forest plot of studies assessing the differences between patients with stable angina pectoris and patients with unstable angina pectoris (upper panel) and comparison of each of these groups with controls (two lower panels).

Unfortunately, those studies that used either DNA damage or total antioxidant capacity (TEAC) to assess oxidative stress in patients with CVD in comparison to healthy controls (Fig. 2) were too few and too small to enable any conclusions.

#### **4. Discussion**

418 Atherogenesis

pectoris (SAP) and (iii) healthy controls. As seen in Fig. 3, the MDA concentrations of patients with SAP are not different from matched controls, whereas UAP patients have significantly higher MDA concentrations than both healthy controls and patients with SAP. We also stratified the results from two types of studies, those that had acute coronary conditions (MI, UAP) and those CVD patients who had chronic coronary conditions (SAP and occlusions). As seen in Fig. 4, patients with acute coronary conditions had marked and significantly higher MDA concentrations (SMD = 2.30, 0.93 to 3.67) than matched controls. By contrast, patients with chronic coronary conditions had only slightly higher MDA

The concentrations of beta-carotene, as evaluated by Singh et al. (Singh et al., 1995), were significantly lower in CVD patients than in matched controls. The difference between the concentrations all other low molecular weight antioxidants (LMWA) in patients with CVD and healthy controls were not statistically significant (Fig. 2). In six of the seven studies vitamin E

Significantly lower concentrations were observed only by Singh et al. Furthermore, pooling the results from all the seven studies failed to achieve statistically significant differences (SMD = -0.19, -0.67 to 0.29), although the heterogeneity index substantially increased from 1.35 to 11.51. The differences between the results of Singh et al. and all other studies cannot be attributed either to publication bias or to small-sample bias. A viable possibility is that the difference is due to selection bias, particularly by a biased selection of controls. Indeed, the major difference between the results obtained by Singh et al. and the results observed in all the other studies assessing LMWA was that Singh et al. selected their control group to exclude most, if not all of the CVD-related risk factors (smoking, DM and glucose

Fig. 3. A forest plot of studies assessing the differences between patients with stable angina pectoris and patients with unstable angina pectoris (upper panel) and comparison of each of

these groups with controls (two lower panels).

concentrations (SMD = 0.60, 0.18 to 1.01) as compared with matched controls.

concentration in CVD patients and controls was similar (SMD = 0.02, -0.17 to 0.21).

intolerance), whereas the CVD patients were not devoid of these risk factors.

Based on many lines of indirect evidence, oxidative stress has long been associated with CVD. The following findings have been considered to lend support to the oxidative theory of CVD: (i) oxidized LDL may cause formation of foam cell in-vitro (Chisolm & Steinberg, 2000), (ii) the development of atherosclerosis is preceded by an increase of the levels of many indicators of oxidative stress in lab animals (Chisolm & Steinberg, 2000) and (iii) the incidence of CVD in individuals with low concentrations of antioxidants is relatively high (Chisolm and Steinberg, 2000). These (and other) findings and clinical trials led researchers to two assumptions: (i) oxidative stress plays a pivotal role in the formation of atherogenic plaque and (ii) individuals with prevalent CVD are likely to be under oxidative stress and therefore have high plasma concentrations of lipid peroxidation products and loss of LMWA.

The first assumption has long been disputed and is currently under scrutiny (Williams & Fisher, 2005). The second assumption is weakened by the results of our current metaanalysis, which shows that the prevalence of CVD is only slightly associated with OS, as defined on the basis MDA concentration. By contrast, no evidence is available for association between CVD and OS, as determined on the basis of all other indices of oxidative stress. Specifically, the concentrations of almost all the micronutrients in the plasma of CVD patients are within normal ranges (Fig. 2), the activities of relevant enzymes did not differ from those observed in matched controls (Fig. 2). The same results were observed for most of the indices of lipid peroxidation. The only index that is significantly different with the prevalence of CVD is the plasma concentration of MDA. Even if we choose to ignore the reservations regarding the use of MDA as an index of lipid peroxidation (Draper et al., 1993), the possibility of publication bias should not be ignored. Hence, we think that the existing evidence for association between oxidative stress and the prevalence of CVD is quite weak. In other words, it appears that the role of oxidative stress in atherogenesis has been overestimated. In accordance with this conclusion, is the viewpoint of the recent review, regarding association of several more OS indices (circulation levels of oxidized LDL and myeloperoxidase) with CVD (Strobel et al., 2011). The authors conclude that "results of studies using Ox-LDL have been equivocal" and that "the ability of oxidative stress biomarkers to predict CVD has yet to be established".

The latter considerations accord with the current trend in cardiovascular disease research, which views atherosclerosis as mostly an inflammatory disease (Ross, 1999) and implies that oxidative stress is a result and not the cause for atherosclerosis. This trend also accords with and therefore is strengthened by two of our findings: (i) we observed that patients with UAP, a condition commonly associated with acute inflammation, are under higher "oxidative stress", as assessed by the serum concentration of MDA, than both patients with SAP and matched controls (Fig. 3) and (ii) we observed that studies including more acute conditions are under higher "oxidative stress" than matched controls (Fig. 4).

In our opinion, these findings are of little relevance to the possibility that many individuals may benefit from antioxidant supplementation.

Are CVD Patients Under Oxidative Stress? 421

association is questionable due to (i) poor reliability of the laboratory assay of MDA and (ii)

Most of the clinical trials that were designed to reduce OS by means of antioxidant supplementation yielded disappointing results. The latter results are consistent with the results of our meta-analysis. Hence, oxidative stress is only weakly associated with the

We thank the Lady Davis foundation and Israel Science Foundation (research grant 362/02- 18) for financial support. We would also like to thank Dr. Yariv Gerber and Prof. Uri Goldburt for their sound guidance and Mrs. Ariela Bor for her skillful technical assistance.

Abuja, P.M. and Albertini, R., 2001. Methods for monitoring oxidative stress, lipid peroxidation and oxidation resistance of lipoproteins. Clin.Chim.Acta 306, 1-17. Akkus, I., Saglam, N.I., Caglayan, O., Vural, H., Kalak, S., and Saglam, M., 1996.

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publication bias.

**7. References** 

prevalence of cardiovascular disease.

**6. Acknowledgments** 

Our ongoing research is aimed at developing such an assay (or assays) that can serve as a basis for selective antioxidant supplementation. Our working hypothesis is a two step approach of identifying those individuals that may benefit from antioxidant supplementation. The first step is an initial, short-term treatment with vitamin E. The second step should be to assess plasma concentrations of lipid peroxidation products and to continue treatment with vitamin E only to individuals, responding to treatment by reducing significantly plasma concentration of lipid peroxidation products. In conclusion, we have no evidence that justify indiscriminate supplementation of vitamin E, nor do we have sufficient evidence to ban it, as recommended by the authors of the Cache County Study (Hayden et al., 2007). At present we have no assay that can be used to identify patients that are likely to benefit from Vitamin E supplementation.


Fig. 4. A forest plot comparing two strata of studies. The first stratum compares the differences between patients with either stable angina pectoris or mild occlusions of the coronary arteries and healthy controls. The second stratum compares the differences between patients with either unstable angina pectoris or MI and healthy controls. Notably, while the first stratum of patients with SAP varies slightly from healthy controls, the second stratum of patients with UAP shows a significant difference. The two strata differ significantly (p = 0.02).

#### **5. Conclusion**

Our meta-analysis shows that the commonly accepted paradigm regarding the role of OS in the pathogenesis of CVD appears to be overestimated. CVD is associated with OS only when the evaluation of OS is based on plasma concentrations of MDA. Notably, even this association is questionable due to (i) poor reliability of the laboratory assay of MDA and (ii) publication bias.

Most of the clinical trials that were designed to reduce OS by means of antioxidant supplementation yielded disappointing results. The latter results are consistent with the results of our meta-analysis. Hence, oxidative stress is only weakly associated with the prevalence of cardiovascular disease.

#### **6. Acknowledgments**

We thank the Lady Davis foundation and Israel Science Foundation (research grant 362/02- 18) for financial support. We would also like to thank Dr. Yariv Gerber and Prof. Uri Goldburt for their sound guidance and Mrs. Ariela Bor for her skillful technical assistance.

#### **7. References**

420 Atherogenesis

Our ongoing research is aimed at developing such an assay (or assays) that can serve as a basis for selective antioxidant supplementation. Our working hypothesis is a two step approach of identifying those individuals that may benefit from antioxidant supplementation. The first step is an initial, short-term treatment with vitamin E. The second step should be to assess plasma concentrations of lipid peroxidation products and to continue treatment with vitamin E only to individuals, responding to treatment by reducing significantly plasma concentration of lipid peroxidation products. In conclusion, we have no evidence that justify indiscriminate supplementation of vitamin E, nor do we have sufficient evidence to ban it, as recommended by the authors of the Cache County Study (Hayden et al., 2007). At present we have no assay that can be used to identify patients that are likely to

Fig. 4. A forest plot comparing two strata of studies. The first stratum compares the differences between patients with either stable angina pectoris or mild occlusions of the coronary arteries and healthy controls. The second stratum compares the differences between patients with either unstable angina pectoris or MI and healthy controls. Notably, while the first stratum of patients with SAP varies slightly from healthy controls, the second

stratum of patients with UAP shows a significant difference. The two strata differ

Our meta-analysis shows that the commonly accepted paradigm regarding the role of OS in the pathogenesis of CVD appears to be overestimated. CVD is associated with OS only when the evaluation of OS is based on plasma concentrations of MDA. Notably, even this

benefit from Vitamin E supplementation.

significantly (p = 0.02).

**5. Conclusion** 


Are CVD Patients Under Oxidative Stress? 423

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**20** 

Adrian Manea1,2

*Romania* 

**Vascular Biology of Reactive Oxygen Species** 

**and NADPH Oxidases: Role in Atherogenesis** 

Eukaryotic cells face constantly the formation of reactive oxygen species (ROS) as a result of their aerobic metabolism. ROS play an important role in the regulation of signal transduction pathways and gene expression but its over-production is acutely harmful to cells, particularly in cardiovascular diseases (CVD) by a mechanism that is not fully understood. Most CVD (the leading cause of mortality in developed countries) entail the focal development of atherosclerotic plaques in response to various deleterious insults that affect the artery wall's cells (Simionescu, 2007). Atheroma may occlude partially or totally the arterial lumen and ultimately, rupture of the vulnerable plaques results in thrombus formation and obstruction of the vessels of vital organs like heart, brain, lung, and kidney. Atheroma formation is characterized by progressive lipid accumulation in the vessel's intima, dysfunctions of endothelial cells (EC) and smooth muscle cells (SMC), and a strong inflammatory reaction with the participation of extravasated immune cells (Fearon & Faux, 2009). Compelling evidence (including ours) revealed that oxidative stress and NADPH oxidase - derived ROS play the key role in all stages of atherosclerosis and that genetic ablation of various oxidase components protects the cells against the detrimental effects of oxidative stress (Simionescu et al., 2009). Therefore, understanding the molecular mechanisms of ROS formation and function is a prerequisite of an effective anti-oxidative

As the name indicates, ROS are a class of highly reactive molecules derived from chemical conversion of molecular oxygen (O2). ROS are formed in all the aerobic cells and organisms as by-products of metabolic and respiration processes, under the influence of ionizing radiation or produced deliberately by specialized enzyme systems. ROS formation is initiated by reduction of O2 with one electron leading to the formation of short-lived and

interaction with various converting enzymes gives rise to a large spectrum of molecules with diverse physicochemical characteristics such as H2O2 and HO•. The dismutation of

•- to H2O2 can be either spontaneous or catalyzed by specialized enzymes namely, members of the superoxide dismutase family. H2O2 may be completely reduced to H2O by

**2. Reactive oxygen species formation in the vasculature** 

highly reactive superoxide anion (O2

**1. Introduction** 

stress therapy.

O2

*2"Nicolae Simionescu" Institute of Cellular Biology and Pathology, Bucharest* 

*1"Petru Poni"Institute of Macromolecular Chemistry, Iasi* 

•-). Successive reduction of O2•-, protonation or


### **Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis**

Adrian Manea1,2

*1"Petru Poni"Institute of Macromolecular Chemistry, Iasi 2"Nicolae Simionescu" Institute of Cellular Biology and Pathology, Bucharest Romania* 

#### **1. Introduction**

424 Atherogenesis

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201.

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randomised trials. Lancet 361, 2017-2023.

disease. Atherosclerosis 168, 99-106.

Eukaryotic cells face constantly the formation of reactive oxygen species (ROS) as a result of their aerobic metabolism. ROS play an important role in the regulation of signal transduction pathways and gene expression but its over-production is acutely harmful to cells, particularly in cardiovascular diseases (CVD) by a mechanism that is not fully understood. Most CVD (the leading cause of mortality in developed countries) entail the focal development of atherosclerotic plaques in response to various deleterious insults that affect the artery wall's cells (Simionescu, 2007). Atheroma may occlude partially or totally the arterial lumen and ultimately, rupture of the vulnerable plaques results in thrombus formation and obstruction of the vessels of vital organs like heart, brain, lung, and kidney. Atheroma formation is characterized by progressive lipid accumulation in the vessel's intima, dysfunctions of endothelial cells (EC) and smooth muscle cells (SMC), and a strong inflammatory reaction with the participation of extravasated immune cells (Fearon & Faux, 2009). Compelling evidence (including ours) revealed that oxidative stress and NADPH oxidase - derived ROS play the key role in all stages of atherosclerosis and that genetic ablation of various oxidase components protects the cells against the detrimental effects of oxidative stress (Simionescu et al., 2009). Therefore, understanding the molecular mechanisms of ROS formation and function is a prerequisite of an effective anti-oxidative stress therapy.

#### **2. Reactive oxygen species formation in the vasculature**

As the name indicates, ROS are a class of highly reactive molecules derived from chemical conversion of molecular oxygen (O2). ROS are formed in all the aerobic cells and organisms as by-products of metabolic and respiration processes, under the influence of ionizing radiation or produced deliberately by specialized enzyme systems. ROS formation is initiated by reduction of O2 with one electron leading to the formation of short-lived and highly reactive superoxide anion (O2•-). Successive reduction of O2 •-, protonation or interaction with various converting enzymes gives rise to a large spectrum of molecules with diverse physicochemical characteristics such as H2O2 and HO•. The dismutation of O2 •- to H2O2 can be either spontaneous or catalyzed by specialized enzymes namely, members of the superoxide dismutase family. H2O2 may be completely reduced to H2O by

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 427

transcription factors, peptides, ion channels and transporters, lipids, carbohydrates, and other oxygen-based species, a process that influence dramatically the cell behavior (Shao & Heinecke, 2009). The affinity of ROS for a specific substrate is dictated by both physicochemical features of the reactive oxygen intermediates and also of the targeted molecules. In addition, the occurrence and the abundance of specific functional groups, such as iron-sulfur centers, disulfide-bonds, amino and hydroxyl groups or fatty acids doublebonds, greatly influence the chemical interactions between ROS and redox-sensitive biological molecules. As initially showed in microorganisms, eukaryotic cells respond to

largely antioxidant enzymes and molecules implicated in the preservation of cellular homeostasis, self-renewal, and reparatory processes. In terms of selectivity, O2•- reacts preferentially with the transcription factors and electron transporters in respiratory chains containing iron-sulfur clusters. In contrast, H2O2 reacts mainly with the disulfide-bonds present on the protein kinases/phosphatases, transcription factors, and ion channels. Additional compelling evidence highlights that the redox-regulation of cell function represents an evolutionary conserved mechanism that alter directly or indirectly the

Protein tyrosine phosphatases (PTPs) are probably the best characterized signaling molecules targeted directly by ROS, especially of H2O2, owing to the existence of a highly conserved 230-amino-acid domain that contains reactive cysteine, which catalyzes the hydrolysis of protein phosphotyrosine residues. Consequently, a key mechanism whereby H2O2 controls various cellular processes is determined by the reversible oxidation of PTPs catalytic cysteines that blocks protein dephosphorylation causing transient inhibition of

Notably, several members of the protein tyrosine kinases (PTKs) family, including nonreceptor protein tyrosine kinases (i.e., Src, Jak, Pyk) have been shown to be activated in response to cellular redox variations (Tonks, 2006). Nevertheless, the precise molecular mechanisms of PTKs redox regulation are not entirely understood, and it is not clear if the PTK activities are directly correlated with the alterations induced by ROS action on enzyme structures Still, most of the available date suggests that the majority of the effects are

Another important class of molecules regulated by redox-dependent mechanisms is represented by the mitogen-activated protein kinase (MAPKs) family, that control key physiological processes such as mitosis, differentiation, proliferation, cell survival, and apoptosis. MAPKs are serine/threonine-specific protein kinases which activities are tightly regulated by complex phosphorylation pathways. Emerging evidence demonstrates that in the cardiovascular system, the functions of MAPKs are also influenced by extracellular and intracellular ROS by yet incompletely defined mechanisms (Wu et al., 2008). Apparently, the upstream regulators of MAPKs, namely MAPK kinases (MEKs), PTKs, and PTPs, might be the actual molecular targets of ROS and the genuine sensors of the intracellular redox state

Similar to PTPs, MAPK phosphatases (MKPs) display a highly conserved redox-sensitive cysteine in their catalytic core. Thus, the oxidative inhibition of MKPs may results in the persistent activation of MAPKs, as observed in various developmental or pathological states. Taken together, redox-dependent and as well as redox-independent activation of MAPKs cascades congregate to activate downstream signaling pathway in response to

attributable to PTP inhibition by ROS rather than PTK oxidation (Tabet et al., 2008).

activities of a large spectrum of signaling molecules (Liu et al., 2005).

•- and H2O2 by the up-regulation of various gene products,

increased generation of O2

changes (Sedeek et al., 2009).

PTPs.

means of various peroxidases such as catalase and glutathione peroxidase or partially reduced to HO•, one of the most powerful oxidizing agent identified in biological systems. The generation of HO• is mediated by various free transition metal ions (e.g., Fe2+, Cu2+) via the Haber-Weiss reaction (Manea, 2010).

Apart from the aforementioned chemical processing of O2, superoxide can react with other molecular species including nitrogen species such as nitric oxide (NO) or polyunsaturated fatty acids. The reaction between O2•- and NO is tightly controlled by the rate of diffusion of both radicals, and result in the formation of ONOO- a potent oxidant. Alternative reactions may led to the generation of mixed reactive oxygen and nitrogen radicals such as nitrogen dioxide radical (•NO2) and nitryl chloride (NO2Cl) (Turrens, 2003).

Lipid peroxidation products formation represents an important mechanism whereby ROS elicit physiological and pathophysiological function in the living cells. ROS (especially HO•, •NO2, and ONOO-) may react with polyunsaturated fatty acids present on biological membranes or circulating/infiltrated lipoproteins, a condition that facilitate the formation of fatty acid peroxyl radical (R-COO-) that can further attack adjacent fatty acid chains and trigger the production of other lipid radicals by a chain reaction mechanism (Negre-Salvayre et al., 2010; Riahi et al., 2010; Shao & Heinecke, 2009).

Tyrosyl radicals produced by myeloperoxidase (MPO) have also been shown to be involved in the initiation of lipid peroxidation (Hazen et al., 1997). *In vitro* studies revealed that lipid peroxidation occurred only in the presence of free L-tyrosine suggesting that tyrosyl radicals formation by MPO are essential mediators for the initiation of lipid peroxidation and subsequent LDL oxidation by activated human neutrophils, which contain abundant MPO and H2O2 (Savenkova et al., 1994). Tyrosyl radicals have also been shown to play a role in LDL oxidation *in vivo* and in atherogenesis. Analysis of LDL isolated from human vascular tissue demonstrated that *o,o'*-dityrosine levels were 100 times greater than that observed in circulating LDL. Similarly, *o,o'*-dityrosine formation was found to be robust increased in atherosclerotic fatty streaks and in advanced atheromas compared to normal aortic tissue, indicating that tyrosyl radical formation was capable of protein damage *in vivo*  (Leeuwenburgh et al., 1997).

#### **3. Molecular targets of ROS**

The biological function of ROS is highly regulated by their basic physicochemical properties, cellular compartmentalization and the formation rate. Since O<sup>2</sup> •- is a short-lived charged species, it cannot diffuse through biological membranes and acts closeness of the formation site. Nevertheless, an anion channel-dependent plasma membrane transport mechanisms has been demonstrated to play an important role in mediating cell-to-cell communication. Notably, O<sup>2</sup> •- is water-soluble and functions either as an oxidizing agent (e.g., one-electron reduction of O2•- yields H2O2) or as a reducing agent (e.g., ONOO- formation). HO• is extremely reactive and does not diffuse more that a few molecular diameters from its site of formation (Touyz, 2003). In contrast, H2O2 is highly stable under physiological conditions. Being an uncharged molecule, H2O2 is membrane-permeable and able of activating downstream signalling molecules relatively far from the site of formation.

At low, physiological concentration, ROS modulate key signalling processes initiated by hormones, cytokines, vasoactive agents, blood coagulation factors, and hemodynamic shear stress. Reactive oxygen intermediates react at near-diffusion rate and influence the activity of numerous of signalling molecules including receptors, protein kinases/phosphatases,

means of various peroxidases such as catalase and glutathione peroxidase or partially reduced to HO•, one of the most powerful oxidizing agent identified in biological systems. The generation of HO• is mediated by various free transition metal ions (e.g., Fe2+, Cu2+) via

Apart from the aforementioned chemical processing of O2, superoxide can react with other molecular species including nitrogen species such as nitric oxide (NO) or polyunsaturated fatty acids. The reaction between O2•- and NO is tightly controlled by the rate of diffusion of both radicals, and result in the formation of ONOO- a potent oxidant. Alternative reactions may led to the generation of mixed reactive oxygen and nitrogen radicals such as

Lipid peroxidation products formation represents an important mechanism whereby ROS elicit physiological and pathophysiological function in the living cells. ROS (especially HO•, •NO2, and ONOO-) may react with polyunsaturated fatty acids present on biological membranes or circulating/infiltrated lipoproteins, a condition that facilitate the formation of fatty acid peroxyl radical (R-COO-) that can further attack adjacent fatty acid chains and trigger the production of other lipid radicals by a chain reaction mechanism (Negre-Salvayre

Tyrosyl radicals produced by myeloperoxidase (MPO) have also been shown to be involved in the initiation of lipid peroxidation (Hazen et al., 1997). *In vitro* studies revealed that lipid peroxidation occurred only in the presence of free L-tyrosine suggesting that tyrosyl radicals formation by MPO are essential mediators for the initiation of lipid peroxidation and subsequent LDL oxidation by activated human neutrophils, which contain abundant MPO and H2O2 (Savenkova et al., 1994). Tyrosyl radicals have also been shown to play a role in LDL oxidation *in vivo* and in atherogenesis. Analysis of LDL isolated from human vascular tissue demonstrated that *o,o'*-dityrosine levels were 100 times greater than that observed in circulating LDL. Similarly, *o,o'*-dityrosine formation was found to be robust increased in atherosclerotic fatty streaks and in advanced atheromas compared to normal aortic tissue, indicating that tyrosyl radical formation was capable of protein damage *in vivo* 

The biological function of ROS is highly regulated by their basic physicochemical properties, cellular compartmentalization and the formation rate. Since O2•- is a short-lived charged species, it cannot diffuse through biological membranes and acts closeness of the formation site. Nevertheless, an anion channel-dependent plasma membrane transport mechanisms has been demonstrated to play an important role in mediating cell-to-cell communication.

reduction of O2•- yields H2O2) or as a reducing agent (e.g., ONOO- formation). HO• is extremely reactive and does not diffuse more that a few molecular diameters from its site of formation (Touyz, 2003). In contrast, H2O2 is highly stable under physiological conditions. Being an uncharged molecule, H2O2 is membrane-permeable and able of activating

At low, physiological concentration, ROS modulate key signalling processes initiated by hormones, cytokines, vasoactive agents, blood coagulation factors, and hemodynamic shear stress. Reactive oxygen intermediates react at near-diffusion rate and influence the activity of numerous of signalling molecules including receptors, protein kinases/phosphatases,

downstream signalling molecules relatively far from the site of formation.

•- is water-soluble and functions either as an oxidizing agent (e.g., one-electron

nitrogen dioxide radical (•NO2) and nitryl chloride (NO2Cl) (Turrens, 2003).

the Haber-Weiss reaction (Manea, 2010).

et al., 2010; Riahi et al., 2010; Shao & Heinecke, 2009).

(Leeuwenburgh et al., 1997).

Notably, O<sup>2</sup>

**3. Molecular targets of ROS** 

transcription factors, peptides, ion channels and transporters, lipids, carbohydrates, and other oxygen-based species, a process that influence dramatically the cell behavior (Shao & Heinecke, 2009). The affinity of ROS for a specific substrate is dictated by both physicochemical features of the reactive oxygen intermediates and also of the targeted molecules. In addition, the occurrence and the abundance of specific functional groups, such as iron-sulfur centers, disulfide-bonds, amino and hydroxyl groups or fatty acids doublebonds, greatly influence the chemical interactions between ROS and redox-sensitive biological molecules. As initially showed in microorganisms, eukaryotic cells respond to increased generation of O2•- and H2O2 by the up-regulation of various gene products, largely antioxidant enzymes and molecules implicated in the preservation of cellular homeostasis, self-renewal, and reparatory processes. In terms of selectivity, O2•- reacts preferentially with the transcription factors and electron transporters in respiratory chains containing iron-sulfur clusters. In contrast, H2O2 reacts mainly with the disulfide-bonds present on the protein kinases/phosphatases, transcription factors, and ion channels. Additional compelling evidence highlights that the redox-regulation of cell function represents an evolutionary conserved mechanism that alter directly or indirectly the activities of a large spectrum of signaling molecules (Liu et al., 2005).

Protein tyrosine phosphatases (PTPs) are probably the best characterized signaling molecules targeted directly by ROS, especially of H2O2, owing to the existence of a highly conserved 230-amino-acid domain that contains reactive cysteine, which catalyzes the hydrolysis of protein phosphotyrosine residues. Consequently, a key mechanism whereby H2O2 controls various cellular processes is determined by the reversible oxidation of PTPs catalytic cysteines that blocks protein dephosphorylation causing transient inhibition of PTPs.

Notably, several members of the protein tyrosine kinases (PTKs) family, including nonreceptor protein tyrosine kinases (i.e., Src, Jak, Pyk) have been shown to be activated in response to cellular redox variations (Tonks, 2006). Nevertheless, the precise molecular mechanisms of PTKs redox regulation are not entirely understood, and it is not clear if the PTK activities are directly correlated with the alterations induced by ROS action on enzyme structures Still, most of the available date suggests that the majority of the effects are attributable to PTP inhibition by ROS rather than PTK oxidation (Tabet et al., 2008).

Another important class of molecules regulated by redox-dependent mechanisms is represented by the mitogen-activated protein kinase (MAPKs) family, that control key physiological processes such as mitosis, differentiation, proliferation, cell survival, and apoptosis. MAPKs are serine/threonine-specific protein kinases which activities are tightly regulated by complex phosphorylation pathways. Emerging evidence demonstrates that in the cardiovascular system, the functions of MAPKs are also influenced by extracellular and intracellular ROS by yet incompletely defined mechanisms (Wu et al., 2008). Apparently, the upstream regulators of MAPKs, namely MAPK kinases (MEKs), PTKs, and PTPs, might be the actual molecular targets of ROS and the genuine sensors of the intracellular redox state changes (Sedeek et al., 2009).

Similar to PTPs, MAPK phosphatases (MKPs) display a highly conserved redox-sensitive cysteine in their catalytic core. Thus, the oxidative inhibition of MKPs may results in the persistent activation of MAPKs, as observed in various developmental or pathological states. Taken together, redox-dependent and as well as redox-independent activation of MAPKs cascades congregate to activate downstream signaling pathway in response to

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 429

while GPx3 is particularly abundant in the plasma. GPx4 has as substrates lipid hydroperoxides and is present at a low level in nearly every cell type. Thioredoxins (TRx) and glutaredoxins (GRx) are proteins that function as antioxidants by enabling the reduction of other proteins by cysteine thioldisulfide exchange. Apart from being involved in antioxidant defense, different isoforms of the TRx and GRx families have been shown to play an important role in regulation of gene expression by redox-dependent processes. Peroxiredoxins (PRx) represent a ubiquitous family of antioxidant enzymes whose activities are tightly regulated by phosphorylation cascades and by changes in the redox and oligomerization states. PRx controls intracellular peroxide levels and mediate signal

Physiological production of ROS contribute to the preservation of vascular homeostasis by regulating important biological processes such as cell growth, proliferation, differentiation, apoptosis, cytoskeletal organization, and cell migration. Still, in the last few decades, it has become apparent that overproduction of ROS correlated with alterations of the antioxidant system, vascular inflammation and metabolic dysfunction are key pathological initiators of cardiovascular disorders. Generated in excess, ROS react randomly with all of biological molecules inducing the irreversible alterations of DNA, proteins, carbohydrates, and lipids components, thus altering cell functions (Martinet et al., 2001). As a result, extensive studies have concentrated on the role of oxidative stress-induced cellular dysfunction, redox control of vascular response to inflammatory and metabolic insults, the molecular mechanisms of ROS generation and the means that this class of molecules contributes to vascular damage. Oxidative stress represents a pathological condition characterized by the incapacity of antioxidant mechanisms to neutralize the deleterious effects of ROS and their metabolites. The means of oxidative stress onset and progression in vascular pathological states, include the overproduction of ROS, changes in the endogenous antioxidant system, and the production of various oxygen intermediates such as ONOO- and HO• that cannot be efficiently buffered by the naturally occurring antioxidant mechanisms. In addition, spatial and temporal co-expression and co-localization of various enzymatic and non-enzymatic ROS-producing sources at the site of vascular insults may potentially exacerbate predispose

transduction in cardiovascular cells (Woo et al., 2010).

**5. Role of oxidative stress in atherogenesis** 

to vascular insults and dysfunction (Kondo et al., 2009; Lee et al., 2009).

hypochlorous acid (HOCl).

The importance of oxidative stress in onset and development of atherosclerosis is commonly accepted (Fearon & Faux, 2009). Still, numerous clinical trials failed to demonstrate that the antioxidant therapy improve the health of patients with cardiovascular diseases (Yusuf et al., 2000). Consequently, many questions arise relative to our current knowledge of the molecular processes implicated in ROS formation and action. Hitherto, different pharmacological approaches have been employed to counteract oxidative stress-induced injury in the cardiovascular system i.e. antioxidant supplements containing vitamins C and E, polyphenols or selective inhibitors of distinct sources of ROS (Olukman et al., 2010). Nevertheless, these pharmacological interventions have many disadvantages such as inadequate concentration of active compounds at the site of ROS formation, or vitamins themselves becoming radicals with pro-oxidant activity or not being effective scavengers for various reactive oxygen/nitrogen intermediates, namely hydrogen peroxide (H2O2), peroxynitrite anion (ONOO-), hydroxyl anion (HO•), and

hormones, growth factors, pro-inflammatory mediators, and vasoactive agents. Besides MAPKs, the activity of serine/threonine protein kinases Akt and Rho has been indicated to be redox-sensitive and to play a central role in cellular survival pathways (Lee & Griendling, 2008). Apart from protein kinases/phosphatases and transcription factors, ROS are important regulators calcium homeostasis, by mechanisms that engages reversible thiol oxidation of the cysteine residues present on ion channels and transporters. In the vascular cells ROS, particularly O2 •- and H2O2, also enhance intracellular Ca2+ concentrations by increasing the extracellular influx through the plasma membrane channels and mobilization from intracellular stores, and by the inhibition of Ca2+-ATPases located in the plasma membrane and endoplasmic reticulum. The plasma membrane K+ channels have been shown to be redox-sensitive, a process that mediates hyperpolarization-dependent vascular relaxation (Belia et al., 2009; Briones &Touyz, 2010). These data indicates that the redox status of ion channels and transporters plays an essential role in cell physiology and represents an important determinant of vascular pathology under conditions of the altered production of ROS.

#### **4. Antioxidant mechanisms in the cardiovascular cells**

ROS are physiologically produced at low concentration during metabolic processes in nonphagocytic cells, by the mitochondrial respiratory chain, cyclooxygenases, lipoxygesases, cytochrome P450 reductase, xanthine oxidase.

Almost three decades ago, the commonly accepted assumption was that the antioxidant system has developed to defend the cells against the damaging and unavoidable effects of ROS, which are capable to produce irreversible, structural, and functional oxidative damage of DNA, proteins, lipids, and carbohydrates. This theory was supported by many experimental evidence regarding the strategic tissular distribution, expression/concentration levels, and localization of the antioxidants within cellular compartments. Nevertheless, soon after the discovery of enzyme systems that deliberately generates ROS (e.g., NADPH oxidases) under physiological and pathological states, it has become apparent that ROS are not just the by-products of aerobic metabolism, but also important signalling molecules (Forman et al., 2010; Go & Jones 2010). Therefore, the subtle relationship among oxidizing and reducing agents permits ROS to function as second messengers and to regulate various cellular functions. Thus, besides neutralization of ROS, the antioxidant system has emerged as a critical regulator of the redox-sensitive processes.

The concentration of various oxygen-based reactive intermediates is maintained in physiological range by a very complex antioxidant system comprising both enzymatic ROS scavengers, namely superoxide dismutase, catalase, glutathione peroxidase, thioredoxin, glutaredoxin, peroxiredoxin, heme oxygenase, and paraoxonase, and non-enzymatic ROS quenchers, such as glutathione, vitamins, lipoate, urate, and ubiquinone (Zadák et al., 2009). Superoxide dismutases (SOD) represent a family of enzymes that catalyze the dismutation of O2•- into O2 and H2O2. Three SOD isoforms are expressed concomitantly in different cellular compartments, including the cytosol (SOD1; Cu/ZnSOD), mitochondria (SOD2; MnSOD), and the extracellular space (SOD3; ecSOD) (Valdivia et al., 2009). Catalase (CAT) is found in peroxisomes where it decomposes H2O2 to H2O and O2. Glutathione peroxidases (GPx) represent a family of isoenzymes encoded by separate genes that differ in cellular distribution pattern and substrate specificity. GPx1, the most abundant isoform, is expressed in the cytosol and has H2O2 as its main substrate. GPx2 is an extracellular space enzyme, while GPx3 is particularly abundant in the plasma. GPx4 has as substrates lipid hydroperoxides and is present at a low level in nearly every cell type. Thioredoxins (TRx) and glutaredoxins (GRx) are proteins that function as antioxidants by enabling the reduction of other proteins by cysteine thioldisulfide exchange. Apart from being involved in antioxidant defense, different isoforms of the TRx and GRx families have been shown to play an important role in regulation of gene expression by redox-dependent processes.

Peroxiredoxins (PRx) represent a ubiquitous family of antioxidant enzymes whose activities are tightly regulated by phosphorylation cascades and by changes in the redox and oligomerization states. PRx controls intracellular peroxide levels and mediate signal transduction in cardiovascular cells (Woo et al., 2010).

#### **5. Role of oxidative stress in atherogenesis**

428 Atherogenesis

hormones, growth factors, pro-inflammatory mediators, and vasoactive agents. Besides MAPKs, the activity of serine/threonine protein kinases Akt and Rho has been indicated to be redox-sensitive and to play a central role in cellular survival pathways (Lee & Griendling, 2008). Apart from protein kinases/phosphatases and transcription factors, ROS are important regulators calcium homeostasis, by mechanisms that engages reversible thiol oxidation of the cysteine residues present on ion channels and transporters. In the vascular cells ROS, particularly O2•- and H2O2, also enhance intracellular Ca2+ concentrations by increasing the extracellular influx through the plasma membrane channels and mobilization from intracellular stores, and by the inhibition of Ca2+-ATPases located in the plasma membrane and endoplasmic reticulum. The plasma membrane K+ channels have been shown to be redox-sensitive, a process that mediates hyperpolarization-dependent vascular relaxation (Belia et al., 2009; Briones &Touyz, 2010). These data indicates that the redox status of ion channels and transporters plays an essential role in cell physiology and represents an important determinant of vascular pathology under conditions of the altered

ROS are physiologically produced at low concentration during metabolic processes in nonphagocytic cells, by the mitochondrial respiratory chain, cyclooxygenases, lipoxygesases,

Almost three decades ago, the commonly accepted assumption was that the antioxidant system has developed to defend the cells against the damaging and unavoidable effects of ROS, which are capable to produce irreversible, structural, and functional oxidative damage of DNA, proteins, lipids, and carbohydrates. This theory was supported by many experimental evidence regarding the strategic tissular distribution, expression/concentration levels, and localization of the antioxidants within cellular compartments. Nevertheless, soon after the discovery of enzyme systems that deliberately generates ROS (e.g., NADPH oxidases) under physiological and pathological states, it has become apparent that ROS are not just the by-products of aerobic metabolism, but also important signalling molecules (Forman et al., 2010; Go & Jones 2010). Therefore, the subtle relationship among oxidizing and reducing agents permits ROS to function as second messengers and to regulate various cellular functions. Thus, besides neutralization of ROS, the antioxidant system has emerged as a critical regulator of the redox-sensitive processes. The concentration of various oxygen-based reactive intermediates is maintained in physiological range by a very complex antioxidant system comprising both enzymatic ROS scavengers, namely superoxide dismutase, catalase, glutathione peroxidase, thioredoxin, glutaredoxin, peroxiredoxin, heme oxygenase, and paraoxonase, and non-enzymatic ROS quenchers, such as glutathione, vitamins, lipoate, urate, and ubiquinone (Zadák et al., 2009). Superoxide dismutases (SOD) represent a family of enzymes that catalyze the dismutation

•- into O2 and H2O2. Three SOD isoforms are expressed concomitantly in different cellular compartments, including the cytosol (SOD1; Cu/ZnSOD), mitochondria (SOD2; MnSOD), and the extracellular space (SOD3; ecSOD) (Valdivia et al., 2009). Catalase (CAT) is found in peroxisomes where it decomposes H2O2 to H2O and O2. Glutathione peroxidases (GPx) represent a family of isoenzymes encoded by separate genes that differ in cellular distribution pattern and substrate specificity. GPx1, the most abundant isoform, is expressed in the cytosol and has H2O2 as its main substrate. GPx2 is an extracellular space enzyme,

**4. Antioxidant mechanisms in the cardiovascular cells** 

cytochrome P450 reductase, xanthine oxidase.

production of ROS.

of O2

Physiological production of ROS contribute to the preservation of vascular homeostasis by regulating important biological processes such as cell growth, proliferation, differentiation, apoptosis, cytoskeletal organization, and cell migration. Still, in the last few decades, it has become apparent that overproduction of ROS correlated with alterations of the antioxidant system, vascular inflammation and metabolic dysfunction are key pathological initiators of cardiovascular disorders. Generated in excess, ROS react randomly with all of biological molecules inducing the irreversible alterations of DNA, proteins, carbohydrates, and lipids components, thus altering cell functions (Martinet et al., 2001). As a result, extensive studies have concentrated on the role of oxidative stress-induced cellular dysfunction, redox control of vascular response to inflammatory and metabolic insults, the molecular mechanisms of ROS generation and the means that this class of molecules contributes to vascular damage.

Oxidative stress represents a pathological condition characterized by the incapacity of antioxidant mechanisms to neutralize the deleterious effects of ROS and their metabolites. The means of oxidative stress onset and progression in vascular pathological states, include the overproduction of ROS, changes in the endogenous antioxidant system, and the production of various oxygen intermediates such as ONOO- and HO• that cannot be efficiently buffered by the naturally occurring antioxidant mechanisms. In addition, spatial and temporal co-expression and co-localization of various enzymatic and non-enzymatic ROS-producing sources at the site of vascular insults may potentially exacerbate predispose to vascular insults and dysfunction (Kondo et al., 2009; Lee et al., 2009).

The importance of oxidative stress in onset and development of atherosclerosis is commonly accepted (Fearon & Faux, 2009). Still, numerous clinical trials failed to demonstrate that the antioxidant therapy improve the health of patients with cardiovascular diseases (Yusuf et al., 2000). Consequently, many questions arise relative to our current knowledge of the molecular processes implicated in ROS formation and action. Hitherto, different pharmacological approaches have been employed to counteract oxidative stress-induced injury in the cardiovascular system i.e. antioxidant supplements containing vitamins C and E, polyphenols or selective inhibitors of distinct sources of ROS (Olukman et al., 2010). Nevertheless, these pharmacological interventions have many disadvantages such as inadequate concentration of active compounds at the site of ROS formation, or vitamins themselves becoming radicals with pro-oxidant activity or not being effective scavengers for various reactive oxygen/nitrogen intermediates, namely hydrogen peroxide (H2O2), peroxynitrite anion (ONOO-), hydroxyl anion (HO•), and hypochlorous acid (HOCl).

2009).

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 431

result, the Nox-derived O2•- reacts with NO thus producing ONOO-, an extremely cytotoxic chemical species which directly affect and oxidize biological molecules in invading microorganisms, resulting in molecular alteration and microbial death (El-Benna et al., 2007). The phagocyte-type Nox consists of five subunits: a membrane-associated cytochrome b558, comprising a heavily glycosylated 91-kDa protein (gp91phox; Nox2) and nonglycosylated 22-kDa subunit (p22phox), and three cytosolic regulatory components, p40phox, p47phox*,* and p67phox. Besides "Phox" components, assembly of Nox in an active complex requires the contribution of a low-molecular-weight GTP-binding protein, Rac1/2 or Rap 1A. In latent cells, the Nox complex is dissociated but is rapidly assembled and activated following the exposure to pathogens or inflammatory mediators. Serine phosphorylation of p47phox represents the limiting step of Nox activation and triggers complex formation of cytosolic subunits followed by translocation to the membrane and association with cytochrome b558 (Hoyal et al., 2003; Lassègue & Griendling 2002; Li & Shah 2003;). Other than Nox2, macrophages also express Nox1 and Nox4 as inducible isoforms that, reportedly mediate LDL oxidation in the vascular wall (Lee et al., 2009; Maitra et al.,

The expression of functionally active Nox subtypes has been reported in non-phagocytes, including cardiovascular cells. Thus far, the members of the Nox enzyme family consists of seven isoforms (Nox1-5, Duox1/2), each with a particular cell and tissue distribution. Nox enzymes are broadly divided into three major categories, as a function of the extra catalytic domains to the phagocyte-type subunit Nox2. The first group includes Nox1, Nox3, and Nox4 isoforms, which display a number of similarities with Nox2, for instance their structural organization and molecular weight. Besides Nox2-type catalytic core, Nox5, the second group of the Nox family, possess an extra amino-terminal calmodulin-like domain that contains four Ca2+-binding EF-hands structures (Lambeth, 2007). Thus far, four splice variants of Nox5, namely Nox5α, Nox5β, Nox5γ, and Nox5δ, have been identified in humans. In particular, the *Nox5* gene is not present in the rodents' genome. A third class of of Nox is represented by the Nox5-like dual oxidases (Duox) which possess, in addition to the Nox5-type structure, an extracellular peroxidase domain that uses the H2O2 generated by its Nox catalytic core. For their function, all the Nox1-4 subtypes necessitate the p22phox component, while Nox5 and Duox are activated directly by calcium. As shown in aortic SMCs, activation of Nox1 requires the participation of a ClC-3 anion transporter. The anion transporter co-expresses with Nox1 in early endosomes and is required for charge neutralization of the electron flow generated by Nox1 across the membrane of signalling vesicles (Miller et al., 2007). Nox4 is constitutively active and its activity is supported by the association with p22phox, required for the electron transfer, and polymerase delta interacting-protein 2 (Polidp2), that apparently may serve to stabilize the enzymatic complex (Lyle et al., 2009). The activities of Nox1, Nox2, and Nox3 isoforms are highly controlled by phosphorylation reactions involving regulatory subunits that initiate the assembly of Nox into an active enzymatic complex. Other than p40phox, p47phox, and p67phox cytosolic regulatory components, two different structurally related proteins have been discovered in non-phagocyte, specifically Nox organizer 1 (Noxo1), which is an analog of p47phox, and Nox activator 1 (Noxa1), which is an analog of p67phox. Despite the structural similarities, dissimilar functional aspects are involved in the regulation of enzyme activity. For instance, different to p47phox, which in the resting cells is located in the cytosol, Noxo1 is pre-localized at the membrane jointly with Nox1 and p22phox (Lambeth,

Excessive ROS formation in atherogenesis triggers a chain of critical events such as EC dysfunction, oxidation of macromolecules especially LDL and extracellular matrix constituents, phenotypic alterations of SMC and macrophage/SMC-derived foam cell and modulate the function of signalling molecules in fibroblasts, which promotes inflammation of vascular adventitia (Sima et al., 2009). Vascular resident cells and transvasated immune cells are important sources of ROS within the atheroma (Heistad et al., 2009). These particularities show that atherosclerosis represents a multifactorial vascular disorder characterized by complex interactions and cross talk between the resident cells of the vascular wall, the cells of the immune system and the factors they produce.

As shown in various animal models of atherosclerosis, oxidative stress is a primary occurrence and a key contributor to endothelial dysfunction portrayed by diminished endothelial NO bioavailability, enhanced endothelial transcytosis, up-regulation of proinflammatory molecules, and the alteration of EC fibrinolytic activity (Dejana et al., 2009; Vendrov et al., 2007). In addition, oxidation of macromolecules especially of LDL (oxLDL) plays a key role in all stages of atherogenesis such as fatty streak formation, development of complex lesion, and plaque rupture. Of particular importance is that oxidative stress contributes, at least in part in the modulation of SMC phenotype switching and ultimately contributes to artery wall thickening. In atherosclerosis, SMCs undergo hypertrophy, produce excess extracellular matrix and inflammatory cytokines, proliferate and migrate from the media towards the vessel's intima.

Clinical evidence highlights that oxidative stress is a characteristic feature of many pathological conditions that predispose to atherosclerotic lesion formation such as hypercholesterolemia, hypertension, and diabetes. However, the precise pathological mechanisms accountable for the installation of oxidative stress are still an unsettled subject. In this context, although not completely validated in humans, oxidative stress may not be the sole causative effect of atherosclerosis and one has to consider the diversity of enzymatic and non-enzymatic sources of ROS, their vascular distribution pattern and subcellular compartmentalization, and complex regulation during various stages of the disease progression (Förstermann, 2008).

#### **6. Vascular sources of ROS: Role of NADPH oxidases**

Various pathways of ROS generation that can potentially contribute to oxidative stress have been described in the cardiovascular system including non-enzymatic decomposition of various compounds and metabolites (e.g., glucose autoxidation), production of ROS as byproducts of cellular respiration and metabolism (i.e., mitochondrial respiratory chain, lipo-/cyclooxygenases, dysfunctional nitric oxide (NO) synthases, cytochrome P450 reductases, xanthine oxidase), lysosomal enzymes or generated in a highly regulated manner by specialized enzymes (e.g., NADPH oxidases) (Gu et al., 2001; Harrison et al., 2003; Madamanchi et al., 2005; Martinez-Hervas et al., 2010; Zalba et al., 2007).

NADPH oxidases (Nox) represent a family of multi-component enzymes, whose unique biological function is the production of ROS both in physiological and pathological states (Lambeth, 2004). Nox was originally identified and characterized as being a "burst" enzyme in professional phagocytes such as neutrophils and macrophages. In phagocytes, in cooperation with MPO, Nox plays a major role in host defense process against invading pathogens through the production of toxic hypochlorous acid (HOCl), a highly reactive oxidant. During phagocytosis, macrophages also generate significant amounts of NO. As a

Excessive ROS formation in atherogenesis triggers a chain of critical events such as EC dysfunction, oxidation of macromolecules especially LDL and extracellular matrix constituents, phenotypic alterations of SMC and macrophage/SMC-derived foam cell and modulate the function of signalling molecules in fibroblasts, which promotes inflammation of vascular adventitia (Sima et al., 2009). Vascular resident cells and transvasated immune cells are important sources of ROS within the atheroma (Heistad et al., 2009). These particularities show that atherosclerosis represents a multifactorial vascular disorder characterized by complex interactions and cross talk between the resident cells of the

As shown in various animal models of atherosclerosis, oxidative stress is a primary occurrence and a key contributor to endothelial dysfunction portrayed by diminished endothelial NO bioavailability, enhanced endothelial transcytosis, up-regulation of proinflammatory molecules, and the alteration of EC fibrinolytic activity (Dejana et al., 2009; Vendrov et al., 2007). In addition, oxidation of macromolecules especially of LDL (oxLDL) plays a key role in all stages of atherogenesis such as fatty streak formation, development of complex lesion, and plaque rupture. Of particular importance is that oxidative stress contributes, at least in part in the modulation of SMC phenotype switching and ultimately contributes to artery wall thickening. In atherosclerosis, SMCs undergo hypertrophy, produce excess extracellular matrix and inflammatory cytokines, proliferate and migrate

Clinical evidence highlights that oxidative stress is a characteristic feature of many pathological conditions that predispose to atherosclerotic lesion formation such as hypercholesterolemia, hypertension, and diabetes. However, the precise pathological mechanisms accountable for the installation of oxidative stress are still an unsettled subject. In this context, although not completely validated in humans, oxidative stress may not be the sole causative effect of atherosclerosis and one has to consider the diversity of enzymatic and non-enzymatic sources of ROS, their vascular distribution pattern and subcellular compartmentalization, and complex regulation during various stages of the disease

Various pathways of ROS generation that can potentially contribute to oxidative stress have been described in the cardiovascular system including non-enzymatic decomposition of various compounds and metabolites (e.g., glucose autoxidation), production of ROS as byproducts of cellular respiration and metabolism (i.e., mitochondrial respiratory chain, lipo-/cyclooxygenases, dysfunctional nitric oxide (NO) synthases, cytochrome P450 reductases, xanthine oxidase), lysosomal enzymes or generated in a highly regulated manner by specialized enzymes (e.g., NADPH oxidases) (Gu et al., 2001; Harrison et al.,

NADPH oxidases (Nox) represent a family of multi-component enzymes, whose unique biological function is the production of ROS both in physiological and pathological states (Lambeth, 2004). Nox was originally identified and characterized as being a "burst" enzyme in professional phagocytes such as neutrophils and macrophages. In phagocytes, in cooperation with MPO, Nox plays a major role in host defense process against invading pathogens through the production of toxic hypochlorous acid (HOCl), a highly reactive oxidant. During phagocytosis, macrophages also generate significant amounts of NO. As a

2003; Madamanchi et al., 2005; Martinez-Hervas et al., 2010; Zalba et al., 2007).

vascular wall, the cells of the immune system and the factors they produce.

from the media towards the vessel's intima.

**6. Vascular sources of ROS: Role of NADPH oxidases** 

progression (Förstermann, 2008).

result, the Nox-derived O2•- reacts with NO thus producing ONOO-, an extremely cytotoxic chemical species which directly affect and oxidize biological molecules in invading microorganisms, resulting in molecular alteration and microbial death (El-Benna et al., 2007). The phagocyte-type Nox consists of five subunits: a membrane-associated cytochrome b558, comprising a heavily glycosylated 91-kDa protein (gp91phox; Nox2) and nonglycosylated 22-kDa subunit (p22phox), and three cytosolic regulatory components, p40phox, p47phox*,* and p67phox. Besides "Phox" components, assembly of Nox in an active complex requires the contribution of a low-molecular-weight GTP-binding protein, Rac1/2 or Rap 1A. In latent cells, the Nox complex is dissociated but is rapidly assembled and activated following the exposure to pathogens or inflammatory mediators. Serine phosphorylation of p47phox represents the limiting step of Nox activation and triggers complex formation of cytosolic subunits followed by translocation to the membrane and association with cytochrome b558 (Hoyal et al., 2003; Lassègue & Griendling 2002; Li & Shah 2003;). Other than Nox2, macrophages also express Nox1 and Nox4 as inducible isoforms that, reportedly mediate LDL oxidation in the vascular wall (Lee et al., 2009; Maitra et al., 2009).

The expression of functionally active Nox subtypes has been reported in non-phagocytes, including cardiovascular cells. Thus far, the members of the Nox enzyme family consists of seven isoforms (Nox1-5, Duox1/2), each with a particular cell and tissue distribution. Nox enzymes are broadly divided into three major categories, as a function of the extra catalytic domains to the phagocyte-type subunit Nox2. The first group includes Nox1, Nox3, and Nox4 isoforms, which display a number of similarities with Nox2, for instance their structural organization and molecular weight. Besides Nox2-type catalytic core, Nox5, the second group of the Nox family, possess an extra amino-terminal calmodulin-like domain that contains four Ca2+-binding EF-hands structures (Lambeth, 2007). Thus far, four splice variants of Nox5, namely Nox5α, Nox5β, Nox5γ, and Nox5δ, have been identified in humans. In particular, the *Nox5* gene is not present in the rodents' genome. A third class of of Nox is represented by the Nox5-like dual oxidases (Duox) which possess, in addition to the Nox5-type structure, an extracellular peroxidase domain that uses the H2O2 generated by its Nox catalytic core. For their function, all the Nox1-4 subtypes necessitate the p22phox component, while Nox5 and Duox are activated directly by calcium. As shown in aortic SMCs, activation of Nox1 requires the participation of a ClC-3 anion transporter. The anion transporter co-expresses with Nox1 in early endosomes and is required for charge neutralization of the electron flow generated by Nox1 across the membrane of signalling vesicles (Miller et al., 2007). Nox4 is constitutively active and its activity is supported by the association with p22phox, required for the electron transfer, and polymerase delta interacting-protein 2 (Polidp2), that apparently may serve to stabilize the enzymatic complex (Lyle et al., 2009). The activities of Nox1, Nox2, and Nox3 isoforms are highly controlled by phosphorylation reactions involving regulatory subunits that initiate the assembly of Nox into an active enzymatic complex. Other than p40phox, p47phox, and p67phox cytosolic regulatory components, two different structurally related proteins have been discovered in non-phagocyte, specifically Nox organizer 1 (Noxo1), which is an analog of p47phox, and Nox activator 1 (Noxa1), which is an analog of p67phox. Despite the structural similarities, dissimilar functional aspects are involved in the regulation of enzyme activity. For instance, different to p47phox, which in the resting cells is located in the cytosol, Noxo1 is pre-localized at the membrane jointly with Nox1 and p22phox (Lambeth,

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 433

development of atherosclerosis and microvascular diseases (retinopathy, neuropathy, and nephropathy). In addition, Nox1, Nox2, and Nox4 are activated and up-regulated in the

Hyperglycemia, the primary clinical manifestation of diabetes, contributes at least in part to diabetic complications by inducing Nox and the ensuing oxidative stress. Moreover, advanced glycation end-products (AGEs), a direct consequence of the high and persistent blood glucose level, are also important inducers of Nox-derived ROS in vascular cells in diabetes. Besides hyperglycemia, hyperinsulinemia contribute to aberrant ROS production and vascular wall dysfunction. Since Nox is one of the main triggers of oxidative stress, it has a prominent role in the pathology of diabetes-induced vasculopathy (Gao & Mann, 2009). These data make Nox enzymes potential therapeutic targets to counteract the

Hypertension represents a major risk factor for atherosclerosis and its complications and several reports highlight that oxidative stress is both cause and consequence of hypertension (Briones & Touyz, 2010). Nox1 deficiency in mice reduces angiotensin II (Ang II) -dependent blood pressure, media hypertrophy, and extracellular matrix deposition, but not cell proliferation (Gavazzi et al., 2007; Matsuno et al., 2005). In agreement with these data, AngII-treated mice overexpressing Nox1 in vascular smooth muscle cells exhibit an increase of blood pressure, a condition that is associated with medial hypertrophy and significant production of ROS (Dikalova et al., 2005). Furthermore, overexpression of Nox1 in vascular SMCs leads to enhanced responsiveness to Ang II causing up-regulation of ROS, eNOS uncoupling and the consequent decline in NO bioavailability, followed by impaired

Consistent with these reports, compared with wild-type mice, Nox2 ablation (Nox2-/-) diminishes robustly ROS-mediated protein oxidation, neointimal formation, SMCs proliferation and leukocyte accumulation, indicating that Nox2-mediated signalling and

The role of Nox4 and Nox5 enzymes in atherogenesis is less investigated and consequently, not entirely elucidated, since there are few atherosclerosis-related studies conducted on Nox4 deficient mice, and the *Nox5* gene is not present in the rodent's genome. Thus, most of the current data are provided by studies performed *in vitro* on various cell-types and

Recently, a Nox4 deficient mouse model and a cardiomyocyte-targeted Nox4-transgenic model have been developed to investigate the role of Nox4 during cardiac stress. (Zhang et al., 2010). One of the main breakthroughs of this study is that in contrast to the effects of generated by activated Nox1 or Nox2, the up-regulation cardiomyocyte Nox4 results in protection against pressure overload-induced adverse cardiac remodeling. The authors conclude that Nox4 facilitates the maintenance of myocardial capillary density during pressure overload by regulating stress-induced cardiomyocyte hypoxia inducible factor-1 activation and release of vascular endothelial growth factor, resulting in increased paracrine angiogenic activity. In addition, unlike Nox1, Nox2 or Nox5, it seems that Nox4 produces directly H2O2 and thus is incapable of scavenging NO or producing

The beneficial effects of Nox4-derived ROS were also reported by means of a newly developed transgenic mouse with endothelial-specific Nox4 overexpression (Ray et al., 2011). The authors showed that vascular segments and endothelial cells of these animals had

oxidation has a requisite role in the cell response to injury (Chen et al., 2004).

blood vessels of diabetic animals (Ding et al., 2007; Xu et al., 2007).

deleterious effects of ROS in diabetes.

vascular relaxation (Dikalova et al., 2010).

isolated tissues.

ONOO-.

2004). Different subtypes of the Nox enzyme family along with their regulatory proteins are expressed in the cardiovascular cells (i.e., ECs, SMCs, vascular and cardiac fibroblasts, cardiac myocytes, and pericytes), and in circulating immune cells interacting with the blood vessels (i.e., monocytes/macrophages, neutrophils, lymphocytes, platelets, dendritic cells) (Manea et al., 2005).

Nox subtypes are differentially located within the cellular compartments, suggesting a specific correlation between Nox subtypes, subcellular distribution and their specific function to control precise ROS-mediated signal transduction cascades. For instance, Nox1 and Nox2 were detected in caveolae, in the plasma membrane, and endosomes. Nox4 has been detected in focal adhesions, mitochondria endoplasmic reticulum, and the nucleus (Ago et al., 2010; Kuroda & Sadoshima, 2010). Nox5 is present in the perinuclear regions, endoplasmic reticulum, and in the plasma membrane (BelAiba et al., 2007; Fulton, 2009).

#### **7. Involvement of Nox enzymes in atherogenresis**

Studies in cell culture and transgenic/knockout mice provided most of the existing data concerning the role of Nox-dependent oxidative stress in atherosclerosis. Nox activity is upregulated by numerous factors linked to atherosclerotic lesion formation and progression namely, inflammatory cytokines (tumor necrosis factor α, interferon γ), vasoactive agents (angiotensin II, endothelin 1), metabolic factors (high glucose, modified proteins/lipoproteins/lipids, homocysteine), growth factors (platelet-derived growth factor), coagulation factors (thrombin), and pathological shear stress (Chung et al., 2010; Hwang et al., 2003). Apart from direct detrimental effects, compelling data exists that Noxderived ROS interact and stimulate other enzymatic sources of oxygen/nitrogen reactive intermediates, and generally amplify the initial response to insults (Cohena & Tong, 2010; Schrader & Fahimi, 2006).

It is generally accepted that Nox-derived ROS cooperate, and act in concert with other pathological factors leading to vascular inflammation and injury, and that genetic ablation of various Nox subunits (i.e., p47phox, Nox1, Nox2) defends the vascular cells against the harmful effects of oxidative stress. ApoE-/- mice, which develop atherosclerotic lesions that cover the entire range of human lesions (i.e., fatty streaks, intermediate lesions, fibrous plaques, and vulnerable plaques exhibiting necrotic core and intra-plaque hemorrhage) have been extensively used to investigate the role of Nox enzymes in atherogenesis (Nakashima et al., 1994). Using this animal model, it has become evident that enhances in Nox activity and expression occur early in atherogenesis, and hyperactivity of Nox associated with the up-regulation of various isoforms marks all the stages of the plaque formation (Fenyo et al., 2011).

In contrast, ApoE/p47phox double-knockout mice display significantly less atherosclerotic lesions compared with ApoE-/- mice. In the same line, aortic O2 •- levels have been shown to be are lower in p47phox-/- mice than in wild-type mice. In addition, aortic SMCs from p47phox-/- mice exhibit a decreased proliferative response to growth factors compared with that of the SMCs of wild-type mice (Vendrov et al., 2007).

Accelerated atherosclerosis represents a major vascular complication of diabetes mellitus and is responsible for 70-80% of deaths in diabetic patients in developed and developing countries. Numerous reports revealed that the Nox expression and activity are significantly up-regulated in the vasculature of diabetic subjects, and are associated with the

2004). Different subtypes of the Nox enzyme family along with their regulatory proteins are expressed in the cardiovascular cells (i.e., ECs, SMCs, vascular and cardiac fibroblasts, cardiac myocytes, and pericytes), and in circulating immune cells interacting with the blood vessels (i.e., monocytes/macrophages, neutrophils, lymphocytes, platelets, dendritic cells)

Nox subtypes are differentially located within the cellular compartments, suggesting a specific correlation between Nox subtypes, subcellular distribution and their specific function to control precise ROS-mediated signal transduction cascades. For instance, Nox1 and Nox2 were detected in caveolae, in the plasma membrane, and endosomes. Nox4 has been detected in focal adhesions, mitochondria endoplasmic reticulum, and the nucleus (Ago et al., 2010; Kuroda & Sadoshima, 2010). Nox5 is present in the perinuclear regions, endoplasmic reticulum, and in the plasma membrane (BelAiba et al., 2007; Fulton, 2009).

Studies in cell culture and transgenic/knockout mice provided most of the existing data concerning the role of Nox-dependent oxidative stress in atherosclerosis. Nox activity is upregulated by numerous factors linked to atherosclerotic lesion formation and progression namely, inflammatory cytokines (tumor necrosis factor α, interferon γ), vasoactive agents (angiotensin II, endothelin 1), metabolic factors (high glucose, modified proteins/lipoproteins/lipids, homocysteine), growth factors (platelet-derived growth factor), coagulation factors (thrombin), and pathological shear stress (Chung et al., 2010; Hwang et al., 2003). Apart from direct detrimental effects, compelling data exists that Noxderived ROS interact and stimulate other enzymatic sources of oxygen/nitrogen reactive intermediates, and generally amplify the initial response to insults (Cohena & Tong, 2010;

It is generally accepted that Nox-derived ROS cooperate, and act in concert with other pathological factors leading to vascular inflammation and injury, and that genetic ablation of various Nox subunits (i.e., p47phox, Nox1, Nox2) defends the vascular cells against the harmful effects of oxidative stress. ApoE-/- mice, which develop atherosclerotic lesions that cover the entire range of human lesions (i.e., fatty streaks, intermediate lesions, fibrous plaques, and vulnerable plaques exhibiting necrotic core and intra-plaque hemorrhage) have been extensively used to investigate the role of Nox enzymes in atherogenesis (Nakashima et al., 1994). Using this animal model, it has become evident that enhances in Nox activity and expression occur early in atherogenesis, and hyperactivity of Nox associated with the up-regulation of various isoforms marks all the stages of the plaque formation (Fenyo et al.,

In contrast, ApoE/p47phox double-knockout mice display significantly less atherosclerotic lesions compared with ApoE-/- mice. In the same line, aortic O2•- levels have been shown to be are lower in p47phox-/- mice than in wild-type mice. In addition, aortic SMCs from p47phox-/- mice exhibit a decreased proliferative response to growth factors compared with

Accelerated atherosclerosis represents a major vascular complication of diabetes mellitus and is responsible for 70-80% of deaths in diabetic patients in developed and developing countries. Numerous reports revealed that the Nox expression and activity are significantly up-regulated in the vasculature of diabetic subjects, and are associated with the

**7. Involvement of Nox enzymes in atherogenresis** 

that of the SMCs of wild-type mice (Vendrov et al., 2007).

(Manea et al., 2005).

Schrader & Fahimi, 2006).

2011).

development of atherosclerosis and microvascular diseases (retinopathy, neuropathy, and nephropathy). In addition, Nox1, Nox2, and Nox4 are activated and up-regulated in the blood vessels of diabetic animals (Ding et al., 2007; Xu et al., 2007).

Hyperglycemia, the primary clinical manifestation of diabetes, contributes at least in part to diabetic complications by inducing Nox and the ensuing oxidative stress. Moreover, advanced glycation end-products (AGEs), a direct consequence of the high and persistent blood glucose level, are also important inducers of Nox-derived ROS in vascular cells in diabetes. Besides hyperglycemia, hyperinsulinemia contribute to aberrant ROS production and vascular wall dysfunction. Since Nox is one of the main triggers of oxidative stress, it has a prominent role in the pathology of diabetes-induced vasculopathy (Gao & Mann, 2009). These data make Nox enzymes potential therapeutic targets to counteract the deleterious effects of ROS in diabetes.

Hypertension represents a major risk factor for atherosclerosis and its complications and several reports highlight that oxidative stress is both cause and consequence of hypertension (Briones & Touyz, 2010). Nox1 deficiency in mice reduces angiotensin II (Ang II) -dependent blood pressure, media hypertrophy, and extracellular matrix deposition, but not cell proliferation (Gavazzi et al., 2007; Matsuno et al., 2005). In agreement with these data, AngII-treated mice overexpressing Nox1 in vascular smooth muscle cells exhibit an increase of blood pressure, a condition that is associated with medial hypertrophy and significant production of ROS (Dikalova et al., 2005). Furthermore, overexpression of Nox1 in vascular SMCs leads to enhanced responsiveness to Ang II causing up-regulation of ROS, eNOS uncoupling and the consequent decline in NO bioavailability, followed by impaired vascular relaxation (Dikalova et al., 2010).

Consistent with these reports, compared with wild-type mice, Nox2 ablation (Nox2-/-) diminishes robustly ROS-mediated protein oxidation, neointimal formation, SMCs proliferation and leukocyte accumulation, indicating that Nox2-mediated signalling and oxidation has a requisite role in the cell response to injury (Chen et al., 2004).

The role of Nox4 and Nox5 enzymes in atherogenesis is less investigated and consequently, not entirely elucidated, since there are few atherosclerosis-related studies conducted on Nox4 deficient mice, and the *Nox5* gene is not present in the rodent's genome. Thus, most of the current data are provided by studies performed *in vitro* on various cell-types and isolated tissues.

Recently, a Nox4 deficient mouse model and a cardiomyocyte-targeted Nox4-transgenic model have been developed to investigate the role of Nox4 during cardiac stress. (Zhang et al., 2010). One of the main breakthroughs of this study is that in contrast to the effects of generated by activated Nox1 or Nox2, the up-regulation cardiomyocyte Nox4 results in protection against pressure overload-induced adverse cardiac remodeling. The authors conclude that Nox4 facilitates the maintenance of myocardial capillary density during pressure overload by regulating stress-induced cardiomyocyte hypoxia inducible factor-1 activation and release of vascular endothelial growth factor, resulting in increased paracrine angiogenic activity. In addition, unlike Nox1, Nox2 or Nox5, it seems that Nox4 produces directly H2O2 and thus is incapable of scavenging NO or producing ONOO-.

The beneficial effects of Nox4-derived ROS were also reported by means of a newly developed transgenic mouse with endothelial-specific Nox4 overexpression (Ray et al., 2011). The authors showed that vascular segments and endothelial cells of these animals had

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 435

stimulation is mediated at least in part, by pro-inflammatory transcription factors

In previous studies, we have shown that in human aortic SMCs exposed angiotensin II or tumor necrosis factor-α, the pro-inflammatory transcription factor AP-1 is an essential regulator of the genes coding for p22phox, Nox1, and Nox4 components (Manea et al., 2008; Raicu & Manea, 2010). Other than, activator protein -1 (AP-1), the vascular inflammationrelated and growth-promoting transcription factors signal transducer and activator of transcription (STAT1 and STAT3) proteins physically interact with the promoters of human Nox1 and Nox4 genes in SMCs exposed to interferon (IFN) γ and a Jauns kinase

Moreover, the promoter activities of the genes coding for p22phox, p47phox, and p67phox, have been demonstrated to be considerably augmented in SMCs overexpressing STAT1/STAT3, a result that suggests the existence of functionally gamma activated sequence (GAS)/interferon-stimulated response element (ISRE) consensus sequences (Manea et al., 2010a). In human aortic SMCs, Ets1, a critical mediator of vascular inflammation and remodelling, regulates p47phox expression in response to AngII (Ni et al., 2007). Similar observations were made in A7r5 cells and primary mouse aortic SMCs, in which the growth-promoting transcription factor E2F actually interacts and controls the

In atherosclerosis, and other major cardiovascular disorders, nuclear factor kB (NF-kB) signalling represents a critical regulating mechanism involved in disease onset and progression, including inflammation, cell proliferation, migration, differentiation and apoptosis. Several line of evidence indicate that NF-kB is a redox-sensitive transcription factor which is robustly activated by ROS possible generated by activated Nox. Interestingly, a positive feed-back loop of Nox activation by NF-kB has been proposed in several studies. Thus, a new integrative concept has emergedthe "vicious cycle", to describe the interconnection between metabolic dysfunction, inflammation, and oxidative stress that converges to vascular disorders (Manea, 2010). In murine monocytes, the expression of the Nox2 is induced by NF-kB. Moreover, the up-regulation of p47phox and p22phox expression by LPS/IFNγ was blunted in IkBα-overexpressing cells suggesting the involvement of the NF-kB signaling in the regulation of the Nox components (Anrather et al. 2006). Similar finding were reported in human monocytes/macrophages exposed to TNFα (Gauss et al., 2007). Moreover, in previous studies we have shown that, NF-kB is an important transcriptional regulator of the genes coding for p22phox, Nox1, and Nox4, and has a profound impact in the up-regulation of Nox activity in TNFα-treated human aortic

The molecular mechanisms that facilitate hypoxia sensing and related signalling events are critical for the maintenance of vascular cell homeostasis. Compelling data depicts that hypoxic conditions up-regulate the expression and activity various Nox subtypes (Goyal et al., 2004). It has been demonstrated that persistent hypoxia induces Nox4 gene and protein expression levels in pulmonary artery SMCs and in pulmonary vessels in mice exposed to hypoxic conditions (Diebold et al., 2010). Mechanistically, the response is dependent on hypoxia inducible factor-1α (HIF-1α), which interacts with the corresponding elements in the Nox4 promoter. As a result, the HIF-1α dependent upregulation of Nox4 by may be an essential mechanism to preserve ROS level after hypoxia and the hypoxia-induced proliferation of pulmonary artery SMCs. Furthermore,

•- production.

CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ (Maitra et al., 2009).

(Jak)/STAT-dependent mechanisms are implicated in the ensuing O2

Nox4 transcriptional program (Zhang et al., 2008).

SMCs (Manea et al., 2007; Manea et al., 2010b).

a significant increase in H2O2 generation rather that O2•- and a significant augmentation of the pro-oxidative status. Despite of these aspects, the blood pressure of the animals was lower under basal conditions and after angiotensin II treatment. Interestingly, endotheliumdependent relaxation was significantly improved compared with wild-type animals. Notably, these effects were sensitive to the *ex vivo* addition of catalase and *in vivo* administration of *N*-acetylcysteine, indicating that they were mediated by peroxide-type, namely H2O2, mechanisms. Mechanistically, it seems that an H2O2 action on potassium channels may be responsible for the elaboration of endothelium-derived hyperpolarizing factor (EDHF) type. Thus, one has to ponder that the type of ROS released in the vascular system determines their biological function.

As mentioned above, less is know about the role of Nox5 in atherogenesis, and this is due mainly to the absence of *Nox5* gene in the rodents' genome and therefore to the lack of a reliable animal model., Still, it has been reported a significant correlation between Nox5 expression and atherosclerotic lesion progression. Interestingly, a specific expression pattern was reported; with Nox5 being expressed mainly by the endothelium in the early stages of the disease while its expression is significantly increased in SMCs underlying fibro-lipid atherosclerotic lesions (Guzik et al., 2008).

#### **8. Mechanisms of Nox regulation**

#### **8.1 Phosphorylation pathways and transcription factors**

Nox activity and expression is highly regulated at multiple levels by various physiologic and pathological factors which, by this means, dictate the enzyme complex function. The activities of both Nox1 and Nox2 isoforms are primarily regulated by complex networking of phosphorylation cascades involving regulatory components (i.e., p40phox, p47phox, p67phox, Noxo1, Noxa1) which induce the assembly of the enzyme complex. Nox4 is constitutively active and does not necessitate phosphorylation of regulatory proteins, whereas Nox5 has been demonstrated to be Ca2+-responsivenes. Yet, activation mechanisms involving protein kinase C and the proto-oncogenic tyrosine kinase c-Abl phosphorylation of Nox5 have been reported (El Jamali et al., 2008; Serrander et al., 2007).

The phosphorylation mechanisms responsible for Nox1 and Nox2 activation consist of a large spectrum of signalling molecules such as protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), GTP-binding proteins (Ras, Rac1/2), members of the mitogen-activated protein kinase (MAPK) family (p38MAPK, ERK1/2), phospholipases (PLCβ/γ, PLD), arachidonic acid metabolites, and non-receptor protein tyrosine kinases (Kilpatrick et al., 2010; Yamamori et al., 2004). Besides the aforementioned kinases, chaperone proteins (e.g., protein disulfide isomerase) have been proved to be important regulators on Nox function (Janiszewski et al., 2005).

In addition to the phosphorylation of cytosolic regulatory subunits, alterations of the Nox isoforms expression have been shown to be critical for their activity. Multiple transcription factors are coordinately implicated in the modulation of Nox expression and function. PU.1, Elf-1, IRF-1 (interferon regulatory factor-1), and ICSBP (interferon consensus sequence binding protein) are important transcriptional regulators of Nox2 in the myelomonocytic cell lineage (Kakar et al., 2005). In human colon epithelial Caco-2 cells, GATA-binding factors are critical for Nox1 transcriptional activity (Brewer et al., 2006), whereas in murine macrophages, the up-regulation of Nox1 in response to lipopolysaccharide (LPS)

a significant increase in H2O2 generation rather that O2•- and a significant augmentation of the pro-oxidative status. Despite of these aspects, the blood pressure of the animals was lower under basal conditions and after angiotensin II treatment. Interestingly, endotheliumdependent relaxation was significantly improved compared with wild-type animals. Notably, these effects were sensitive to the *ex vivo* addition of catalase and *in vivo* administration of *N*-acetylcysteine, indicating that they were mediated by peroxide-type, namely H2O2, mechanisms. Mechanistically, it seems that an H2O2 action on potassium channels may be responsible for the elaboration of endothelium-derived hyperpolarizing factor (EDHF) type. Thus, one has to ponder that the type of ROS released in the vascular

As mentioned above, less is know about the role of Nox5 in atherogenesis, and this is due mainly to the absence of *Nox5* gene in the rodents' genome and therefore to the lack of a reliable animal model., Still, it has been reported a significant correlation between Nox5 expression and atherosclerotic lesion progression. Interestingly, a specific expression pattern was reported; with Nox5 being expressed mainly by the endothelium in the early stages of the disease while its expression is significantly increased in SMCs underlying fibro-lipid

Nox activity and expression is highly regulated at multiple levels by various physiologic and pathological factors which, by this means, dictate the enzyme complex function. The activities of both Nox1 and Nox2 isoforms are primarily regulated by complex networking of phosphorylation cascades involving regulatory components (i.e., p40phox, p47phox, p67phox, Noxo1, Noxa1) which induce the assembly of the enzyme complex. Nox4 is constitutively active and does not necessitate phosphorylation of regulatory proteins, whereas Nox5 has been demonstrated to be Ca2+-responsivenes. Yet, activation mechanisms involving protein kinase C and the proto-oncogenic tyrosine kinase c-Abl phosphorylation of Nox5 have been reported (El Jamali et al., 2008; Serrander et al.,

The phosphorylation mechanisms responsible for Nox1 and Nox2 activation consist of a large spectrum of signalling molecules such as protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), GTP-binding proteins (Ras, Rac1/2), members of the mitogen-activated protein kinase (MAPK) family (p38MAPK, ERK1/2), phospholipases (PLCβ/γ, PLD), arachidonic acid metabolites, and non-receptor protein tyrosine kinases (Kilpatrick et al., 2010; Yamamori et al., 2004). Besides the aforementioned kinases, chaperone proteins (e.g., protein disulfide isomerase) have been proved to be important regulators on Nox function

In addition to the phosphorylation of cytosolic regulatory subunits, alterations of the Nox isoforms expression have been shown to be critical for their activity. Multiple transcription factors are coordinately implicated in the modulation of Nox expression and function. PU.1, Elf-1, IRF-1 (interferon regulatory factor-1), and ICSBP (interferon consensus sequence binding protein) are important transcriptional regulators of Nox2 in the myelomonocytic cell lineage (Kakar et al., 2005). In human colon epithelial Caco-2 cells, GATA-binding factors are critical for Nox1 transcriptional activity (Brewer et al., 2006), whereas in murine macrophages, the up-regulation of Nox1 in response to lipopolysaccharide (LPS)

system determines their biological function.

atherosclerotic lesions (Guzik et al., 2008).

**8. Mechanisms of Nox regulation** 

2007).

(Janiszewski et al., 2005).

**8.1 Phosphorylation pathways and transcription factors** 

stimulation is mediated at least in part, by pro-inflammatory transcription factors CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ (Maitra et al., 2009).

In previous studies, we have shown that in human aortic SMCs exposed angiotensin II or tumor necrosis factor-α, the pro-inflammatory transcription factor AP-1 is an essential regulator of the genes coding for p22phox, Nox1, and Nox4 components (Manea et al., 2008; Raicu & Manea, 2010). Other than, activator protein -1 (AP-1), the vascular inflammationrelated and growth-promoting transcription factors signal transducer and activator of transcription (STAT1 and STAT3) proteins physically interact with the promoters of human Nox1 and Nox4 genes in SMCs exposed to interferon (IFN) γ and a Jauns kinase (Jak)/STAT-dependent mechanisms are implicated in the ensuing O2 •- production. Moreover, the promoter activities of the genes coding for p22phox, p47phox, and p67phox, have been demonstrated to be considerably augmented in SMCs overexpressing STAT1/STAT3, a result that suggests the existence of functionally gamma activated sequence (GAS)/interferon-stimulated response element (ISRE) consensus sequences (Manea et al., 2010a). In human aortic SMCs, Ets1, a critical mediator of vascular inflammation and remodelling, regulates p47phox expression in response to AngII (Ni et al., 2007). Similar observations were made in A7r5 cells and primary mouse aortic SMCs, in which the growth-promoting transcription factor E2F actually interacts and controls the Nox4 transcriptional program (Zhang et al., 2008).

In atherosclerosis, and other major cardiovascular disorders, nuclear factor kB (NF-kB) signalling represents a critical regulating mechanism involved in disease onset and progression, including inflammation, cell proliferation, migration, differentiation and apoptosis. Several line of evidence indicate that NF-kB is a redox-sensitive transcription factor which is robustly activated by ROS possible generated by activated Nox. Interestingly, a positive feed-back loop of Nox activation by NF-kB has been proposed in several studies. Thus, a new integrative concept has emergedthe "vicious cycle", to describe the interconnection between metabolic dysfunction, inflammation, and oxidative stress that converges to vascular disorders (Manea, 2010). In murine monocytes, the expression of the Nox2 is induced by NF-kB. Moreover, the up-regulation of p47phox and p22phox expression by LPS/IFNγ was blunted in IkBα-overexpressing cells suggesting the involvement of the NF-kB signaling in the regulation of the Nox components (Anrather et al. 2006). Similar finding were reported in human monocytes/macrophages exposed to TNFα (Gauss et al., 2007). Moreover, in previous studies we have shown that, NF-kB is an important transcriptional regulator of the genes coding for p22phox, Nox1, and Nox4, and has a profound impact in the up-regulation of Nox activity in TNFα-treated human aortic SMCs (Manea et al., 2007; Manea et al., 2010b).

The molecular mechanisms that facilitate hypoxia sensing and related signalling events are critical for the maintenance of vascular cell homeostasis. Compelling data depicts that hypoxic conditions up-regulate the expression and activity various Nox subtypes (Goyal et al., 2004). It has been demonstrated that persistent hypoxia induces Nox4 gene and protein expression levels in pulmonary artery SMCs and in pulmonary vessels in mice exposed to hypoxic conditions (Diebold et al., 2010). Mechanistically, the response is dependent on hypoxia inducible factor-1α (HIF-1α), which interacts with the corresponding elements in the Nox4 promoter. As a result, the HIF-1α dependent upregulation of Nox4 by may be an essential mechanism to preserve ROS level after hypoxia and the hypoxia-induced proliferation of pulmonary artery SMCs. Furthermore,

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 437

Fig. 1. Schematic depiction of the major mechanisms responsible for the up-regulation of Nox enzymes and installation of oxidative stress in atherosclerosis. In response to cardiovascular risk factors, vascular cells through their receptors activate a range of signalling pathways that up-regulate Nox expression, activity, and the ensuing ROS

production. This triggers a chain of critical events that generally amplify the initial response to vascular insults (i.e., activation of other cellular sources of ROS and redox-sensitive signalling effectors). Persistent Nox activation leads to oxidative stress that is a major contributor to the initiation and the development of atherosclerotic lesions. The diagram highlights that genetic, epigenetic, as well as genetic/non-epigenetic-independent

mechanisms linked to Nox up-regulation and hyperactivity in atherosclerosis may be used to target and control pharmacologically the Nox-derived oxidative stress (green text).

The Nox-derived ROS may have both beneficial and deleterious effects. Thus, we can safely assume that these effects are function of the expression pattern and regulation of various Nox isoforms, their subcellular compartmentalization, and the rate of ROS generation. Despite of the numerous existing data, the precise mechanisms of Nox regulation in atherosclerosis and the stream of signalling molecules (up-, or down-regulated) responsible for the increased oxidative stress that is associated with the onset and development of cardiovascular dysfunction, is poorly understood. Thus, a complex interplay of genetic, epigenetic and non-epigenetic factors, transcription factors, co-activators, and/or corepressors may be coordinately involved in the up-regulation of Nox activity in

**9. Conclusion** 

activating transcription factor-1 (ATF-1), a transcription factor of the CREB (CRE-binding protein)/ATF family, proved to play a key role in the induction of Nox1 in rat vascular SMCs (Katsuyama et al., 2005).

Nuclear factor (erythroid-derived 2)-like 2, also known as Nrf2 represents a master modulator of the antioxidant responses by inducing genes (e.g., *Sod* genes*)* with important function in combating oxidative stress. Interestingly, it has been demonstrated that Nrf2, also controls Nox4 expression in mouse lung and human lung endothelium in response to hyperoxia (Pendyala & Natarajan, 2010).

#### **8.2 Genetic and epigenetic mechanisms of Nox regulation**

Genetic studies highlight that several Nox-related polymorphisms are closely associated with an increased susceptibility for cardiovascular disorders. One of the most investigated genes from the Nox complex is *CYBA* which encodes the p22phox essential subunit. The p22phox is ubiquitously expressed in cardiovascular cells and forms stable and functional heterodimers with Nox1, Nox2 or Nox4, a critical structure for enzyme activity as shown by studies employing siRNA technology to knock-down p22phox expression (Kawahara et al., 2005). Moreover, it has been demonstrated that p22phox is more abundant in advanced atherosclerotic plaques than in nonatherosclerotic arteries, suggesting a correlation between p22phox expression, O2•- production, and the severity of atherosclerosis (Azumi et al., 1999).

The occurrence of particular polymorphisms of the *CYBA* gene has been shown to predispose to oxidative stress and to be independently correlated with cardiovascular risk factors and disease occurrence namely hypertension, coronary artery disease, myocardial infarction, cerebrovascular disease, diabetic and non-diabetic nephropathy) (San José et al., 2008). Various *CYBA* allelic variants were detected in both exonic sequences such as C242T, A640G, C549T (Dinauer et al., 1990; Guzik et al., 2000; Inoue et al., 1998), and promoter regions namely -930A*/*G, -675A*/*T, -852C*/*G, -536C*/*T (Lim et al., 2006; Moreno et al., 2007), which potentially affect the p22phox expression and consequently the Nox activity. Thus far, data indicating the existence of functional Nox1-5 polymorphisms with a relevant impact on vascular pathology are not available yet.

Emerging evidence demonstrates that epigenetic events such as DNA methylation and modifications of histone tails are important processes of oxidative stress onset. DNA methylation mechanisms of the promoter CpG islands has been shown to be involved in the up-regulation of 15-lipoxygenase, a pro-oxidative enzyme with implications in plaque formation and vulnerability, and down-regulation of superoxide dismutase 3, endothelial NO synthase, and various anti-proliferative genes (estrogen receptor-α), a condition that leads to oxidative stress, impaired vascular relaxation, and aberrant SMC hyperplasia (Fernandez et al., 2010). Hitherto, data about the role of epigenetics in the regulation of Nox subtypes are missing. Nevertheless, using both *in vitro* (e,g., human aortic SMCs exposed to pro-inflammatory conditions) and *in vivo* (ApoE-/- mice fed a high fat, cholesterol rich diet) models, we have found recently that an aberrant methylation of the Nox1 promoter may be responsible for the up-regulated expression and activity of this enzyme (Manea et al., unpublished data).

A schematic representation of the key molecular pathways implicated in the up-regulation of Nox enzymes as well as the potential pharmacological targets intended to counteract oxidative stress are presented in the figure below.

Fig. 1. Schematic depiction of the major mechanisms responsible for the up-regulation of Nox enzymes and installation of oxidative stress in atherosclerosis. In response to cardiovascular risk factors, vascular cells through their receptors activate a range of signalling pathways that up-regulate Nox expression, activity, and the ensuing ROS production. This triggers a chain of critical events that generally amplify the initial response to vascular insults (i.e., activation of other cellular sources of ROS and redox-sensitive signalling effectors). Persistent Nox activation leads to oxidative stress that is a major contributor to the initiation and the development of atherosclerotic lesions. The diagram highlights that genetic, epigenetic, as well as genetic/non-epigenetic-independent mechanisms linked to Nox up-regulation and hyperactivity in atherosclerosis may be used to target and control pharmacologically the Nox-derived oxidative stress (green text).

#### **9. Conclusion**

436 Atherogenesis

activating transcription factor-1 (ATF-1), a transcription factor of the CREB (CRE-binding protein)/ATF family, proved to play a key role in the induction of Nox1 in rat vascular

Nuclear factor (erythroid-derived 2)-like 2, also known as Nrf2 represents a master modulator of the antioxidant responses by inducing genes (e.g., *Sod* genes*)* with important function in combating oxidative stress. Interestingly, it has been demonstrated that Nrf2, also controls Nox4 expression in mouse lung and human lung endothelium in response to

Genetic studies highlight that several Nox-related polymorphisms are closely associated with an increased susceptibility for cardiovascular disorders. One of the most investigated genes from the Nox complex is *CYBA* which encodes the p22phox essential subunit. The p22phox is ubiquitously expressed in cardiovascular cells and forms stable and functional heterodimers with Nox1, Nox2 or Nox4, a critical structure for enzyme activity as shown by studies employing siRNA technology to knock-down p22phox expression (Kawahara et al., 2005). Moreover, it has been demonstrated that p22phox is more abundant in advanced atherosclerotic plaques than in nonatherosclerotic arteries, suggesting a correlation between p22phox expression, O2•- production, and the severity of

The occurrence of particular polymorphisms of the *CYBA* gene has been shown to predispose to oxidative stress and to be independently correlated with cardiovascular risk factors and disease occurrence namely hypertension, coronary artery disease, myocardial infarction, cerebrovascular disease, diabetic and non-diabetic nephropathy) (San José et al., 2008). Various *CYBA* allelic variants were detected in both exonic sequences such as C242T, A640G, C549T (Dinauer et al., 1990; Guzik et al., 2000; Inoue et al., 1998), and promoter regions namely -930A*/*G, -675A*/*T, -852C*/*G, -536C*/*T (Lim et al., 2006; Moreno et al., 2007), which potentially affect the p22phox expression and consequently the Nox activity. Thus far, data indicating the existence of functional Nox1-5 polymorphisms with a relevant

Emerging evidence demonstrates that epigenetic events such as DNA methylation and modifications of histone tails are important processes of oxidative stress onset. DNA methylation mechanisms of the promoter CpG islands has been shown to be involved in the up-regulation of 15-lipoxygenase, a pro-oxidative enzyme with implications in plaque formation and vulnerability, and down-regulation of superoxide dismutase 3, endothelial NO synthase, and various anti-proliferative genes (estrogen receptor-α), a condition that leads to oxidative stress, impaired vascular relaxation, and aberrant SMC hyperplasia (Fernandez et al., 2010). Hitherto, data about the role of epigenetics in the regulation of Nox subtypes are missing. Nevertheless, using both *in vitro* (e,g., human aortic SMCs exposed to pro-inflammatory conditions) and *in vivo* (ApoE-/- mice fed a high fat, cholesterol rich diet) models, we have found recently that an aberrant methylation of the Nox1 promoter may be responsible for the up-regulated expression and activity of this enzyme (Manea et al.,

A schematic representation of the key molecular pathways implicated in the up-regulation of Nox enzymes as well as the potential pharmacological targets intended to counteract

SMCs (Katsuyama et al., 2005).

hyperoxia (Pendyala & Natarajan, 2010).

atherosclerosis (Azumi et al., 1999).

unpublished data).

impact on vascular pathology are not available yet.

oxidative stress are presented in the figure below.

**8.2 Genetic and epigenetic mechanisms of Nox regulation** 

The Nox-derived ROS may have both beneficial and deleterious effects. Thus, we can safely assume that these effects are function of the expression pattern and regulation of various Nox isoforms, their subcellular compartmentalization, and the rate of ROS generation. Despite of the numerous existing data, the precise mechanisms of Nox regulation in atherosclerosis and the stream of signalling molecules (up-, or down-regulated) responsible for the increased oxidative stress that is associated with the onset and development of cardiovascular dysfunction, is poorly understood. Thus, a complex interplay of genetic, epigenetic and non-epigenetic factors, transcription factors, co-activators, and/or corepressors may be coordinately involved in the up-regulation of Nox activity in

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#### **10. Acknowledgement**

This work was supported by grants from the Romanian Ministry of Education, and Research (CNCSIS-UEFISCSU project numbers PNII-IDEI 1005/2009 and PNII-TE 65/2010), and from the European Foundation for the Study of Diabetes - New Horizons. The financial support of European Social Fund – "Cristofor I. Simionescu" Postdoctoral Fellowship Programme (ID POSDRU/89/1.5/S/55216), Sectoral Operational Programme Human Resources Development 2007 – 2013 is acknowledged.

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**21** 

*Spain* 

**Modified Forms of LDL in Plasma** 

José Luis Sánchez-Quesada1 and Sandra Villegas2 *1Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona* 

*2Biochemistry and Molecular Biology Department, Universitat Autonoma de Barcelona* 

High blood concentration of low-density lipoprotein (LDL) cholesterol is a major risk factor for early development of atherosclerosis. Massive accumulation of cholesterol in the arterial wall and formation of lipid-laden cells (foam cells) typical of the atheromatous plaque occur after LDL entrapment in the subendothelial space. However, there is a general agreement that native non-modified LDL does not present any of the typical features of an atherogenic lipoprotein; native LDL does not promote foam cell formation and has no inflammatory, proliferative or apoptotic capacity. It is therefore assumed that when LDL is trapped in the arterial wall it is modified by several mechanisms such as lipoperoxidation, non-enzymatic glycosylation, enzymatic lipolysis and/or proteolysis. As a consequence, modified LDL particles acquire inflammatory and apoptotic capacity and are recognizable by scavenger receptors to promote foam cell formation. Although most studies on LDL modification have focused on mechanisms that could occur in the vessel wall, modified LDL particles have been reported in blood. The concentration of these particles is increased in subjects with high cardiovascular risk. The present chapter reviews the possible mechanisms leading to LDL modification. It then focuses on a subfraction of modified LDL particles detected in

Pioneering studies by Goldstein and Brown demonstrated the involvement of LDL receptor (LDLr) in the plasma clearance of LDL and its major role in the development of atherosclerosis (Goldstein & Brown, 1985). However, these authors' outstanding observations gave rise to what was called the "cholesterol paradox" (Brown & Goldstein, 1983). Patients with homozygous familial hypercholesterolemia lack functional LDLr. Moreover, LDLr expression is negatively regulated by the intracellular cholesterol content, which results in a tight control of the concentration of esterified cholesterol in the cytoplasm (Brown & Goldstein, 1986). However, atherosclerosis is characterized by an abundance of foam cells loaded with esterified cholesterol droplets. Hence, there must be an alternative pathway leading to the massive accumulation of cholesterol in foam cells of the atherosclerotic lesion. This alternative pathway was discovered through the study of the scavenger receptors (SR) expressed by monocyte-derived macrophages (Krieger, 1992). These receptors have the ability to bind an unusual variety of ligands with the common

**1. Introduction** 

plasma, named electronegative LDL.

**2. Modification of LDL as a key event for atherosclerosis** 


### **Modified Forms of LDL in Plasma**

José Luis Sánchez-Quesada1 and Sandra Villegas2 *1Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona 2Biochemistry and Molecular Biology Department, Universitat Autonoma de Barcelona Spain* 

#### **1. Introduction**

446 Atherogenesis

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18121-18126, ISSN 0027-8424

NADPH oxidase NOX4 promoter in vascular smooth muscle cells by E2F. *Free* 

Yu, B., Dong, X., Walker, S.J., Brandes, R.P. & Shah, A.M. (2010). NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis. *Proceedings of the National Academy of Sciences USA,* 107,

> High blood concentration of low-density lipoprotein (LDL) cholesterol is a major risk factor for early development of atherosclerosis. Massive accumulation of cholesterol in the arterial wall and formation of lipid-laden cells (foam cells) typical of the atheromatous plaque occur after LDL entrapment in the subendothelial space. However, there is a general agreement that native non-modified LDL does not present any of the typical features of an atherogenic lipoprotein; native LDL does not promote foam cell formation and has no inflammatory, proliferative or apoptotic capacity. It is therefore assumed that when LDL is trapped in the arterial wall it is modified by several mechanisms such as lipoperoxidation, non-enzymatic glycosylation, enzymatic lipolysis and/or proteolysis. As a consequence, modified LDL particles acquire inflammatory and apoptotic capacity and are recognizable by scavenger receptors to promote foam cell formation. Although most studies on LDL modification have focused on mechanisms that could occur in the vessel wall, modified LDL particles have been reported in blood. The concentration of these particles is increased in subjects with high cardiovascular risk. The present chapter reviews the possible mechanisms leading to LDL modification. It then focuses on a subfraction of modified LDL particles detected in plasma, named electronegative LDL.

#### **2. Modification of LDL as a key event for atherosclerosis**

Pioneering studies by Goldstein and Brown demonstrated the involvement of LDL receptor (LDLr) in the plasma clearance of LDL and its major role in the development of atherosclerosis (Goldstein & Brown, 1985). However, these authors' outstanding observations gave rise to what was called the "cholesterol paradox" (Brown & Goldstein, 1983). Patients with homozygous familial hypercholesterolemia lack functional LDLr. Moreover, LDLr expression is negatively regulated by the intracellular cholesterol content, which results in a tight control of the concentration of esterified cholesterol in the cytoplasm (Brown & Goldstein, 1986). However, atherosclerosis is characterized by an abundance of foam cells loaded with esterified cholesterol droplets. Hence, there must be an alternative pathway leading to the massive accumulation of cholesterol in foam cells of the atherosclerotic lesion. This alternative pathway was discovered through the study of the scavenger receptors (SR) expressed by monocyte-derived macrophages (Krieger, 1992). These receptors have the ability to bind an unusual variety of ligands with the common

Modified Forms of LDL in Plasma 449

been described in the last two decades (SRBI, CD36, LOX-1, CD68, LRP1, TLR4) (Adachi & Tsujimoto, 2006; Llorente-Cortes & Badimon, 2005; Miller, 2003; Van Berkel, 2000). These receptors play a role in innate immunity, scavenging a number of ligands and extracellular debris. Some of these are also expressed in endothelial cells or in smooth muscle cells (Adachi & Tsujimoto, 2006). The derivatization of lysines has an additional effect on LDL; since a lysine-rich cluster is the LDLr binding site of apoB (residues 3359- 3369), the loss of their positive charge abolishes the interaction between oxLDL and LDLr

Besides lipid accumulation, other phenomena occur during the evolution of the disease. Atherosclerosis is a chronic inflammatory process that begins with leukocyte recruitment to the lesion area (Ross, 1999). A number of inflammation mediators, such as cytokines (IL6), chemokines (IL8, MCP1) or vascular adhesion molecules (VCAM, ICAM1, eselectin) are hyperexpressed in response to oxLDL by all cells involved in atherogenesis, including endothelial cells, monocytes/macrophages, lymphocytes and smooth muscle cells (Berliner, 1997; Tedgui & Mallat, 2006). Related to the inflammatory activity of oxLDL is its ability to promote the expression of growth factors and colony stimulating factors. This proliferative activity is mediated by oxidation-derived lipids, and in smooth muscle cells it induces the change of the normal contractile phenotype to a proliferative phenotype (Auge, 2002). The proliferative phenotype of smooth muscle cells is characterized by the high production of collagen that will contribute decisively to the

Another hallmark of the atheromatous plaque in its advanced stages is the presence of a necrotic core formed by debris from dead cells (Stary, 2000). OxLDL also contributes to the formation of this necrotic core, because when the content of esterified cholesterol exceeds the storage capacity of foam cells apoptotic processes are induced (Hessler, 1983). Another mechanism of cytotoxicity is the high content of lysophosphatidylcholine in oxLDL that disrupts the integrity of cytoplasmatic membrane and promotes cell death

OxLDL is immunogenic, and autoantibodies against oxidation-specific epitopes are detected in normal and hyperlipemic subjects (Lopes-Virella & Virella, 2010). The role of these autoantibodies is not well defined and published results are divergent; positive or negative associations between autoantibodies and atherosclerosis have been reported. In general, antibodies of IgG class are presumed to be pro-atherogenic, whereas IgM antibodies would play a protective role (Frostegard, 2010). Further deleterious properties of oxLDL are its capacity to increase the tissue factor activity (Petit, 1999), favouring thrombosis, or the

The lipoperoxidative process is sequential and lipids degrade to produce new products which in turn form other molecules (Quehenberger, 1988). As a consequence, oxLDL comprises an extremely heterogeneous group of particles that have different atherogenic characteristics depending on the relative content of each molecule (Esterbauer, 1993). For instance, minimally oxidized LDL (mmLDL) has a high inflammatory activity due to its high content in oxidized phospholipids formed during the early phases of oxidation. However, mmLDL has normal affinity to the LDLr and is not recognized by SR. In contrast, extensively oxidized LDL has relatively low inflammatory activity but it is a major inductor of cytotoxicity due to its high content of oxysterols. Indeed, the more oxidized the LDL the

inhibition of the release of nitric oxide (Minuz, 1995), promoting vasoconstriction.

(Boren, 1998a).

(Naito, 1994).

thickening of the arterial wall (Negishi, 2004).

more these particles promote foam cell formation.

characteristic of high electronegative charge. The first SR described was the type A SR (SRA) (Goldstein, 1979). SRA do not recognize native LDL but is able to bind modified forms of LDL; indeed, SRA expression is not regulated by the cytoplasm concentration of cholesterol. Early studies were performed using acetylated LDL (acLDL), a good ligand for SRA. However, acetylation does not occur spontaneously "in vivo". Soon, researchers looked for further modifications of LDL that would increase its negative charge and were able to occur "in vivo". The finding that oxidized LDL (oxLDL) could be internalized through SR and promote the formation of foam cells opened a vast field of research (Steinberg, 1989).

#### **2.1 Oxidative modification of LDL**

Oxidative modifications of lipid and proteins are frequent in many pathophysiological processes "in vivo" and it is now well-established that LDL undergoes oxidative modifications that confer to these modified particles a number of atherogenic properties (Witztum & Steinberg, 1991). In the early 80s several researchers independently observed that the incubation of LDL with endothelial cells in culture drastically altered these particles, transforming them into more electronegative LDL, after which they become a ligand for macrophages SR (Henriksen, 1981; Hessler, 1979). These studies concluded that the changes induced by endothelial cells on LDL were due to free radical modification. This was the origin of the oxidative modification hypothesis of atherogenesis (Steinberg, 1989). This hypothesis was gradually strengthened by subsequent findings regarding the properties of oxLDL obtained over the ensuing years. Table 1 summarizes the atherogenic properties observed in oxLDL and their contribution to atherosclerosis.


Table 1. Atherogenic characteristics of oxLDL

Oxidation of LDL primarily attacks the double bonds of unsaturated fatty acids in phospholipids and generates a plethora of lipid-derived compounds, including oxidized cholesterol (oxysterols), oxidized-fragmented phospholipids, lysophosphatidylcholine, hydroperoxids, aldehydes and ketones (Esterbauer, 1990). The protein moiety of LDL can be modified by reacting with lipid-oxidation products. Both aldehydes and ketones, for example, malondialdehyde (MDA) or 4-hydroxynonenal (HNE), have the ability to derivatize lysine and arginine residues in apoB (Fogelman, 1980). This reaction forms an adduct that eliminates the positive charge of these aminoacids and increases the negative charge of the particle, favouring its recognition by SRA (Goldstein, 1979). In addition to SRA, further SRs and other receptors which bind different forms of modified LDLs have

characteristic of high electronegative charge. The first SR described was the type A SR (SRA) (Goldstein, 1979). SRA do not recognize native LDL but is able to bind modified forms of LDL; indeed, SRA expression is not regulated by the cytoplasm concentration of cholesterol. Early studies were performed using acetylated LDL (acLDL), a good ligand for SRA. However, acetylation does not occur spontaneously "in vivo". Soon, researchers looked for further modifications of LDL that would increase its negative charge and were able to occur "in vivo". The finding that oxidized LDL (oxLDL) could be internalized through SR and

promote the formation of foam cells opened a vast field of research (Steinberg, 1989).

Oxidative modifications of lipid and proteins are frequent in many pathophysiological processes "in vivo" and it is now well-established that LDL undergoes oxidative modifications that confer to these modified particles a number of atherogenic properties (Witztum & Steinberg, 1991). In the early 80s several researchers independently observed that the incubation of LDL with endothelial cells in culture drastically altered these particles, transforming them into more electronegative LDL, after which they become a ligand for macrophages SR (Henriksen, 1981; Hessler, 1979). These studies concluded that the changes induced by endothelial cells on LDL were due to free radical modification. This was the origin of the oxidative modification hypothesis of atherogenesis (Steinberg, 1989). This hypothesis was gradually strengthened by subsequent findings regarding the properties of oxLDL obtained over the ensuing years. Table 1 summarizes the atherogenic properties

**2.1 Oxidative modification of LDL** 

Increase in the expression of cytokines, chemokines and vascular adhesion

Table 1. Atherogenic characteristics of oxLDL

molecules

observed in oxLDL and their contribution to atherosclerosis.

Recognition by SR Foam cell formation

Inhibition of NO release and function Vasoconstriction Increase of tissue factor activity Thombosis

Promotion of apoptosis and cytotoxicity Formation of necrotic core

**Characteristic Consequence in atherosclerosis**  Impaired binding to the LDL receptor Decreased plasma clearance

Increase in the expression of growth factors Cell proliferation and collagen secretion

Immunogenicity Immunogenic component of atherosclerosis

Oxidation of LDL primarily attacks the double bonds of unsaturated fatty acids in phospholipids and generates a plethora of lipid-derived compounds, including oxidized cholesterol (oxysterols), oxidized-fragmented phospholipids, lysophosphatidylcholine, hydroperoxids, aldehydes and ketones (Esterbauer, 1990). The protein moiety of LDL can be modified by reacting with lipid-oxidation products. Both aldehydes and ketones, for example, malondialdehyde (MDA) or 4-hydroxynonenal (HNE), have the ability to derivatize lysine and arginine residues in apoB (Fogelman, 1980). This reaction forms an adduct that eliminates the positive charge of these aminoacids and increases the negative charge of the particle, favouring its recognition by SRA (Goldstein, 1979). In addition to SRA, further SRs and other receptors which bind different forms of modified LDLs have

Leukocyte recruitment, inflammation

been described in the last two decades (SRBI, CD36, LOX-1, CD68, LRP1, TLR4) (Adachi & Tsujimoto, 2006; Llorente-Cortes & Badimon, 2005; Miller, 2003; Van Berkel, 2000). These receptors play a role in innate immunity, scavenging a number of ligands and extracellular debris. Some of these are also expressed in endothelial cells or in smooth muscle cells (Adachi & Tsujimoto, 2006). The derivatization of lysines has an additional effect on LDL; since a lysine-rich cluster is the LDLr binding site of apoB (residues 3359- 3369), the loss of their positive charge abolishes the interaction between oxLDL and LDLr (Boren, 1998a).

Besides lipid accumulation, other phenomena occur during the evolution of the disease. Atherosclerosis is a chronic inflammatory process that begins with leukocyte recruitment to the lesion area (Ross, 1999). A number of inflammation mediators, such as cytokines (IL6), chemokines (IL8, MCP1) or vascular adhesion molecules (VCAM, ICAM1, eselectin) are hyperexpressed in response to oxLDL by all cells involved in atherogenesis, including endothelial cells, monocytes/macrophages, lymphocytes and smooth muscle cells (Berliner, 1997; Tedgui & Mallat, 2006). Related to the inflammatory activity of oxLDL is its ability to promote the expression of growth factors and colony stimulating factors. This proliferative activity is mediated by oxidation-derived lipids, and in smooth muscle cells it induces the change of the normal contractile phenotype to a proliferative phenotype (Auge, 2002). The proliferative phenotype of smooth muscle cells is characterized by the high production of collagen that will contribute decisively to the thickening of the arterial wall (Negishi, 2004).

Another hallmark of the atheromatous plaque in its advanced stages is the presence of a necrotic core formed by debris from dead cells (Stary, 2000). OxLDL also contributes to the formation of this necrotic core, because when the content of esterified cholesterol exceeds the storage capacity of foam cells apoptotic processes are induced (Hessler, 1983). Another mechanism of cytotoxicity is the high content of lysophosphatidylcholine in oxLDL that disrupts the integrity of cytoplasmatic membrane and promotes cell death (Naito, 1994).

OxLDL is immunogenic, and autoantibodies against oxidation-specific epitopes are detected in normal and hyperlipemic subjects (Lopes-Virella & Virella, 2010). The role of these autoantibodies is not well defined and published results are divergent; positive or negative associations between autoantibodies and atherosclerosis have been reported. In general, antibodies of IgG class are presumed to be pro-atherogenic, whereas IgM antibodies would play a protective role (Frostegard, 2010). Further deleterious properties of oxLDL are its capacity to increase the tissue factor activity (Petit, 1999), favouring thrombosis, or the inhibition of the release of nitric oxide (Minuz, 1995), promoting vasoconstriction.

The lipoperoxidative process is sequential and lipids degrade to produce new products which in turn form other molecules (Quehenberger, 1988). As a consequence, oxLDL comprises an extremely heterogeneous group of particles that have different atherogenic characteristics depending on the relative content of each molecule (Esterbauer, 1993). For instance, minimally oxidized LDL (mmLDL) has a high inflammatory activity due to its high content in oxidized phospholipids formed during the early phases of oxidation. However, mmLDL has normal affinity to the LDLr and is not recognized by SR. In contrast, extensively oxidized LDL has relatively low inflammatory activity but it is a major inductor of cytotoxicity due to its high content of oxysterols. Indeed, the more oxidized the LDL the more these particles promote foam cell formation.

Modified Forms of LDL in Plasma 451

Several studies have focused on the atherogenic effects of LDL modified with different types of secreted phospholipase A2 (sPLA2) (Divchev & Schieffer, 2008). Some sPLA2 have been detected in atherosclerotic lesions and their products, lysophosphatidylcholine (LPC) and NEFA, induce cytotoxicity at relatively high concentrations by disrupting membrane integrity (Dersch, 2005; Naito, 1994). Moreover, both molecules have inflammatory potential by stimulating the expression of cytokines and chemokines (Sonoki, 2003). In addition, extensively lipolyzed LDL with some (type X), but not all (type IIA or V), sPLA2 are able to induce foam cell formation (Curfs, 2008). An interesting approach was performed by Sparrow and coworkers, which combined sPLA2 and lipoxygenase to modify LDL (Sparrow, 1988). This combination of lipoperoxidation and phospholipolysis generated LDL particles with similar properties to those promoted by endothelial cell-induced oxidative modification. Another atherogenic characteristic of sPLA2-modified LDL is its increased affinity for PG binding due to the exposition of an alternative binding site in apoB that specifically recognizes PG (Boren, 1998b). As occurs with SMase-modified LDL, this would

PAF-acetylhydrolase (PAF-AH), another PLA2 with relevance in the metabolism of LDL, merits special mention. This enzyme is transported in plasma bound to lipoproteins (approximately 70% in LDL and 25% in HDL) but, in contrast to sPLA2, its substrate is not native phospholipids but fragmented phospholipids that have been generated by oxidation (Tjoelker & Stafforini, 2000). Thus, LPC content is high in oxLDL due to the action of PAF-AH. However, there is controversy regarding the pro- or anti-atherogenic role of PAF-AH (Tellis & Tselepis, 2009). On one hand, its function should be atheroprotective since it degrades highly-inflammatory fragmented phospholipids. On the other hand, however, the by-products formed are LPC and short-chain NEFA, which also have inflammatory

The hyperexpression of acid and neutral SMases in atherosclerotic lesions has been known for several years (Tabas, 1999). SMase hydrolyzes sphingomyelin (SM) yielding phosphorylcholine and ceramide. Ceramide directly exerts several biological effects, but its main action is to elicit the production of bioactive sphingolipids, such as sphingosine-1 phosphate that plays a major role in apoptosis (van Blitterswijk, 2003). Another action of SMase is to promote extensive LDL aggregation (Oorni, 2000). Aggregation of LDL confers several atherogenic properties. First, aggregated LDL (agLDL) is able to induce foam cell formation, not through SR in this case but through the LDL-receptor related protein 1 (LRP1), a receptor of aggregated lipoproteins highly expressed in smooth muscle cells (Llorente-Cortes & Badimon, 2005). Second, agLDL binds with higher affinity to PG (Oorni, 1998). As discussed below, this binding promotes structural changes in apoB. The association of agLDL to PG precludes the exit of this lipoprotein from the subendothelial

potential, though to a lesser extent than oxidized phospholipids (MacPhee, 1999).

space, and consequently favours that agLDL could undergo further modifications.

Although non-enzymatic glycosylation of LDL occurs in all subjects, it has stronger consequences in people with diabetes mellitus. Glucose can interact with the free amino groups of lysines and arginines in apoB, forming a Schiff base that rearranges to yield an

**2.2.2 Phospholipase A2-modified LDL (PLA2-LDL)** 

lead to increased subendothelial retention.

**2.2.3 Sphingomyelinase-modified LDL (SMase-LDL)** 

**2.2.4 Glycated LDL (glLDL)** 

#### **2.2 Further modifications of LDL occurring in the arterial wall**

Oxidative modification is not the only process affecting the native properties of lipoproteins in the arterial wall. Disappointing results from large-antioxidant trials have led to the concept that alternative mechanisms of modification could be involved (Steinberg, 2009). Current knowledge indicates that although oxidation is still relevant, other processes could contribute substantially to the development of atherosclerosis. A number of modifications have been studied over the past three decades.

Enzymes that are hyperexpressed in the microenvironment of the lesion area, such as lipases (cholesterol esterase (CEase), sphingomyelinase (SMase) or secretary phospholipase A2 (sPLA2)) or proteases (matrix metalloproteases or cathepsins) can modify LDL (Pentikainen, 2000). In the case of diabetes, non-enzymatic glycosylation could have a major role in LDL modification (Witztum, 1997). The interaction of LDL with the proteoglycans (PG) could modify apoB conformation, destabilizing its conformation and promoting aggregation (Pentikainen, 1997). Other putative physiological processes that could modify LDL are carbamylation (Basnakian, 2010) or desialylation (Tertov, 1990). Table 2 summarizes several possible physiological mechanisms leading to LDL modification.


Table 2. Atherogenic properties of several modified LDLs. 1 The characteristics of oxLDL, and also those of the other modifications, depend on the extent of modification

#### **2.2.1 Enzymatically-modified LDL (E-LDL)**

In an attempt to find a modified LDL alternative to oxLDL, Bhakdi and coworkers performed a series of studies using LDL that was modified by means of one protease and CEase. They found that this "enzymatically-modified LDL" (E-LDL) acquired a number of atherogenic properties (Bhakdi, 1995). As a result of the enzymatic treatment, E-LDL presents mild apoB fragmentation, and it has high free cholesterol and non-esterified fatty acids (NEFA) content (Klouche, 1998). E-LDL induces inflammation, proliferation and apoptosis (Dersch, 2005; Klouche, 1999; Klouche, 2000). The high content of NEFA seems to be major factor responsible for these atherogenic properties (Suriyaphol, 2002). E-LDL adds further atherogenic characteristics because it binds to C-reactive protein and activates the classical complement pathway (Bhakdi, 2004). The activation of complement by E-LDL concurs with the emerging concept of the innate immune response as a potentially important factor in atherosclerosis (Hartvigsen, 2009).

Oxidative modification is not the only process affecting the native properties of lipoproteins in the arterial wall. Disappointing results from large-antioxidant trials have led to the concept that alternative mechanisms of modification could be involved (Steinberg, 2009). Current knowledge indicates that although oxidation is still relevant, other processes could contribute substantially to the development of atherosclerosis. A number of modifications

Enzymes that are hyperexpressed in the microenvironment of the lesion area, such as lipases (cholesterol esterase (CEase), sphingomyelinase (SMase) or secretary phospholipase A2 (sPLA2)) or proteases (matrix metalloproteases or cathepsins) can modify LDL (Pentikainen, 2000). In the case of diabetes, non-enzymatic glycosylation could have a major role in LDL modification (Witztum, 1997). The interaction of LDL with the proteoglycans (PG) could modify apoB conformation, destabilizing its conformation and promoting aggregation (Pentikainen, 1997). Other putative physiological processes that could modify LDL are carbamylation (Basnakian, 2010) or desialylation (Tertov, 1990). Table 2 summarizes several

**formation** 

Oxidation1 +++ +++ +++

protease -/+ ++ ++ Lipolysis by SMase + - -/+ Lipolysis by PLA2 -/+ ++ + Non-enzymatic glycosylation -/+ -/+ -/+ Binding to PG ++ - - Carbamylation ++ ++ ++ Desialylation ++ + ++ Table 2. Atherogenic properties of several modified LDLs. 1 The characteristics of oxLDL,

and also those of the other modifications, depend on the extent of modification

In an attempt to find a modified LDL alternative to oxLDL, Bhakdi and coworkers performed a series of studies using LDL that was modified by means of one protease and CEase. They found that this "enzymatically-modified LDL" (E-LDL) acquired a number of atherogenic properties (Bhakdi, 1995). As a result of the enzymatic treatment, E-LDL presents mild apoB fragmentation, and it has high free cholesterol and non-esterified fatty acids (NEFA) content (Klouche, 1998). E-LDL induces inflammation, proliferation and apoptosis (Dersch, 2005; Klouche, 1999; Klouche, 2000). The high content of NEFA seems to be major factor responsible for these atherogenic properties (Suriyaphol, 2002). E-LDL adds further atherogenic characteristics because it binds to C-reactive protein and activates the classical complement pathway (Bhakdi, 2004). The activation of complement by E-LDL concurs with the emerging concept of the innate immune response as a potentially

**Inflammation/ proliferation** 

**Cytotoxicity/ apoptosis** 

**2.2 Further modifications of LDL occurring in the arterial wall** 

possible physiological mechanisms leading to LDL modification.

**Mechanisms of modification Foam cell** 

**2.2.1 Enzymatically-modified LDL (E-LDL)** 

important factor in atherosclerosis (Hartvigsen, 2009).

Modification by CEase and

have been studied over the past three decades.

#### **2.2.2 Phospholipase A2-modified LDL (PLA2-LDL)**

Several studies have focused on the atherogenic effects of LDL modified with different types of secreted phospholipase A2 (sPLA2) (Divchev & Schieffer, 2008). Some sPLA2 have been detected in atherosclerotic lesions and their products, lysophosphatidylcholine (LPC) and NEFA, induce cytotoxicity at relatively high concentrations by disrupting membrane integrity (Dersch, 2005; Naito, 1994). Moreover, both molecules have inflammatory potential by stimulating the expression of cytokines and chemokines (Sonoki, 2003). In addition, extensively lipolyzed LDL with some (type X), but not all (type IIA or V), sPLA2 are able to induce foam cell formation (Curfs, 2008). An interesting approach was performed by Sparrow and coworkers, which combined sPLA2 and lipoxygenase to modify LDL (Sparrow, 1988). This combination of lipoperoxidation and phospholipolysis generated LDL particles with similar properties to those promoted by endothelial cell-induced oxidative modification. Another atherogenic characteristic of sPLA2-modified LDL is its increased affinity for PG binding due to the exposition of an alternative binding site in apoB that specifically recognizes PG (Boren, 1998b). As occurs with SMase-modified LDL, this would lead to increased subendothelial retention.

PAF-acetylhydrolase (PAF-AH), another PLA2 with relevance in the metabolism of LDL, merits special mention. This enzyme is transported in plasma bound to lipoproteins (approximately 70% in LDL and 25% in HDL) but, in contrast to sPLA2, its substrate is not native phospholipids but fragmented phospholipids that have been generated by oxidation (Tjoelker & Stafforini, 2000). Thus, LPC content is high in oxLDL due to the action of PAF-AH. However, there is controversy regarding the pro- or anti-atherogenic role of PAF-AH (Tellis & Tselepis, 2009). On one hand, its function should be atheroprotective since it degrades highly-inflammatory fragmented phospholipids. On the other hand, however, the by-products formed are LPC and short-chain NEFA, which also have inflammatory potential, though to a lesser extent than oxidized phospholipids (MacPhee, 1999).

#### **2.2.3 Sphingomyelinase-modified LDL (SMase-LDL)**

The hyperexpression of acid and neutral SMases in atherosclerotic lesions has been known for several years (Tabas, 1999). SMase hydrolyzes sphingomyelin (SM) yielding phosphorylcholine and ceramide. Ceramide directly exerts several biological effects, but its main action is to elicit the production of bioactive sphingolipids, such as sphingosine-1 phosphate that plays a major role in apoptosis (van Blitterswijk, 2003). Another action of SMase is to promote extensive LDL aggregation (Oorni, 2000). Aggregation of LDL confers several atherogenic properties. First, aggregated LDL (agLDL) is able to induce foam cell formation, not through SR in this case but through the LDL-receptor related protein 1 (LRP1), a receptor of aggregated lipoproteins highly expressed in smooth muscle cells (Llorente-Cortes & Badimon, 2005). Second, agLDL binds with higher affinity to PG (Oorni, 1998). As discussed below, this binding promotes structural changes in apoB. The association of agLDL to PG precludes the exit of this lipoprotein from the subendothelial space, and consequently favours that agLDL could undergo further modifications.

#### **2.2.4 Glycated LDL (glLDL)**

Although non-enzymatic glycosylation of LDL occurs in all subjects, it has stronger consequences in people with diabetes mellitus. Glucose can interact with the free amino groups of lysines and arginines in apoB, forming a Schiff base that rearranges to yield an

Modified Forms of LDL in Plasma 453

atherosclerosis development but also a reflection of the presence of unstable and ruptured atherosclerotic plaques (Fraley & Tsimikas, 2006). The fact that oxLDL increases temporarily during the acute phase of myocardial infarction or stroke supports this notion (Fraley & Tsimikas, 2006; Nishi, 2002; Uno, 2003) and some studies have raised the possibility that plasma oxLDL could predict future cardiovascular events (Meisinger, 2005). The concentration of oxLDL in plasma is very low, and data reported by several authors are disperse, ranging from 0.01% to 0.5% of total LDL. The heterogeneous nature of oxLDL is one reason to explain variation in the reports of oxLDL concentration, but another could be the use of several antibodies that recognize different epitopes (Ishigaki, 2009). The fact that in vitro oxidation does not reproduce the same epitopes generated during in vivo

Several studies have shown that besides its utility as a biomarker, oxLDL in plasma acts as a pathogenic factor. It has been reported that oxLDL contributes to increase the systemic inflammatory status by stimulating the activity of the transcription factor NF-kB in peripheral blood mononuclear cells (Cominacini, 2005). More direct demonstration of the implication of oxLDL in blood has been obtained by increasing the expression of SR, such as SRA1 (Whitman, 2002), LOX-1 (Ishigaki, 2008), or the chimerical fusion protein SRA1 growth hormone (Laukkanen, 2000), which favours oxLDL removal from blood. These studies showed an inhibition of atherosclerosis development. It was recently reported that repeated administration of the chimerical fusion protein Fc-CD68 decreases the extent of atherosclerosis in hyperlipemic mice (Zeibig, 2011). However, these studies have been tested to date in animal models only. Regarding humans, several trials have reported that lipidlowering therapy in atherosclerotic patients lowers oxLDL, although this decrease is parallel to that of LDL cholesterol (Ky, 2008). However, no trial with a therapy specifically focused on oxLDL is yet available. The development of novel therapies for lowering oxLDL itself is a

The concentration of glLDL in plasma from patients with diabetes is increased, reflecting the hyperglycemia in these patients. This concentration varies depending on the method used for its quantification (ELISA, affinity chromatography), but it is generally higher than that of oxLDL (Cohen, 1993; Reaven, 1995). The proportion of glLDL in diabetics can reach up to 7- 8% of total LDL and approximately half in normoglycemic subjects. It is important to note that glLDL is present in all individuals, and even in normoglycemic subjects the concentration in plasma is higher than that of oxLDL. This suggests glLDL plays a role in the development of atherosclerosis even in absence of hyperglycemia. Interestingly, glLDL is more abundant in the subfractions of LDL that are smallest and have the highest density, probably because these small-dense LDL particles are prone to non-enzymatic glycosylation (Younis, 2009). These particles are also more susceptible to oxidation and have a relatively low affinity to the LDL receptor. These properties are related to the strong association

Alternative pathways can also result in LDL glycosylation without a direct involvement of glucose. Metabolites of glucose such as glyoxal, methylglyoxal or glycaldehyde have a higher reducing capacity as glycating agents (Rabbani & Thornalley, 2011). Minimal modification by methylglyoxal renders LDL particles with atherogenic properties, including binding to PG and susceptibility to aggregation (Rabbani, 2011). LDL particles with this low

between small-dense LDL and high cardiovascular risk (Krauss, 1995).

level of modification are increased in diabetic patients (Rabbani, 2010).

modification makes it difficult to develop a golden standard.

promising strategy for atherosclerosis treatment.

**3.2 Glycated LDL in plasma** 

Amadori product (Brownlee, 1992). This modification leads to a loss of electropositive charges in glycated LDL (glLDL), decreasing its affinity to the LDLr and consequently prolonging its mean lifetime in plasma (Witztum, 1982). The increase in lifetime can result in further modification of LDL forming advanced glycation end-products (AGE) and producing a form of LDL named AGE-LDL (Menzel, 1997). AGE-LDL can also be internalized by specific receptors (RAGE) (Bucala, 1996). AGE-LDL and glLDL increase chemotactic activity in monocytes, stimulate cell proliferation and enhance platelet aggregation, although the relative contribution that the coexistence of oxidation could have is not well established. The formation of AGE involves oxidative reactions and it has been demonstrated that lipoperoxidation and non-enzymatic glycosylation are mutually potentiated processes (Sobal, 2000). Thus, glycosylation of LDL is not only noxious per se but also because it promotes LDL oxidation.

#### **2.2.5 Proteoglycan-bound LDL (PG-LDL)**

PG are the main constituents of the arterial intima. It has been hypothesized that subendothelial retention of lipoproteins due to the binding of LDL to PG is the initial event in atherogenesis, even prior to endothelial dysfunction or inflammation (Williams & Tabas, 2005). The retention itself increases the time of LDL in the subendothelial space and, therefore, the possibility that further modifications occur. Furthermore, the binding LDL-PG also has a direct effect on LDL modification because this binding promotes changes in the structure of apoB that facilitate processes such as oxidation or lipolysis mediated by SMase or sPLA2 (Hevonoja, 2000). On the other hand, LDL-PG complexes are taken up by cells through different types of SR, promoting foam cell formation.

#### **3. Modified LDL in human plasma**

For many years it was considered that LDL modification was a phenomenon occurring mainly in the intima layer of the arterial wall. Oxidative modification was the most studied process, and most researchers accepted that the abundance in plasma of soluble molecules with antioxidant capacity (albumin, uric acid, bilirubin, glutathione, ascorbic acid) would inhibit oxidation of lipoproteins. Moreover, the binding of oxLDL by SR expressed in circulating monocytes is known to promote a rapid clearance of extensively oxidized LDL. However, progress in enzyme immunoassay procedures has provided direct evidence of oxLDL in circulating plasma. Besides oxLDL, similar methods have been used to detect other forms of modified LDL in blood. These include glLDL, AGE-LDL, carbamylated LDL (ca-LDL), desialylated LDL and electronegative LDL (LDL(-)).

#### **3.1 Oxidized LDL in plasma**

Despite the abundance of antioxidant defences in blood, increased oxidative stress has been described in plasma from patients with atherosclerosis. Early studies reported that increased levels of oxidized lipids in plasma were associated with atherosclerosis development (Avogaro, 1986). In agreement with that, the existence of oxLDL in blood is increased in subjects with high cardiovascular risk (Holvoet, 1999; Holvoet, 1998) or in diseases, such as diabetes, obesity, metabolic syndrome and hyperlipemia (Ishigaki, 2009). Although it cannot be totally ruled out that a part of oxLDL could be formed in blood, it is generally accepted that oxLDL originates primarily in the arterial wall and that the molecules reach the blood from the subendothelial space. For this reason, oxLDL is considered not only a biomarker of

Amadori product (Brownlee, 1992). This modification leads to a loss of electropositive charges in glycated LDL (glLDL), decreasing its affinity to the LDLr and consequently prolonging its mean lifetime in plasma (Witztum, 1982). The increase in lifetime can result in further modification of LDL forming advanced glycation end-products (AGE) and producing a form of LDL named AGE-LDL (Menzel, 1997). AGE-LDL can also be internalized by specific receptors (RAGE) (Bucala, 1996). AGE-LDL and glLDL increase chemotactic activity in monocytes, stimulate cell proliferation and enhance platelet aggregation, although the relative contribution that the coexistence of oxidation could have is not well established. The formation of AGE involves oxidative reactions and it has been demonstrated that lipoperoxidation and non-enzymatic glycosylation are mutually potentiated processes (Sobal, 2000). Thus, glycosylation of LDL is not only noxious per se but also because it promotes LDL oxidation.

PG are the main constituents of the arterial intima. It has been hypothesized that subendothelial retention of lipoproteins due to the binding of LDL to PG is the initial event in atherogenesis, even prior to endothelial dysfunction or inflammation (Williams & Tabas, 2005). The retention itself increases the time of LDL in the subendothelial space and, therefore, the possibility that further modifications occur. Furthermore, the binding LDL-PG also has a direct effect on LDL modification because this binding promotes changes in the structure of apoB that facilitate processes such as oxidation or lipolysis mediated by SMase or sPLA2 (Hevonoja, 2000). On the other hand, LDL-PG complexes are taken up by cells

For many years it was considered that LDL modification was a phenomenon occurring mainly in the intima layer of the arterial wall. Oxidative modification was the most studied process, and most researchers accepted that the abundance in plasma of soluble molecules with antioxidant capacity (albumin, uric acid, bilirubin, glutathione, ascorbic acid) would inhibit oxidation of lipoproteins. Moreover, the binding of oxLDL by SR expressed in circulating monocytes is known to promote a rapid clearance of extensively oxidized LDL. However, progress in enzyme immunoassay procedures has provided direct evidence of oxLDL in circulating plasma. Besides oxLDL, similar methods have been used to detect other forms of modified LDL in blood. These include glLDL, AGE-LDL, carbamylated LDL

Despite the abundance of antioxidant defences in blood, increased oxidative stress has been described in plasma from patients with atherosclerosis. Early studies reported that increased levels of oxidized lipids in plasma were associated with atherosclerosis development (Avogaro, 1986). In agreement with that, the existence of oxLDL in blood is increased in subjects with high cardiovascular risk (Holvoet, 1999; Holvoet, 1998) or in diseases, such as diabetes, obesity, metabolic syndrome and hyperlipemia (Ishigaki, 2009). Although it cannot be totally ruled out that a part of oxLDL could be formed in blood, it is generally accepted that oxLDL originates primarily in the arterial wall and that the molecules reach the blood from the subendothelial space. For this reason, oxLDL is considered not only a biomarker of

**2.2.5 Proteoglycan-bound LDL (PG-LDL)** 

**3. Modified LDL in human plasma** 

**3.1 Oxidized LDL in plasma** 

through different types of SR, promoting foam cell formation.

(ca-LDL), desialylated LDL and electronegative LDL (LDL(-)).

atherosclerosis development but also a reflection of the presence of unstable and ruptured atherosclerotic plaques (Fraley & Tsimikas, 2006). The fact that oxLDL increases temporarily during the acute phase of myocardial infarction or stroke supports this notion (Fraley & Tsimikas, 2006; Nishi, 2002; Uno, 2003) and some studies have raised the possibility that plasma oxLDL could predict future cardiovascular events (Meisinger, 2005). The concentration of oxLDL in plasma is very low, and data reported by several authors are disperse, ranging from 0.01% to 0.5% of total LDL. The heterogeneous nature of oxLDL is one reason to explain variation in the reports of oxLDL concentration, but another could be the use of several antibodies that recognize different epitopes (Ishigaki, 2009). The fact that in vitro oxidation does not reproduce the same epitopes generated during in vivo modification makes it difficult to develop a golden standard.

Several studies have shown that besides its utility as a biomarker, oxLDL in plasma acts as a pathogenic factor. It has been reported that oxLDL contributes to increase the systemic inflammatory status by stimulating the activity of the transcription factor NF-kB in peripheral blood mononuclear cells (Cominacini, 2005). More direct demonstration of the implication of oxLDL in blood has been obtained by increasing the expression of SR, such as SRA1 (Whitman, 2002), LOX-1 (Ishigaki, 2008), or the chimerical fusion protein SRA1 growth hormone (Laukkanen, 2000), which favours oxLDL removal from blood. These studies showed an inhibition of atherosclerosis development. It was recently reported that repeated administration of the chimerical fusion protein Fc-CD68 decreases the extent of atherosclerosis in hyperlipemic mice (Zeibig, 2011). However, these studies have been tested to date in animal models only. Regarding humans, several trials have reported that lipidlowering therapy in atherosclerotic patients lowers oxLDL, although this decrease is parallel to that of LDL cholesterol (Ky, 2008). However, no trial with a therapy specifically focused on oxLDL is yet available. The development of novel therapies for lowering oxLDL itself is a promising strategy for atherosclerosis treatment.

#### **3.2 Glycated LDL in plasma**

The concentration of glLDL in plasma from patients with diabetes is increased, reflecting the hyperglycemia in these patients. This concentration varies depending on the method used for its quantification (ELISA, affinity chromatography), but it is generally higher than that of oxLDL (Cohen, 1993; Reaven, 1995). The proportion of glLDL in diabetics can reach up to 7- 8% of total LDL and approximately half in normoglycemic subjects. It is important to note that glLDL is present in all individuals, and even in normoglycemic subjects the concentration in plasma is higher than that of oxLDL. This suggests glLDL plays a role in the development of atherosclerosis even in absence of hyperglycemia. Interestingly, glLDL is more abundant in the subfractions of LDL that are smallest and have the highest density, probably because these small-dense LDL particles are prone to non-enzymatic glycosylation (Younis, 2009). These particles are also more susceptible to oxidation and have a relatively low affinity to the LDL receptor. These properties are related to the strong association between small-dense LDL and high cardiovascular risk (Krauss, 1995).

Alternative pathways can also result in LDL glycosylation without a direct involvement of glucose. Metabolites of glucose such as glyoxal, methylglyoxal or glycaldehyde have a higher reducing capacity as glycating agents (Rabbani & Thornalley, 2011). Minimal modification by methylglyoxal renders LDL particles with atherogenic properties, including binding to PG and susceptibility to aggregation (Rabbani, 2011). LDL particles with this low level of modification are increased in diabetic patients (Rabbani, 2010).

(Fig. 1).

Modified Forms of LDL in Plasma 455

the content in lipoperoxides decreased (De Castellarnau, 2000; Demuth, 1996; Sevanian, 1997). Discrepancies regarding the increased content in oxidized lipids in LDL(-), continue; some authors do not find differences compared to native LDL (Benitez, 2007b; Demuth, 1996; Sanchez-Quesada, 2003), and others report increased amounts of oxidized lipids in LDL(-) (Asatryan, 2003; Sevanian, 1997; Ziouzenkova, 2002). But in any case, this level of lipoperoxidation is closer to minimally oxidized LDL than to extensively oxidized LDL suggesting that alternative mechanisms should be involved in LDL(-) formation

Fig. 1. Formation of LDL(-). The modification of native LDL by one or several mechanisms

inflammatory, proliferative and apoptotic lipids and non-apoB proteins in LDL(-), as well as

LDL is a heterogeneous mixture of particles that differ in size (24-28 nm of diameter) and density (1.019-1.063 g/ml). Small-dense particles have fewer lipid molecules while largebuoyant particles have more. It is known that LDL particles at both extremes of the density range have increased electronegative charge compared with mid-density particles (Lund-Katz, 1998). It has been proposed that the origin of LDL(-) could be related with the impairment of the catabolic cascade that transforms VLDL-to-IDL-to-LDL in blood (Sanchez-Quesada, 2004). Such impairment leads to the formation of small-dense or largebuoyant LDL particles. In agreement with this, LDL(-) is most abundant (>80% of LDL(-)) in small-dense particles in normolipemic subjects. Interestingly, in hypercholesterolemic and hypertriglyceridemic patients, LDL(-) are also abundant large-buoyant particles (Sanchez-Quesada, 2002). Another consequence of impaired LDL catabolism is the presence of non-apoB proteins in LDL. Theoretically, the protein moiety of LDL consists of a single copy of a very large protein, apoB. However, it is known that some particles of LDL also contain other proteins. In native LDL, the content of non-apoB proteins is less than 1%. In contrast, LDL(-) contains up to 5% of non-apoB proteins (Bancells, 2010a;

alters the composition of LDL surface. These alterations include the increase of

structural abnormalities in apoB.

**4.1.1 Impaired catabolism of LDL** 

Yang, 2003).

A more advanced form of glLDL is AGE-LDL, in which AGE are formed due to autooxidation of Amadori adducts yielding a number of products, such as carboxymethyl lysine or pentosyl lysine (Brownlee, 2000). AGE-LDL has atherogenic characteristics that are similar to oxLDL, probably because their oxidized lipid content is similar. Although it was generally considered that AGE-LDL was generated in the arterial wall this modified form of LDL has also been detected in blood (Lopes-Virella & Virella, 2010). AGE-LDL, like other AGE-containing proteins, is recognized in circulation by RAGE. RAGE activation stimulates cytokyne and growth factors release (Ramasamy, 2009). An excess of stimulation (i.e. an excess of AGE-LDL) plays an essential role in atherogenic alterations.

#### **3.3 Carbamylated LDL (ca-LDL)**

Carbamylation of proteins is a post-translational modification in which amine-containing residues react with cyanate, a compound that derives from urea or from thiocyanate. This modification is relatively frequent in patients with chronic uremia or in heavy smokers. Both situations are closely related to increased cardiovascular risk and carbamylated LDL (ca-LDL) is increased in the plasma in both groups of subjects (Basnakian, 2010). Ca-LDL is recognized by SR, promotes monocyte adhesion to endothelium, stimulates cell proliferation and causes cell injury (Apostolov, 2007; Carracedo, 2011).

#### **3.4 Desialylated LDL (ds-LDL)**

Native LDL has high content of sialic acid in the carbohydrate chains attached to apoB. Tertov and colleagues isolated a fraction of desialylated LDL from plasma (Tertov, 1990). This fraction, which was increased in patients with advanced atherosclerosis, induced foam cell formation in cultured smooth muscle cells and presented inflammatory properties (Orekhov, 1991). They suggested that low sialic acid in LDL was a cardiovascular risk factor (Ruelland, 1993) but this idea was not supported by other authors (Cerne, 2002). Later studies revealed that desialylated LDL was oxidized and that the loss of sialic acid was a consequence of oxidative modification (Tertov, 1995).

#### **4. Electronegative LDL, a pool of modified LDL in blood**

A common characteristic of most of the previously described modifications is an increase of the negative electric charge. Taking advantage of this property, Avogaro and co-workers fractionated total LDL from human plasma by anion-exchange chromatography, into two subfractions, a major subfraction of native LDL and an electronegatively-charged fraction of LDL (LDL(-)) (Avogaro, 1988). In this first report, LDL(-) accounted for 5-20% of total LDL in normolipemic subjects and presented a number of atherogenic characteristics, including impaired binding to LDLr, high aggregation level, capacity to induce cholesterol accumulation in macrophages and higher conjugated diene (a by-product of lipid peroxidation) content. Since then, a number of studies have tried to elucidate the physicochemical and biological characteristics of LDL(-) and its relationship with atherosclerosis.

#### **4.1 Origin of LDL(-) – Discrepancies regarding the oxidative origin**

Early studies focused on the physico-chemical characteristics LDL(-) concluded, in accordance with the high content of oxidized lipids, that LDL(-) was the in vivo counterpart of in vitro oxidized LDL (Cazzolato, 1991). However, as isolation procedures improved and measures to prevent modification increased, the proportion of LDL(-) and

A more advanced form of glLDL is AGE-LDL, in which AGE are formed due to autooxidation of Amadori adducts yielding a number of products, such as carboxymethyl lysine or pentosyl lysine (Brownlee, 2000). AGE-LDL has atherogenic characteristics that are similar to oxLDL, probably because their oxidized lipid content is similar. Although it was generally considered that AGE-LDL was generated in the arterial wall this modified form of LDL has also been detected in blood (Lopes-Virella & Virella, 2010). AGE-LDL, like other AGE-containing proteins, is recognized in circulation by RAGE. RAGE activation stimulates cytokyne and growth factors release (Ramasamy, 2009). An excess of stimulation (i.e. an

Carbamylation of proteins is a post-translational modification in which amine-containing residues react with cyanate, a compound that derives from urea or from thiocyanate. This modification is relatively frequent in patients with chronic uremia or in heavy smokers. Both situations are closely related to increased cardiovascular risk and carbamylated LDL (ca-LDL) is increased in the plasma in both groups of subjects (Basnakian, 2010). Ca-LDL is recognized by SR, promotes monocyte adhesion to endothelium, stimulates cell proliferation

Native LDL has high content of sialic acid in the carbohydrate chains attached to apoB. Tertov and colleagues isolated a fraction of desialylated LDL from plasma (Tertov, 1990). This fraction, which was increased in patients with advanced atherosclerosis, induced foam cell formation in cultured smooth muscle cells and presented inflammatory properties (Orekhov, 1991). They suggested that low sialic acid in LDL was a cardiovascular risk factor (Ruelland, 1993) but this idea was not supported by other authors (Cerne, 2002). Later studies revealed that desialylated LDL was oxidized and that the loss of sialic acid was a

A common characteristic of most of the previously described modifications is an increase of the negative electric charge. Taking advantage of this property, Avogaro and co-workers fractionated total LDL from human plasma by anion-exchange chromatography, into two subfractions, a major subfraction of native LDL and an electronegatively-charged fraction of LDL (LDL(-)) (Avogaro, 1988). In this first report, LDL(-) accounted for 5-20% of total LDL in normolipemic subjects and presented a number of atherogenic characteristics, including impaired binding to LDLr, high aggregation level, capacity to induce cholesterol accumulation in macrophages and higher conjugated diene (a by-product of lipid peroxidation) content. Since then, a number of studies have tried to elucidate the physicochemical and biological characteristics of LDL(-) and its relationship with atherosclerosis.

Early studies focused on the physico-chemical characteristics LDL(-) concluded, in accordance with the high content of oxidized lipids, that LDL(-) was the in vivo counterpart of in vitro oxidized LDL (Cazzolato, 1991). However, as isolation procedures improved and measures to prevent modification increased, the proportion of LDL(-) and

excess of AGE-LDL) plays an essential role in atherogenic alterations.

and causes cell injury (Apostolov, 2007; Carracedo, 2011).

consequence of oxidative modification (Tertov, 1995).

**4. Electronegative LDL, a pool of modified LDL in blood** 

**4.1 Origin of LDL(-) – Discrepancies regarding the oxidative origin** 

**3.3 Carbamylated LDL (ca-LDL)** 

**3.4 Desialylated LDL (ds-LDL)** 

the content in lipoperoxides decreased (De Castellarnau, 2000; Demuth, 1996; Sevanian, 1997). Discrepancies regarding the increased content in oxidized lipids in LDL(-), continue; some authors do not find differences compared to native LDL (Benitez, 2007b; Demuth, 1996; Sanchez-Quesada, 2003), and others report increased amounts of oxidized lipids in LDL(-) (Asatryan, 2003; Sevanian, 1997; Ziouzenkova, 2002). But in any case, this level of lipoperoxidation is closer to minimally oxidized LDL than to extensively oxidized LDL suggesting that alternative mechanisms should be involved in LDL(-) formation (Fig. 1).

Fig. 1. Formation of LDL(-). The modification of native LDL by one or several mechanisms alters the composition of LDL surface. These alterations include the increase of inflammatory, proliferative and apoptotic lipids and non-apoB proteins in LDL(-), as well as structural abnormalities in apoB.

#### **4.1.1 Impaired catabolism of LDL**

LDL is a heterogeneous mixture of particles that differ in size (24-28 nm of diameter) and density (1.019-1.063 g/ml). Small-dense particles have fewer lipid molecules while largebuoyant particles have more. It is known that LDL particles at both extremes of the density range have increased electronegative charge compared with mid-density particles (Lund-Katz, 1998). It has been proposed that the origin of LDL(-) could be related with the impairment of the catabolic cascade that transforms VLDL-to-IDL-to-LDL in blood (Sanchez-Quesada, 2004). Such impairment leads to the formation of small-dense or largebuoyant LDL particles. In agreement with this, LDL(-) is most abundant (>80% of LDL(-)) in small-dense particles in normolipemic subjects. Interestingly, in hypercholesterolemic and hypertriglyceridemic patients, LDL(-) are also abundant large-buoyant particles (Sanchez-Quesada, 2002). Another consequence of impaired LDL catabolism is the presence of non-apoB proteins in LDL. Theoretically, the protein moiety of LDL consists of a single copy of a very large protein, apoB. However, it is known that some particles of LDL also contain other proteins. In native LDL, the content of non-apoB proteins is less than 1%. In contrast, LDL(-) contains up to 5% of non-apoB proteins (Bancells, 2010a; Yang, 2003).

Modified Forms of LDL in Plasma 457

Fig. 2. Atherogenic properties of LDL(-). Modified lipids in LDL(-) surface have

formation, but could mediate the signaling of endothelial apoptosis.

and impairs its plasma clearance.

**4.2.1 Binding to receptors** 

**4.2.2 Inflammatory activity** 

inflammatory, proliferative and apoptotic capacity. Aggregation, which is induced by the formation of ceramide, favours the binding to PGs and increases subendothelial retention. The alteration of apoB conformation in LDL(-) induces a partial loss of affinity to the LDLr

LDL(-) presents a partial loss of affinity to the LDLr, a property that could lengthen its halflife in blood (Benitez, 2004b). It was initially believed that this was due to the derivatization of lysines in apoB involved in receptor recognition, in a mechanism similar to oxLDL (MDA-Lys) or glLDL (glucose-Lys). However, recent studies have shown that lysines in LDL(-) are not derivatized but have an altered ionization state due to differences in the conformation of apoB (Blanco, 2010). Regarding SR, LDL(-) binds differently to distinct types of SR. The increment of electronegativity in LDL(-) is not sufficient to allow its binding to SRA in macrophages (Benitez, 2004b). However, it has been reported that LDL(-) binds to another SR, LOX-1, in endothelial cells (Lu, 2009). This binding does not promote foam cell

LDL(-) has the ability to activate the transcription factors NF-kB, AP-1 and PPAR, inducing the expression of a number of inflammatory molecules in endothelial cells (Abe, 2007; Benitez, 2006; De Castellarnau, 2000; Ziouzenkova, 2003). These molecules include cytokines (IL6) chemokines (IL8, MCP-1, GRO), vascular adhesion molecules (VCAM) and growth factors (GM-CSF, PDGF). Interestingly, LDL(-) also induces the paradoxical expression of the anti-inflammatory cytokine IL10 in lymphocytes and monocytes (Benitez, 2007a). It has been suggested that IL10 production could be a mechanism to control an excessive inflammatory response limiting the extent of injury. LDL(-) could also play a role in angiogenesis modulation since it stimulates vascular endothelial growth factor (VEGF) expression and inhibits the release of the matrix metalloproteinases MMP2 and MMP9 (Lu,

#### **4.1.2 Lipolysis**

Some characteristics of LDL(-), such as high LPC and NEFA content, suggest that a possible mechanism of formation could be mediated by phospholipases. It has been described that in vitro modification of LDL with sPLA2 renders modified particles that mimics some properties of LDL(-) (Asatryan, 2005; Benitez, 2004a; Benitez, 2004b). On the other hand, PAF-AH, which has a 5-10-fold higher activity in LDL(-) than in native LDL, could also play a role in increasing the content of LPC and NEFA and in the generation of LDL(-) (Gaubatz, 2007; Sanchez-Quesada, 2005). Some type of SMase activity could also be involved in LDL(-) generation since a minor subfraction of LDL(-) is aggregated (Bancells, 2010b). It has been reported that LDL(-) has an intrinsic phospholipase C-like (PLC-like) activity that degrades with high affinity both SM and LPC (Bancells, 2008). The origin of such activity is currently unknown, but it could be due to conformational changes in apoB. One or a combination of these phospholipolityc activities could be involved in the formation of LDL(-).

#### **4.1.3 Content of non-esterified fatty acids (NEFA)**

NEFA are transported in blood mainly associated to albumin. However, in some situations that increase NEFA or decrease albumin there is a partition of NEFA towards other proteins, including lipoproteins. In posprandial lipemia or when high energy is required (such as during heavy exercise) the increase of NEFA in blood increases LDL(-) proportion (Benitez, 2002). NEFA content in LDL(-) is three to four-fold higher than in native LDL (Benitez, 2004a; De Castellarnau, 2000; Demuth, 1996). In this context, it has been reported that the main determinant of the electronegativity of LDL(-) is NEFA (Gaubatz, 2007).

#### **4.1.4 Non-enzymatic glycosylation**

It would seem reasonable to consider that glLDL contributes in part to the pool of LDL(-), especially in diabetic patients. However, most glycated LDL particles isolable by affinity chromatography do not have a sufficient negative charge to be isolated with LDL(-) and the content of glLDL in LDL(-) is similar to that in native LDL (Benitez, 2007b; Sanchez-Quesada, 2005).

#### **4.1.5 Hemoglobin derivatization and carbamylation**

Patients with severe renal failure have a high proportion of LDL(-) (Asatryan, 2003). It was reported that LDL from these patients suffered a cross-linking with hemoglobin, rendering a particle with increased negative charge (Ziouzenkova, 2002). Recent studies on carbamylated LDL, however, suggest that carbamylation could underlie the high proportion of LDL(-) in patients with severe renal disease (Apostolov, 2010).

#### **4.2 Biological properties of LDL(-)**

Whatever the mechanism involved in its formation LDL(-), has several potentially atherogenic properties. These include abnormal binding to receptors, inflammatory and cytotoxic properties, high susceptibility to aggregation and increased affinity to PG (Fig. 2).

Some characteristics of LDL(-), such as high LPC and NEFA content, suggest that a possible mechanism of formation could be mediated by phospholipases. It has been described that in vitro modification of LDL with sPLA2 renders modified particles that mimics some properties of LDL(-) (Asatryan, 2005; Benitez, 2004a; Benitez, 2004b). On the other hand, PAF-AH, which has a 5-10-fold higher activity in LDL(-) than in native LDL, could also play a role in increasing the content of LPC and NEFA and in the generation of LDL(-) (Gaubatz, 2007; Sanchez-Quesada, 2005). Some type of SMase activity could also be involved in LDL(-) generation since a minor subfraction of LDL(-) is aggregated (Bancells, 2010b). It has been reported that LDL(-) has an intrinsic phospholipase C-like (PLC-like) activity that degrades with high affinity both SM and LPC (Bancells, 2008). The origin of such activity is currently unknown, but it could be due to conformational changes in apoB. One or a combination of these phospholipolityc activities could be

NEFA are transported in blood mainly associated to albumin. However, in some situations that increase NEFA or decrease albumin there is a partition of NEFA towards other proteins, including lipoproteins. In posprandial lipemia or when high energy is required (such as during heavy exercise) the increase of NEFA in blood increases LDL(-) proportion (Benitez, 2002). NEFA content in LDL(-) is three to four-fold higher than in native LDL (Benitez, 2004a; De Castellarnau, 2000; Demuth, 1996). In this context, it has been reported that the main determinant of the electronegativity of LDL(-) is NEFA

It would seem reasonable to consider that glLDL contributes in part to the pool of LDL(-), especially in diabetic patients. However, most glycated LDL particles isolable by affinity chromatography do not have a sufficient negative charge to be isolated with LDL(-) and the content of glLDL in LDL(-) is similar to that in native LDL (Benitez, 2007b; Sanchez-

Patients with severe renal failure have a high proportion of LDL(-) (Asatryan, 2003). It was reported that LDL from these patients suffered a cross-linking with hemoglobin, rendering a particle with increased negative charge (Ziouzenkova, 2002). Recent studies on carbamylated LDL, however, suggest that carbamylation could underlie the high proportion

Whatever the mechanism involved in its formation LDL(-), has several potentially atherogenic properties. These include abnormal binding to receptors, inflammatory and cytotoxic properties, high susceptibility to aggregation and increased affinity to PG

**4.1.2 Lipolysis** 

(Gaubatz, 2007).

Quesada, 2005).

(Fig. 2).

involved in the formation of LDL(-).

**4.1.4 Non-enzymatic glycosylation** 

**4.2 Biological properties of LDL(-)** 

**4.1.3 Content of non-esterified fatty acids (NEFA)** 

**4.1.5 Hemoglobin derivatization and carbamylation** 

of LDL(-) in patients with severe renal disease (Apostolov, 2010).

Fig. 2. Atherogenic properties of LDL(-). Modified lipids in LDL(-) surface have inflammatory, proliferative and apoptotic capacity. Aggregation, which is induced by the formation of ceramide, favours the binding to PGs and increases subendothelial retention. The alteration of apoB conformation in LDL(-) induces a partial loss of affinity to the LDLr and impairs its plasma clearance.

#### **4.2.1 Binding to receptors**

LDL(-) presents a partial loss of affinity to the LDLr, a property that could lengthen its halflife in blood (Benitez, 2004b). It was initially believed that this was due to the derivatization of lysines in apoB involved in receptor recognition, in a mechanism similar to oxLDL (MDA-Lys) or glLDL (glucose-Lys). However, recent studies have shown that lysines in LDL(-) are not derivatized but have an altered ionization state due to differences in the conformation of apoB (Blanco, 2010). Regarding SR, LDL(-) binds differently to distinct types of SR. The increment of electronegativity in LDL(-) is not sufficient to allow its binding to SRA in macrophages (Benitez, 2004b). However, it has been reported that LDL(-) binds to another SR, LOX-1, in endothelial cells (Lu, 2009). This binding does not promote foam cell formation, but could mediate the signaling of endothelial apoptosis.

#### **4.2.2 Inflammatory activity**

LDL(-) has the ability to activate the transcription factors NF-kB, AP-1 and PPAR, inducing the expression of a number of inflammatory molecules in endothelial cells (Abe, 2007; Benitez, 2006; De Castellarnau, 2000; Ziouzenkova, 2003). These molecules include cytokines (IL6) chemokines (IL8, MCP-1, GRO), vascular adhesion molecules (VCAM) and growth factors (GM-CSF, PDGF). Interestingly, LDL(-) also induces the paradoxical expression of the anti-inflammatory cytokine IL10 in lymphocytes and monocytes (Benitez, 2007a). It has been suggested that IL10 production could be a mechanism to control an excessive inflammatory response limiting the extent of injury. LDL(-) could also play a role in angiogenesis modulation since it stimulates vascular endothelial growth factor (VEGF) expression and inhibits the release of the matrix metalloproteinases MMP2 and MMP9 (Lu,

Modified Forms of LDL in Plasma 459

to-LDL cascade. High NEFA could be a consequence of the increase in NEFA concentration in plasma (posprandial lipemia, intense exercise) (Benitez, 2002; Ursini, 1998), or it could come from lipolysis mediated by phospholipases (Benitez, 2004a). This latter possibility

The most abundant proteins in LDL(-) are apoA-I (0.15 molecules/particle of LDL(-)), apoE (0.22), apoC-III (0.37) and apoA-II (0.14), with a content 3-5 fold higher in LDL(-) than in native LDL (Bancells, 2010a). The role of these proteins is unclear but their relevance is probably low. However, other proteins whose absolute content in LDL(-) is lower, such as apoF (0.06 molecules/particle), apoJ (0.01) or PAF-AH (0.004) (Bancells, 2010a; Yang, 2007), but their relative content compared with native LDL is 10, 20 and 100-fold higher, respectively, could have a much more relevant role. ApoF is the physiological inhibitor of cholesteryl ester transfer protein (Morton, 2008), a protein that regulates the catabolism of the VLDL-IDL-LDL cascade. It could therefore be one cause of impaired LDL(-) maturation. ApoJ (also known as clusterin) is an extracellular chaperone that binds to hydrophobic unfolded proteins, favoring their extracellular clearance (Oda, 1995). ApoJ binds mainly to aggregated LDL(-), supporting the presence of misfolded apoB in this subfraction. The role of PAF-AH in LDL(-) would be to deactivate oxidized phospholipids, but its undesirable effect would be the formation of LPC and short-chain NEFA. It has been suggested that the PLC-like activity present in LDL(-) could act in cooperation with PAF-AH degrading LPC (Bancells, 2010b). Therefore, this would be a mechanism to limit the deleterious effects

The proportion of LDL(-) is increased in a number of pathologic situations having a cardiovascular risk. Familial hypercholesterolemia and hypertriglyceridemia present a proportion of LDL(-) 3-5 fold higher than normolipemic healthy subjects (Sanchez-Quesada, 2002; Sanchez-Quesada, 1999). These results were obtained using ultracentrifugation plus anion-exchange chromatography, both being laborious and time-consuming techniques. This limits their use for routine analysis of lipoprotein profiles. However, more reliable techniques for rapid analysis have recently been developed, including capillary electrophoresis and ELISA (Santo Faulin Tdo, 2008; Zhang, 2009; Zhang, 2008). These methods have confirmed previous data obtained by anion-exchange chromatography. Statin therapy decreases the proportion of LDL(-) but the process is not parallel to the lipidlowering effect. This is because total LDL cholesterol decreases very rapidly (in 2 weeks) whereas LDL(-) decreases more slowly (in up to six months) (Sanchez-Quesada, 1999). This suggests that LDL(-) generation not only depends on lipid metabolism but also on other

Both type 1 and type 2 diabetics have a high proportion of LDL(-) (Moro, 1998; Sanchez-Quesada, 1996; Sanchez-Quesada, 2001; Zhang, 2005). This would suggest that nonenzymatic glycosylation could be involved. However, insulin therapy decreases LDL(-) in type 1 diabetes but not in type 2 diabetes. The different response to insulin treatment has been attributed to differences in the systemic inflammation level, which is higher in type 2 patients. This agrees with the finding that pre-diabetic insulin-resistant subjects with high systemic inflammation also have increased LDL(-) proportion (Zhang, 2005). Therefore,

would be the same mechanism of increased LPC content in LDL(-).

exerted by minimal LDL oxidation on vascular cells.

**4.4 Association of LDL(-) with cardiovascular risk** 

factors such as chronic inflammation.

**4.3.3 Proteins** 

2008; Tai, 2006). The specific molecules that mediate these actions are not well defined; LPC and NEFA probably play a major role, but the action of oxidized lipids remains under discussion (Abe, 2007; Benitez, 2004a; Chen, 2004). If they are present in LDL(-) they could stimulate inflammatory responses, but it is also possible that high PAF-AH activity in LDL (-) degrades readily oxidized phospholipids (Benitez, 2003). Their degradation products, LPC and short-chain NEFA, would therefore be responsible for triggering inflammation.

#### **4.2.3 Cytotoxicity and apoptosis**

LDL(-) induces citotoxicity and apoptosis in endothelial cells and macrophages by different signaling pathways. The mechanisms involved are well defined, especially in endothelial cells, where LOX-1 signaling inhibits fibroblast growth factor 2 (FGF2) transcription and Akt phosphorilation (Chen, 2003; Chen, 2007; Lu, 2008; Tang, 2008; Yang, 2007). In contrast, macrophage apoptosis involves the Fas/FasL signaling pathway and the activation of the transcription factor Nrf2 (Pedrosa, 2010).

#### **4.2.4 Binding to proteoglycans**

LDL(-) and PG present a high affinity for binding (Bancells, 2009). This could favor the subendothelial retention of LDL(-) and trigger the inflammatory response. Some characteristics in LDL(-) are involved in this higher affinity. Aggregation of lipoproteins favors PG binding, and LDL(-) has a high tendency to aggregate (Bancells, 2010b). In fact, a subfraction of aggregated LDL(-) is responsible for the binding to PG (Bancells, 2009). This subfraction has an abnormal conformation that exposes an epitope in apoB, known as site Ib, that is an alternative binding site to PGs (Bancells, 2011).

#### **4.3 Physico-chemical characteristics of LDL(-)**

#### **4.3.1 Structure**

The earliest physical abnormalities reported in LDL(-) were a great heterogeneity in size and density and a high susceptibility to aggregation (Avogaro, 1988). A subpopulation of aggregated LDL(-) has recently been isolated and characterized. This subpopulation, which accounts for only 0.1-0.5 of total LDL in blood, has high affinity to arterial PG (Bancells, 2009). This increased binding seems to be mediated by abnormal conformation of the aminoterminal extreme of apoB (Bancells, 2011). Further evidence of apoB misfolding, in this case affecting LDLr binding, has been obtained by two dimensional nuclear magnetic resonance analyses (Blanco, 2010). LDL(-) is reported to promote aggregation of non-aggregated LDL particles in a process that fits an amyloidogenic model (Parasassi, 2008). It has been reported that the capacity to induce aggregation could be mediated by the PLC-like activity (Bancells, 2010b), although other authors have suggested that plasma sPLA2 could be involved in apoB misfolding (Greco, 2009). Regarding secondary structure of apoB, some authors have reported loss of secondary -helix structures whereas others did not find differences with native LDL (Asatryan, 2005; Bancells, 2009; Benitez, 2004b; Parasassi, 2001).

#### **4.3.2 Lipids**

Although there are contradictory data regarding differences in the lipid content between native LDL and LDL(-) most studies concur in a higher content of triglycerides, NEFA and LPC in LDL(-) (Cazzolato, 1991; De Castellarnau, 2000; Sanchez-Quesada, 2003; Sevanian, 1997; Yang, 2003). The high content of triglycerides reflects impairment of the VLDL-to-IDL-

to-LDL cascade. High NEFA could be a consequence of the increase in NEFA concentration in plasma (posprandial lipemia, intense exercise) (Benitez, 2002; Ursini, 1998), or it could come from lipolysis mediated by phospholipases (Benitez, 2004a). This latter possibility would be the same mechanism of increased LPC content in LDL(-).

#### **4.3.3 Proteins**

458 Atherogenesis

2008; Tai, 2006). The specific molecules that mediate these actions are not well defined; LPC and NEFA probably play a major role, but the action of oxidized lipids remains under discussion (Abe, 2007; Benitez, 2004a; Chen, 2004). If they are present in LDL(-) they could stimulate inflammatory responses, but it is also possible that high PAF-AH activity in LDL (-) degrades readily oxidized phospholipids (Benitez, 2003). Their degradation products, LPC and short-chain NEFA, would therefore be responsible for triggering inflammation.

LDL(-) induces citotoxicity and apoptosis in endothelial cells and macrophages by different signaling pathways. The mechanisms involved are well defined, especially in endothelial cells, where LOX-1 signaling inhibits fibroblast growth factor 2 (FGF2) transcription and Akt phosphorilation (Chen, 2003; Chen, 2007; Lu, 2008; Tang, 2008; Yang, 2007). In contrast, macrophage apoptosis involves the Fas/FasL signaling pathway and the activation of the

LDL(-) and PG present a high affinity for binding (Bancells, 2009). This could favor the subendothelial retention of LDL(-) and trigger the inflammatory response. Some characteristics in LDL(-) are involved in this higher affinity. Aggregation of lipoproteins favors PG binding, and LDL(-) has a high tendency to aggregate (Bancells, 2010b). In fact, a subfraction of aggregated LDL(-) is responsible for the binding to PG (Bancells, 2009). This subfraction has an abnormal conformation that exposes an epitope in apoB, known as site

The earliest physical abnormalities reported in LDL(-) were a great heterogeneity in size and density and a high susceptibility to aggregation (Avogaro, 1988). A subpopulation of aggregated LDL(-) has recently been isolated and characterized. This subpopulation, which accounts for only 0.1-0.5 of total LDL in blood, has high affinity to arterial PG (Bancells, 2009). This increased binding seems to be mediated by abnormal conformation of the aminoterminal extreme of apoB (Bancells, 2011). Further evidence of apoB misfolding, in this case affecting LDLr binding, has been obtained by two dimensional nuclear magnetic resonance analyses (Blanco, 2010). LDL(-) is reported to promote aggregation of non-aggregated LDL particles in a process that fits an amyloidogenic model (Parasassi, 2008). It has been reported that the capacity to induce aggregation could be mediated by the PLC-like activity (Bancells, 2010b), although other authors have suggested that plasma sPLA2 could be involved in apoB misfolding (Greco, 2009). Regarding secondary structure of apoB, some authors have reported loss of secondary -helix structures whereas others did not find differences with

Although there are contradictory data regarding differences in the lipid content between native LDL and LDL(-) most studies concur in a higher content of triglycerides, NEFA and LPC in LDL(-) (Cazzolato, 1991; De Castellarnau, 2000; Sanchez-Quesada, 2003; Sevanian, 1997; Yang, 2003). The high content of triglycerides reflects impairment of the VLDL-to-IDL-

native LDL (Asatryan, 2005; Bancells, 2009; Benitez, 2004b; Parasassi, 2001).

**4.2.3 Cytotoxicity and apoptosis** 

transcription factor Nrf2 (Pedrosa, 2010).

Ib, that is an alternative binding site to PGs (Bancells, 2011).

**4.3 Physico-chemical characteristics of LDL(-)** 

**4.2.4 Binding to proteoglycans** 

**4.3.1 Structure** 

**4.3.2 Lipids** 

The most abundant proteins in LDL(-) are apoA-I (0.15 molecules/particle of LDL(-)), apoE (0.22), apoC-III (0.37) and apoA-II (0.14), with a content 3-5 fold higher in LDL(-) than in native LDL (Bancells, 2010a). The role of these proteins is unclear but their relevance is probably low. However, other proteins whose absolute content in LDL(-) is lower, such as apoF (0.06 molecules/particle), apoJ (0.01) or PAF-AH (0.004) (Bancells, 2010a; Yang, 2007), but their relative content compared with native LDL is 10, 20 and 100-fold higher, respectively, could have a much more relevant role. ApoF is the physiological inhibitor of cholesteryl ester transfer protein (Morton, 2008), a protein that regulates the catabolism of the VLDL-IDL-LDL cascade. It could therefore be one cause of impaired LDL(-) maturation. ApoJ (also known as clusterin) is an extracellular chaperone that binds to hydrophobic unfolded proteins, favoring their extracellular clearance (Oda, 1995). ApoJ binds mainly to aggregated LDL(-), supporting the presence of misfolded apoB in this subfraction. The role of PAF-AH in LDL(-) would be to deactivate oxidized phospholipids, but its undesirable effect would be the formation of LPC and short-chain NEFA. It has been suggested that the PLC-like activity present in LDL(-) could act in cooperation with PAF-AH degrading LPC (Bancells, 2010b). Therefore, this would be a mechanism to limit the deleterious effects exerted by minimal LDL oxidation on vascular cells.

#### **4.4 Association of LDL(-) with cardiovascular risk**

The proportion of LDL(-) is increased in a number of pathologic situations having a cardiovascular risk. Familial hypercholesterolemia and hypertriglyceridemia present a proportion of LDL(-) 3-5 fold higher than normolipemic healthy subjects (Sanchez-Quesada, 2002; Sanchez-Quesada, 1999). These results were obtained using ultracentrifugation plus anion-exchange chromatography, both being laborious and time-consuming techniques. This limits their use for routine analysis of lipoprotein profiles. However, more reliable techniques for rapid analysis have recently been developed, including capillary electrophoresis and ELISA (Santo Faulin Tdo, 2008; Zhang, 2009; Zhang, 2008). These methods have confirmed previous data obtained by anion-exchange chromatography. Statin therapy decreases the proportion of LDL(-) but the process is not parallel to the lipidlowering effect. This is because total LDL cholesterol decreases very rapidly (in 2 weeks) whereas LDL(-) decreases more slowly (in up to six months) (Sanchez-Quesada, 1999). This suggests that LDL(-) generation not only depends on lipid metabolism but also on other factors such as chronic inflammation.

Both type 1 and type 2 diabetics have a high proportion of LDL(-) (Moro, 1998; Sanchez-Quesada, 1996; Sanchez-Quesada, 2001; Zhang, 2005). This would suggest that nonenzymatic glycosylation could be involved. However, insulin therapy decreases LDL(-) in type 1 diabetes but not in type 2 diabetes. The different response to insulin treatment has been attributed to differences in the systemic inflammation level, which is higher in type 2 patients. This agrees with the finding that pre-diabetic insulin-resistant subjects with high systemic inflammation also have increased LDL(-) proportion (Zhang, 2005). Therefore,

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hyperglycemia would not promote the increase of LDL(-) directly but through an increase of systemic inflammation. Another group of subjects with a high proportion of LDL(-) are patients with severe renal disease (Ziouzenkova & Sevanian, 2000). It has been described that -tocopherol supplementation decreases LDL(-) in hemodialysis patients (Mafra, 2009). Regarding patients with established coronary disease, it has been shown that LDL(-) is increased in patients with angiographically documented coronary artery disease (Tomasik, 2003). Moreover, acute coronary syndromes, such as unstable angina or acute myocardial infarction, have higher levels of LDL(-) than chronic coronary syndromes (Mello, 2011). These observations support the partial subendothelial origin of LDL(-) and open the possibility for LDL(-) to be used as a biomarker of the progression of atherosclerotic lesions.

#### **5. Conclusion**

LDL is modified by several mechanisms that confer a number of atherogenic properties to these particles. Although it is believed that such modifications are more frequent in the subendothelial space of the artery wall than in blood, different types of modified LDL have been detected in plasma. LDL(-) is a mixture of modified LDL particles that represent the total pool of modified LDL in plasma. The biological and physico-chemical characteristics of LDL(-) and its association with high cardiovascular risk indicate that this lipoprotein plays a direct role in the development of atherosclerosis. However, although statin and insulin treatment decrease the proportion of LDL(-) , the development of a specific therapy for LDL(-) would be of great interest. Another field of research would be the use of LDL(-) as a biomarker. This could be a promising strategy to evaluate the cardiovascular risk and to monitor the success of distinct therapeutic strategies.

#### **6. Acknowledgment**

JLS-Q and SV are members of the 2009-SGR-1205 and of the 2009-SGR-00761 Research Group from the Generalitat de Catalunya, respectively. JLSQ and SV are funded by PI10/00265, PI10/00975 and CP06/00220 from ISCIII/Spanish Ministry of Health.

#### **7. References**


hyperglycemia would not promote the increase of LDL(-) directly but through an increase of systemic inflammation. Another group of subjects with a high proportion of LDL(-) are patients with severe renal disease (Ziouzenkova & Sevanian, 2000). It has been described that -tocopherol supplementation decreases LDL(-) in hemodialysis patients (Mafra, 2009). Regarding patients with established coronary disease, it has been shown that LDL(-) is increased in patients with angiographically documented coronary artery disease (Tomasik, 2003). Moreover, acute coronary syndromes, such as unstable angina or acute myocardial infarction, have higher levels of LDL(-) than chronic coronary syndromes (Mello, 2011). These observations support the partial subendothelial origin of LDL(-) and open the possibility for LDL(-) to be used as a biomarker of the progression of atherosclerotic lesions.

LDL is modified by several mechanisms that confer a number of atherogenic properties to these particles. Although it is believed that such modifications are more frequent in the subendothelial space of the artery wall than in blood, different types of modified LDL have been detected in plasma. LDL(-) is a mixture of modified LDL particles that represent the total pool of modified LDL in plasma. The biological and physico-chemical characteristics of LDL(-) and its association with high cardiovascular risk indicate that this lipoprotein plays a direct role in the development of atherosclerosis. However, although statin and insulin treatment decrease the proportion of LDL(-) , the development of a specific therapy for LDL(-) would be of great interest. Another field of research would be the use of LDL(-) as a biomarker. This could be a promising strategy to evaluate the cardiovascular risk and to

JLS-Q and SV are members of the 2009-SGR-1205 and of the 2009-SGR-00761 Research Group from the Generalitat de Catalunya, respectively. JLSQ and SV are funded by

Abe, Y., Fornage, M., Yang, C.Y., Bui-Thanh, N.A., Wise, V., Chen, H.H., Rangaraj, G. &

Apostolov, E.O., Ray, D., Savenka, A.V., Shah, S.V. & Basnakian, A.G. (2010) Chronic uremia

Apostolov, E.O., Shah, S.V., Ok, E. & Basnakian, A.G. (2007) Carbamylated low-density

*Vasc Biol*. 27, 4, 826-32, 1524-4636 (Electronic), 1079-5642 (Linking).

mediate mononuclear leukocyte adhesion. *Atherosclerosis*. 192, 1, 56-66, Adachi, H. & Tsujimoto, M. (2006) Endothelial scavenger receptors. *Prog Lipid Res*. 45, 5, 379-

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**22** 

*Romania* 

**Oxidized LDL and NO Synthesis as Biomarkers** 

*2Carol Davila – University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest,* 

Atherosclerosis is a complex, multifactorial disease, developed in the arterial wall in response to various forms of injurious stimuli, resulting in excessive inflammatory and fibro-proliferative reactions. The endothelial cells are involved in all stages of atherogenesis and their dysfunction is a key initial event in the atherosclerotic plaque formation (Simionescu, 2007). The vascular endothelium, with its broad spectrum of paracrine and autocrine functions, can be regarded as a multifunctional organ and "chief governor" of body homeostasis. Occupying a strategic location between the blood and tissues, the endothelial cells exist in a "high-risk position" and react progressively to aggressive factors, at first by modulation of the constitutive functions - permeability and biosynthesis (Simionescu & Antohe, 2006; Sima et al., 2009). Atherogenesis is an intricate process involving hyperlipidemia, oxidative stress and vascular inflammation. Among the diversity of mechanisms implicated in the pathogenesis of atherosclerotic vascular diseases two of them have been discovered in parallel and studied extensively: the oxidation of low-density

lipoprotein (LDL) and the synthesis of endothelium-derived nitric oxide (NO).

**1.1 Relationship between oxidized LDL and NO as biomarkers of oxidative stress and** 

Oxidized LDL and NO are recognized to exert contradictory actions within the vascular endothelium microenvironment and to influence the key events in the development of atherosclerosis such as leukocyte adhesion, platelet aggregation and vascular smoothmuscle cell proliferation and migration. While oxidized LDL (oxLDL) - a biomarker of lipoprotein-associated oxidative stress, is identified as a non-traditional pro-atherogenic emerging cardiovascular risk factor, NO is a free radical signal-transducing molecule that maintains the vasodilating tone, modulates *in vitro* lipid peroxidation reactions and alters

Endothelial dysfunction - known to precede the development of atherosclerosis, is a systemic pathological state of the endothelium defined as an imbalance between

**1. Introduction** 

**endothelial dysfunction** 

Corresponding author

 \*

proinflammatory gene expression (Figure 1).

**of Atherogenesis Correlations with** 

Claudia Borsa1, Cristina Ionescu1 and Daniela Gradinaru1,2\* *1Ana Aslan – National Institute of Gerontology and Geriatrics, Bucharest,* 

**Metabolic Profile in Elderly** 


## **Oxidized LDL and NO Synthesis as Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly**

Claudia Borsa1, Cristina Ionescu1 and Daniela Gradinaru1,2\* *1Ana Aslan – National Institute of Gerontology and Geriatrics, Bucharest, 2Carol Davila – University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania* 

#### **1. Introduction**

472 Atherogenesis

Zhang, B., Miura, S., Yanagi, D., Noda, K., Nishikawa, H., Matsunaga, A., Shirai, K., Iwata,

elevated LDL-C levels: the SPECIAL Study. *Atherosclerosis*. 201, 2, 353-9, Ziouzenkova, O., Asatryan, L., Sahady, D., Orasanu, G., Perrey, S., Cutak, B., Hassell, T.,

Ziouzenkova, O., Asatryan, L., Tetta, C., Wratten, M.L., Hwang, J. & Sevanian, A. (2002)

Ziouzenkova, O. & Sevanian, A. (2000) Oxidative modification of low-density lipoprotein

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76,

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Oxidative stress during ex vivo hemodialysis of blood is decreased by a novel hemolipodialysis procedure utilizing antioxidants. *Free Radic Biol Med*. 33, 2, 248-58,

(LDL) in HD patients: role in electronegative LDL formation. *Blood Purif*. 18, 3, 169-

Atherosclerosis is a complex, multifactorial disease, developed in the arterial wall in response to various forms of injurious stimuli, resulting in excessive inflammatory and fibro-proliferative reactions. The endothelial cells are involved in all stages of atherogenesis and their dysfunction is a key initial event in the atherosclerotic plaque formation (Simionescu, 2007). The vascular endothelium, with its broad spectrum of paracrine and autocrine functions, can be regarded as a multifunctional organ and "chief governor" of body homeostasis. Occupying a strategic location between the blood and tissues, the endothelial cells exist in a "high-risk position" and react progressively to aggressive factors, at first by modulation of the constitutive functions - permeability and biosynthesis (Simionescu & Antohe, 2006; Sima et al., 2009). Atherogenesis is an intricate process involving hyperlipidemia, oxidative stress and vascular inflammation. Among the diversity of mechanisms implicated in the pathogenesis of atherosclerotic vascular diseases two of them have been discovered in parallel and studied extensively: the oxidation of low-density lipoprotein (LDL) and the synthesis of endothelium-derived nitric oxide (NO).

#### **1.1 Relationship between oxidized LDL and NO as biomarkers of oxidative stress and endothelial dysfunction**

Oxidized LDL and NO are recognized to exert contradictory actions within the vascular endothelium microenvironment and to influence the key events in the development of atherosclerosis such as leukocyte adhesion, platelet aggregation and vascular smoothmuscle cell proliferation and migration. While oxidized LDL (oxLDL) - a biomarker of lipoprotein-associated oxidative stress, is identified as a non-traditional pro-atherogenic emerging cardiovascular risk factor, NO is a free radical signal-transducing molecule that maintains the vasodilating tone, modulates *in vitro* lipid peroxidation reactions and alters proinflammatory gene expression (Figure 1).

Endothelial dysfunction - known to precede the development of atherosclerosis, is a systemic pathological state of the endothelium defined as an imbalance between

<sup>\*</sup> Corresponding author

Oxidized LDL and NO Synthesis as

2001; Parthasarathy et al., 1999, 2008).

et al., 2000).

**ageing** 

inflammation and oxidation.

Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly 475

The initial event in atherogenesis is the increased transcytosis of low-density lipoprotein, and its subsequent deposition, retention and oxidative modification in the subendothelium. It is followed by the infiltration of activated inflammatory cells from the coronary

The oxLDL is a byproduct of exposure to reactive oxygen species (ROS), and several potential mechanisms have been proposed for LDL oxidation: cell-mediated lipoxygenase and myeloperoxidase activities, non-enzymatic metal ion-mediated oxidation (iron, copper), superoxide generators (xanthine oxidase, NADPH-oxidase), thiol-dependent oxidation,

Oxidatively modified lipoproteins lead to progression of atherosclerosis through macrophages engulfing oxLDL at the level of scavenger receptors, intracellular depositing of cholesterol esters and at last macrophages transformation into foam cells. Also, oxLDL can induce an immune response leading to anti-oxLDL autoantibodies production, which will determine formation of immune complexes (Steinberg et al., 1989; Tsimikas & Witztum,

Endothelium relaxant factor is a central molecule in vascular homeostasis as a modulator of endothelial tone and reactivity, exerting pleiotropic positive effects on the cardiovascular system. Important for the cardiovascular biology is the consumption of NO by reactive oxygen species. Oxidative modification of NO not only leads to reduced bioavailability but

imbalance of protective and aggressive factors (Cai & Harrison, 2000; Schnabel & Blankenberg, 2007). Subsequently, LDL oxidative modifications are made possible through

A key determinant of the pro-oxidant *versus* oxidant-protective influences of NO is the underlying oxidative status of tissue. When NO is in excess of surrounding oxidants, lipid oxidation and monocyte margination into the vascular wall are attenuated, producing antiatherogenic effects. However, when endogenous tissue rates of oxidant production are accelerated or when tissue oxidant defenses become depleted, NO gives rise to secondary oxidizing species that can increase membrane and lipoprotein lipid oxidation as well as foam cell formation in the vasculature, thus promoting proatherogenic effects (Bloodsworth

Therefore, targeting particularly upstream targets – substrates for oxidation and inflammation, will be important to better understand interactions of hyperlipidemia,

Current evidence suggests that endothelial function is an integrative marker of the net effects of damage from traditional and emerging risk factors on the arterial wall and its intrinsic capacity for repair. This endothelial-dependent vascular biology is critical, not only in the initiation and progression of atherosclerosis, but also in the transition from a stable to an unstable disease state with attendant risks. As a result, study of endothelial function has

emerged as an important endpoint in clinical research (Deanfield et al., 2007).

**1.2 Oxidized LDL and NO endothelial synthesis as factors affecting the vascular** 

Diseases of the vascular system have long been considered to be age-related in terms of their onset and progression. Longevity is a vascular question. More than 50 years ago, a famous anatomist – Rudolf Altschul stated that we have the age of our blood vessels: "a man is as

), which further aggravates the

peroxynitrite and other radical generation compounds (Parthasarathy et al., 2008).

circulation into the arterial wall (Hulsmans & Holvoet, 2010).

also produces the toxic oxidant peroxynitrite (ONOO-

simultaneous NO and superoxide anion radical (O2.-) actions.

vasodilating and vasoconstricting substances produced by (or acting on) the endothelium (Deanfield et al., 2007), leading to a reduced vasodilation, and even a proinflammatory and prothrombotic state (Cottone & Cerasola, 2008).

The most important of the vasodilating substances is nitric oxide, characterized as a noneicosanoid component of endothelial-derived relaxation factor (EDRF), which is continuously synthesized by the endothelium under the action of different neurohumoral mediators such as acetylcholine, histamine, bradikinine, vasopressine, thrombine and serotonine (Rubbo et al., 1996).

Fig. 1. Antiatherogenic effects and role of nitric oxide (NO) *versus* proatherogenic actions of oxidized LDL (oxLDL) exerted on vascular endothelium.

NO is produced by a variety of mammalian cells including: vascular endothelial cells, neurons, smooth muscle cells, macrophages, neutrophils, platelets, cardiomyocytes and pulmonary epithelium. The family of three enzymes responsible for the synthesis of NO, nitric oxide synthases (NOSs): endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) require calmodulin binding for their activities. The inducible nitric oxide synthases are transcriptionally regulated by cytokines and redox-sensitive transcriptional factors. Bacterial and parasitic antigens, which potently induce the expression of cytokines, also lead to induction of iNOS gene expression (Rubbo et al., 1996; Lundberg & Weitzberg, 2005).

In the endothelial microenvironment, concurrently, a variety of substances that adversely influence endothelial function have been recognized, including free fatty acids, cytokines such as TNF-, and prooxidant molecules - including oxidized low-density lipoprotein (oxLDL). There are strong evidences for the role of oxidative stress in all stages of atherogenesis. Among different molecular targets affected by oxidative stress associated with hyperlipidemia and hyperglycemia, LDL is one of the most significant because is the major cholesterol carrier in the blood and contains also a relevant amount of polyunsaturated fatty acids (PUFAs) - the major substrate for lipid peroxidation.

vasodilating and vasoconstricting substances produced by (or acting on) the endothelium (Deanfield et al., 2007), leading to a reduced vasodilation, and even a proinflammatory and

The most important of the vasodilating substances is nitric oxide, characterized as a noneicosanoid component of endothelial-derived relaxation factor (EDRF), which is continuously synthesized by the endothelium under the action of different neurohumoral mediators such as acetylcholine, histamine, bradikinine, vasopressine, thrombine and

Fig. 1. Antiatherogenic effects and role of nitric oxide (NO) *versus* proatherogenic actions of

NO is produced by a variety of mammalian cells including: vascular endothelial cells, neurons, smooth muscle cells, macrophages, neutrophils, platelets, cardiomyocytes and pulmonary epithelium. The family of three enzymes responsible for the synthesis of NO, nitric oxide synthases (NOSs): endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) require calmodulin binding for their activities. The inducible nitric oxide synthases are transcriptionally regulated by cytokines and redox-sensitive transcriptional factors. Bacterial and parasitic antigens, which potently induce the expression of cytokines, also lead to

induction of iNOS gene expression (Rubbo et al., 1996; Lundberg & Weitzberg, 2005).

polyunsaturated fatty acids (PUFAs) - the major substrate for lipid peroxidation.

In the endothelial microenvironment, concurrently, a variety of substances that adversely influence endothelial function have been recognized, including free fatty acids, cytokines such as TNF-, and prooxidant molecules - including oxidized low-density lipoprotein (oxLDL). There are strong evidences for the role of oxidative stress in all stages of atherogenesis. Among different molecular targets affected by oxidative stress associated with hyperlipidemia and hyperglycemia, LDL is one of the most significant because is the major cholesterol carrier in the blood and contains also a relevant amount of

oxidized LDL (oxLDL) exerted on vascular endothelium.

prothrombotic state (Cottone & Cerasola, 2008).

serotonine (Rubbo et al., 1996).

The initial event in atherogenesis is the increased transcytosis of low-density lipoprotein, and its subsequent deposition, retention and oxidative modification in the subendothelium. It is followed by the infiltration of activated inflammatory cells from the coronary circulation into the arterial wall (Hulsmans & Holvoet, 2010).

The oxLDL is a byproduct of exposure to reactive oxygen species (ROS), and several potential mechanisms have been proposed for LDL oxidation: cell-mediated lipoxygenase and myeloperoxidase activities, non-enzymatic metal ion-mediated oxidation (iron, copper), superoxide generators (xanthine oxidase, NADPH-oxidase), thiol-dependent oxidation, peroxynitrite and other radical generation compounds (Parthasarathy et al., 2008).

Oxidatively modified lipoproteins lead to progression of atherosclerosis through macrophages engulfing oxLDL at the level of scavenger receptors, intracellular depositing of cholesterol esters and at last macrophages transformation into foam cells. Also, oxLDL can induce an immune response leading to anti-oxLDL autoantibodies production, which will determine formation of immune complexes (Steinberg et al., 1989; Tsimikas & Witztum, 2001; Parthasarathy et al., 1999, 2008).

Endothelium relaxant factor is a central molecule in vascular homeostasis as a modulator of endothelial tone and reactivity, exerting pleiotropic positive effects on the cardiovascular system. Important for the cardiovascular biology is the consumption of NO by reactive oxygen species. Oxidative modification of NO not only leads to reduced bioavailability but also produces the toxic oxidant peroxynitrite (ONOO- ), which further aggravates the imbalance of protective and aggressive factors (Cai & Harrison, 2000; Schnabel & Blankenberg, 2007). Subsequently, LDL oxidative modifications are made possible through simultaneous NO and superoxide anion radical (O2.-) actions.

A key determinant of the pro-oxidant *versus* oxidant-protective influences of NO is the underlying oxidative status of tissue. When NO is in excess of surrounding oxidants, lipid oxidation and monocyte margination into the vascular wall are attenuated, producing antiatherogenic effects. However, when endogenous tissue rates of oxidant production are accelerated or when tissue oxidant defenses become depleted, NO gives rise to secondary oxidizing species that can increase membrane and lipoprotein lipid oxidation as well as foam cell formation in the vasculature, thus promoting proatherogenic effects (Bloodsworth et al., 2000).

Therefore, targeting particularly upstream targets – substrates for oxidation and inflammation, will be important to better understand interactions of hyperlipidemia, inflammation and oxidation.

Current evidence suggests that endothelial function is an integrative marker of the net effects of damage from traditional and emerging risk factors on the arterial wall and its intrinsic capacity for repair. This endothelial-dependent vascular biology is critical, not only in the initiation and progression of atherosclerosis, but also in the transition from a stable to an unstable disease state with attendant risks. As a result, study of endothelial function has emerged as an important endpoint in clinical research (Deanfield et al., 2007).

#### **1.2 Oxidized LDL and NO endothelial synthesis as factors affecting the vascular ageing**

Diseases of the vascular system have long been considered to be age-related in terms of their onset and progression. Longevity is a vascular question. More than 50 years ago, a famous anatomist – Rudolf Altschul stated that we have the age of our blood vessels: "a man is as

Oxidized LDL and NO Synthesis as

NO release and FMD (Davignon & Ganz, 2004).

autooxidizes to yield nitrite (NO2-

**elderly with hyperlipidemia** 

effect on processes involved in atherogenesis.

(NO3-

**2.1 Purpose** 

**2.2 Materials and methods** 

**2.2.1 Study design** 

Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly 477

The NO activity is assessed representatively using a variety of clinical invasive and noninvasive methods among which, the use of acetylcholine that induces endotheliumdependent dilation and smooth muscle–mediated constriction. The coronary artery diameter is compared by quantitative angiography before and after infusion of acetylcholine. The functional status of the coronary microvasculature can also be assessed using intracoronary Doppler ultrasound to measure blood flow in resistance vessels in response to substances that produce either endothelial-dependent or endothelial-independent vasodilation. Another noninvasive method of detecting endothelial dysfunction uses high-resolution ultrasound to measure the brachial artery diameter in response to reactive hyperemia, which stimulates

NO present in the circulation is originating from endothelial, smooth muscle cells, thrombocytes, leukocytes and cardiomiocytes. NO activity is the net result of a balance between its production and its inactivation by oxygen free radicals. NO released "in vivo" by nitric oxide synthase (NOS) activity in endothelial cells and platelets, rapidly

). Because nitrite plus nitrate are relatively stable compounds in blood, their levels may be a biochemical index of systemic NO production. This is convenient because direct *in vivo* measurements of NO can be very difficult due to the extremely low levels and its short half life. When combined measurements of nitrate and nitrite are conducted, this is usually

denoted by the term NOx (Lundberg & Weitzberg, 2005; Hirata et al., 2010).

**2. Study on correlations of oxLDL and NOx with the metabolic profile in** 

The LDL oxidation and nitric oxide are the key mediators involved in all stages of atherosclerosis: initiation, progression and complications. Their role is antagonistic: oxLDL have pro-atherogenic and NO antiatherogenic functions on vascular endothelium (Figure 1). A reduction in NO production or activity has been proposed as major mechanisms of endothelial dysfunction and a contributor to atherosclerosis. The endothelial dysfunction is considered an early marker for atherosclerosis and can be detected before structural changes in the vascular wall. An impairment of NO bioactivity or synthesis will reduce its braking

In the present study we evaluated the levels of circulating oxidized LDL (oxLDL) and the basal plasma levels of the NO metabolic pathway products, NOx (NO2- + NO3-

examined their relationships with the global metabolic profile in a group of elderly patients with hyperlipidemia. We explored the determinants of oxLDL and NOx, as well as the relation between oxLDL and NOx in order to investigate whether the oxLDL/NOx and

The study population included 170 subjects (72 men and 98 women) aged 60 - 70 years, of the patients hospitalized at the Ana Aslan- National Institute of Gerontology and Geriatrics (NIGG), Bucharest, Romania, who were selected according to clinical and biochemical

oxLDL/HDL-cholesterol ratios are more informative than the individual variables.

), which interacts with oxyhemoglobin yielding nitrate

), and

old as his arteries". Senescent cells undergo distinct changes in gene expression that may cause an impairment of cellular function. In endothelial cells these changes result in a phenotype that is pro-inflammatory, pro-atherosclerotic, and prothrombotic. Endothelial cell senescence can be induced by a number of factors implicated in vascular pathologies, particularly by sustained cell replication and oxidative stress (Erusalimski, 2009).

Oxidative stress and inflammation are major determinants of arterial and biological ageing. Recent studies underscore the association between white blood cell (WBC) telomere length, as index of systemic aging, oxidized LDL, and human vascular aging, expressed by the distensibility of the carotid artery. Results showed that higher levels of oxidized LDL are associated with shorter WBC telomeres and increased stiffness of the carotid artery (Nawrot & Staessen, 2008; Nawrot et al., 2010).

Ageing is characterized not only by a reduced arterial compliance and alteration of the contractile properties of the vascular wall, but also by endothelial dysfunction (Alvarez de Sotomayor, et al., 2005; Brandes et al., 2006). At present, there are several reasons to believe that *in vivo* NO synthesis from L-arginine could indeed be impaired in atherosclerosis, hypertension, dyslipidemia, diabetes, obesity, insulin resistance, metabolic syndrome, as well as in ageing (Lind, 2002; Laroia et al., 2003; Hsueh & Quinones, 2003; Holvoet et al., 2003, 2008a; Vickers et al., 2009; Park et al., 2009; Njajou et al., 2009; Huang, 2009; Park et al., 2011; Tabit et al., 2010).

Recent studies support the fact that advancing age increases the LDL susceptibility to oxidation and decreases the nitric oxide availability and bioactivity (Heffernan et al., 2008). Not only LDL but also very low-density lipoprotein (VLDL), beta–VLDL and even HDL undergo oxidative modification that must be taken into consideration in the complex process of atherosclerosis (Parthasarathy et al., 2008). In elderly, higher oxLDL levels were associated with high coronary risk before any clinical manifestation of CHD (Holvoet et al., 2003), and with higher arterial stiffness, independent of cardiovascular disease risk factors (Brinkley et al., 2009). The oxLDL/Apo-B100 ratio and to a lesser extent the oxLDL/LDL-C ratio were significantly negative associated with the flow-mediated-dilation (FMD) of the brachial artery (van der Zwan et al., 2009).

#### **1.3 Methods for measuring the circulating oxidized LDL and NO endothelial synthesis**

Oxidative biomarkers are now showing strong associations with progression of coronary artery disease (CAD) and predict cardiovascular events, suggesting that they may serve as surrogates and may complement diagnostic investigations. Both *in vitro* and *in vivo*, lowdensity lipoprotein (LDL) particles are susceptible to oxidation and peroxidation by all of the causes of oxidative stress. Therefore, oxidized LDL are included among the "downstream markers" of oxidative stress. During the last decade, several monoclonal antibodies have been generated, each recognizing at least a substantial subset of the whole spectrum of oxLDL particles, leading to a myriad of new reports on the relation between circulating ox-LDL and cardiovascularpathological processes (Itabe and Ueda, 2007; Tsimikas, 2006).

Currently used assays for oxLDL detect minimally oxidized LDL particles. In addition, concentrations of oxLDL depend on the sensitivity of LDL to oxidation; small dense LDLs contain smaller amounts of antioxidants and are, therefore, more prone to oxidation. The widely applied sensitive immunoassay quantifying the circulating levels of oxLDL uses a monoclonal antibody – 4E6, directed against oxidized apolipoprotein B-100 moiety of LDL (Rietzschel et al., 2008; Holvoet et al., 2008b).

The NO activity is assessed representatively using a variety of clinical invasive and noninvasive methods among which, the use of acetylcholine that induces endotheliumdependent dilation and smooth muscle–mediated constriction. The coronary artery diameter is compared by quantitative angiography before and after infusion of acetylcholine. The functional status of the coronary microvasculature can also be assessed using intracoronary Doppler ultrasound to measure blood flow in resistance vessels in response to substances that produce either endothelial-dependent or endothelial-independent vasodilation. Another noninvasive method of detecting endothelial dysfunction uses high-resolution ultrasound to measure the brachial artery diameter in response to reactive hyperemia, which stimulates NO release and FMD (Davignon & Ganz, 2004).

NO present in the circulation is originating from endothelial, smooth muscle cells, thrombocytes, leukocytes and cardiomiocytes. NO activity is the net result of a balance between its production and its inactivation by oxygen free radicals. NO released "in vivo" by nitric oxide synthase (NOS) activity in endothelial cells and platelets, rapidly autooxidizes to yield nitrite (NO2- ), which interacts with oxyhemoglobin yielding nitrate (NO3 - ). Because nitrite plus nitrate are relatively stable compounds in blood, their levels may be a biochemical index of systemic NO production. This is convenient because direct *in vivo* measurements of NO can be very difficult due to the extremely low levels and its short half life. When combined measurements of nitrate and nitrite are conducted, this is usually denoted by the term NOx (Lundberg & Weitzberg, 2005; Hirata et al., 2010).

#### **2. Study on correlations of oxLDL and NOx with the metabolic profile in elderly with hyperlipidemia**

The LDL oxidation and nitric oxide are the key mediators involved in all stages of atherosclerosis: initiation, progression and complications. Their role is antagonistic: oxLDL have pro-atherogenic and NO antiatherogenic functions on vascular endothelium (Figure 1). A reduction in NO production or activity has been proposed as major mechanisms of endothelial dysfunction and a contributor to atherosclerosis. The endothelial dysfunction is considered an early marker for atherosclerosis and can be detected before structural changes in the vascular wall. An impairment of NO bioactivity or synthesis will reduce its braking effect on processes involved in atherogenesis.

#### **2.1 Purpose**

476 Atherogenesis

old as his arteries". Senescent cells undergo distinct changes in gene expression that may cause an impairment of cellular function. In endothelial cells these changes result in a phenotype that is pro-inflammatory, pro-atherosclerotic, and prothrombotic. Endothelial cell senescence can be induced by a number of factors implicated in vascular pathologies,

Oxidative stress and inflammation are major determinants of arterial and biological ageing. Recent studies underscore the association between white blood cell (WBC) telomere length, as index of systemic aging, oxidized LDL, and human vascular aging, expressed by the distensibility of the carotid artery. Results showed that higher levels of oxidized LDL are associated with shorter WBC telomeres and increased stiffness of the carotid artery (Nawrot

Ageing is characterized not only by a reduced arterial compliance and alteration of the contractile properties of the vascular wall, but also by endothelial dysfunction (Alvarez de Sotomayor, et al., 2005; Brandes et al., 2006). At present, there are several reasons to believe that *in vivo* NO synthesis from L-arginine could indeed be impaired in atherosclerosis, hypertension, dyslipidemia, diabetes, obesity, insulin resistance, metabolic syndrome, as well as in ageing (Lind, 2002; Laroia et al., 2003; Hsueh & Quinones, 2003; Holvoet et al., 2003, 2008a; Vickers et al., 2009; Park et al., 2009; Njajou et al., 2009; Huang, 2009; Park et al.,

Recent studies support the fact that advancing age increases the LDL susceptibility to oxidation and decreases the nitric oxide availability and bioactivity (Heffernan et al., 2008). Not only LDL but also very low-density lipoprotein (VLDL), beta–VLDL and even HDL undergo oxidative modification that must be taken into consideration in the complex process of atherosclerosis (Parthasarathy et al., 2008). In elderly, higher oxLDL levels were associated with high coronary risk before any clinical manifestation of CHD (Holvoet et al., 2003), and with higher arterial stiffness, independent of cardiovascular disease risk factors (Brinkley et al., 2009). The oxLDL/Apo-B100 ratio and to a lesser extent the oxLDL/LDL-C ratio were significantly negative associated with the flow-mediated-dilation (FMD) of the

**1.3 Methods for measuring the circulating oxidized LDL and NO endothelial synthesis**  Oxidative biomarkers are now showing strong associations with progression of coronary artery disease (CAD) and predict cardiovascular events, suggesting that they may serve as surrogates and may complement diagnostic investigations. Both *in vitro* and *in vivo*, lowdensity lipoprotein (LDL) particles are susceptible to oxidation and peroxidation by all of the causes of oxidative stress. Therefore, oxidized LDL are included among the "downstream markers" of oxidative stress. During the last decade, several monoclonal antibodies have been generated, each recognizing at least a substantial subset of the whole spectrum of oxLDL particles, leading to a myriad of new reports on the relation between circulating ox-LDL and

Currently used assays for oxLDL detect minimally oxidized LDL particles. In addition, concentrations of oxLDL depend on the sensitivity of LDL to oxidation; small dense LDLs contain smaller amounts of antioxidants and are, therefore, more prone to oxidation. The widely applied sensitive immunoassay quantifying the circulating levels of oxLDL uses a monoclonal antibody – 4E6, directed against oxidized apolipoprotein B-100 moiety of LDL

cardiovascularpathological processes (Itabe and Ueda, 2007; Tsimikas, 2006).

particularly by sustained cell replication and oxidative stress (Erusalimski, 2009).

& Staessen, 2008; Nawrot et al., 2010).

brachial artery (van der Zwan et al., 2009).

(Rietzschel et al., 2008; Holvoet et al., 2008b).

2011; Tabit et al., 2010).

In the present study we evaluated the levels of circulating oxidized LDL (oxLDL) and the basal plasma levels of the NO metabolic pathway products, NOx (NO2- + NO3-), and examined their relationships with the global metabolic profile in a group of elderly patients with hyperlipidemia. We explored the determinants of oxLDL and NOx, as well as the relation between oxLDL and NOx in order to investigate whether the oxLDL/NOx and oxLDL/HDL-cholesterol ratios are more informative than the individual variables.

#### **2.2 Materials and methods**

#### **2.2.1 Study design**

The study population included 170 subjects (72 men and 98 women) aged 60 - 70 years, of the patients hospitalized at the Ana Aslan- National Institute of Gerontology and Geriatrics (NIGG), Bucharest, Romania, who were selected according to clinical and biochemical

Oxidized LDL and NO Synthesis as

control (Table 1).

\* p < 0.01; \*\* p < 0.001

lipid profile in the two groups of our interest.

(NOx) compared to the normolipidemic group (Table 1).

**Variables Control Group** 

Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly 479

systemic level with the oxidative stress and endothelial function parameters, patients were divided into two groups: a group with normal lipid profile (TC<200 mg/dL, LDL<130 mg/dL, TG<150 mg/dL; n=45), and a group with high cardiovascular risk lipid profile

We firstly collected anthropometric data (body weight, height), clinical parameters, including systolic blood pressure, body mass index (BMI), fasting plasma glucose and the

Both cardiovascular disease risk and healthy control subjects showed not significantly different values of weight and body mass index, but systolic blood pressure, fasting plasma glucose and triglycerides were significantly higher in hyperlipidemic patients. The HDLcholesterol concentrations of the two groups were comparable. The atherogenic index and the TC /HDL-cholesterol ratio were significantly higher in hyperlipidemic group *versus*

Also, the increased lipid profile group had a significantly higher circulating levels of oxLDL associated with a significant decrease of the plasma nitric oxide metabolic pathway products

**(n = 45)** 

Age (years) 653 664 Sex (males/females) 15/30 55/70 Systolic blood pressure (mmHg) 115.616.0 129.021.5\*\* Diastolic blood pressure (mmHg) 74.69.7 73.512.7 Body mass index (kg/m2 ) 22.52.9 23.25.3 Glucose (mg/dL) 9112 10012\*\* Total cholesterol (mg/dL) 18222 28532\*\* Triglycerides (mg/dL) 7722 10348\*\* LDL-cholesterol (mg/dL) 10524 21435\*\* HDL-cholesterol (mg/dL) 5611 549

Total cholesterol/HDL-C ratio 3.400.83 5.341.11\*\* Atherogenic index (Ai) 0.130.15 0.240.19\*\* Uric acid(mg/dL) 5.872.07 6.091.94 oxLDL (U/L) 71.5113.11 85.5020\*\* NOx (µmol/L) 32.5210.63 23.528.66\*\* oxLDL/HDL cholesterol ratio 1.350.46 1.620.58\* oxLDL/NOx ratio 2.440.92 4.131.87\*\*

Values are expressed as meansstandard deviation LDL, low-density lipoprotein; HDL-C high-density lipoprotein cholesterol; oxLDL, oxidized low-density lipoprotein; NOx, nitric oxide metabolic pathway

Table 1. Clinical characteristics and metabolic variables in control and hyperlipidemic subjects.

products \* p values derived from Student *t* test: significantly different *vs.* control group;

**Hyperlipidemic** 

**Group (n = 125)** 

(TC>200 mg/dL, LDL>130 mg/dL, TG < or > 150 mg/dL; n = 125).

criteria. The subjects did not have diabetes or any liver, kidney, hematological or oncological overt diseases. We selected in a first group 125 subjects with a high cardiovascular risk lipid profile characterized by hypercholesterolemia [serum total cholesterol (TC) > 200 mg/dL and LDL-cholesterol (LDL-C) > 130 mg/dL], associated or not with hypertriglyceridemia [serum triglycerides (TG) < or > 150 mg/dL]. Subjects were not previously diagnosed with cardiovascular disease and were not under treatment with any vasoactive or cardiovascular drugs. None of the patients used lipid-lowering therapy or antioxidants. The second group considered as the control group included 45 apparently healthy subjects with normal lipid profile (TC < 200 mg/dL, LDL-C < 130 mg/dL and TG < 150 mg/dL). Anthropometric and clinical characteristics were collected after a complete clinical examination. All the participants in this study gave their written informed consent, and the study protocol was approved by the Ana Aslan - NIGG ethics committee. All the procedures followed were in accordance with the institutional guidelines. Venous blood samples were drawn after an overnight fast and 24-hours refraining from smoking, caffeinated foods and beverages.

#### **2.2.2 Biochemical methods**

Total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), triglycerides (TG) and glycemia (G) were determinated by standard enzymatic methods. Results were expressed in mg/dL.

The circulating plasma oxLDL was evaluated by a competitive ELISA kit with the monoclonal antibody 4E6 (kit 10-1158-01, Mercodia, Sweden) directed against an epitope in the apolipoprotein B-100 moiety of oxLDL, formed from substitution of lysine residues of apoB-100 with aldehydes (Holvoet et al., 2008). Results were expressed in U/L plasma.

The total amount of plasma stable metabolic pathway products of NO, [NOx, the sum of nitrites and nitrates (NO2- + NO3-)] was determined using the Griess reagent, following the quantitative conversion of nitrates (NO3-) to nitrites (NO2-), with nitrate reductase (kit 23479, SIGMA). Results were expressed in µmols NOx/L plasma. All the biochemical and immunoenzymatic tests were performed on a ChemWell 2190 Analyser (Awareness Technology, USA).

The plasma atherogenic index (Ai) was calculated by the logarithmically transformed ratio of triglycerides on HDL-cholesterol (TG/HDL-C) (1) (Dobiasova, 2006; Dobiasova et al., 2011).

$$\text{Ai} = \log(\text{TG/HDL-C}) \tag{1}$$

#### **2.2.3 Statistical analysis**

Data are expressed as meansSD. The subjects clinical characteristics were compared using the Mann Whitney Wilcoxon non-parametric test. Differences in means of studied parameters between the groups (hyperlipidemic *vs.* control group) were assessed by Student's paired *t* test. The Pearson's correlation test was used to perform bivariate correlation analysis. Multiple regression analysis was performed to evaluate the independent relation between studied parameters using the Statistical Package for Social Sciences software (SPSS) version 15. Significance was defined at the 0.05 level of confidence.

#### **2.3 Results**

The study population included 170 subjects aged 60-70 years. In order to establish the link of the traditional markers for the evaluation of the cardiovascular risk (TC, TG and LDL-C) at

criteria. The subjects did not have diabetes or any liver, kidney, hematological or oncological overt diseases. We selected in a first group 125 subjects with a high cardiovascular risk lipid profile characterized by hypercholesterolemia [serum total cholesterol (TC) > 200 mg/dL and LDL-cholesterol (LDL-C) > 130 mg/dL], associated or not with hypertriglyceridemia [serum triglycerides (TG) < or > 150 mg/dL]. Subjects were not previously diagnosed with cardiovascular disease and were not under treatment with any vasoactive or cardiovascular drugs. None of the patients used lipid-lowering therapy or antioxidants. The second group considered as the control group included 45 apparently healthy subjects with normal lipid profile (TC < 200 mg/dL, LDL-C < 130 mg/dL and TG < 150 mg/dL). Anthropometric and clinical characteristics were collected after a complete clinical examination. All the participants in this study gave their written informed consent, and the study protocol was approved by the Ana Aslan - NIGG ethics committee. All the procedures followed were in accordance with the institutional guidelines. Venous blood samples were drawn after an overnight fast and 24-hours refraining from smoking, caffeinated foods and beverages.

Total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), triglycerides (TG) and glycemia (G) were determinated by standard enzymatic methods. Results were

The circulating plasma oxLDL was evaluated by a competitive ELISA kit with the monoclonal antibody 4E6 (kit 10-1158-01, Mercodia, Sweden) directed against an epitope in the apolipoprotein B-100 moiety of oxLDL, formed from substitution of lysine residues of apoB-100 with aldehydes (Holvoet et al., 2008). Results were expressed in U/L plasma. The total amount of plasma stable metabolic pathway products of NO, [NOx, the sum of

quantitative conversion of nitrates (NO3-) to nitrites (NO2-), with nitrate reductase (kit 23479, SIGMA). Results were expressed in µmols NOx/L plasma. All the biochemical and immunoenzymatic tests were performed on a ChemWell 2190 Analyser (Awareness

The plasma atherogenic index (Ai) was calculated by the logarithmically transformed ratio of triglycerides on HDL-cholesterol (TG/HDL-C) (1) (Dobiasova, 2006; Dobiasova et al., 2011).

Data are expressed as meansSD. The subjects clinical characteristics were compared using the Mann Whitney Wilcoxon non-parametric test. Differences in means of studied parameters between the groups (hyperlipidemic *vs.* control group) were assessed by Student's paired *t* test. The Pearson's correlation test was used to perform bivariate correlation analysis. Multiple regression analysis was performed to evaluate the independent relation between studied parameters using the Statistical Package for Social Sciences software (SPSS) version 15. Significance was defined at the 0.05 level of confidence.

The study population included 170 subjects aged 60-70 years. In order to establish the link of the traditional markers for the evaluation of the cardiovascular risk (TC, TG and LDL-C) at

)] was determined using the Griess reagent, following the

Ai = log(TG/HDL-C) (1)

**2.2.2 Biochemical methods** 

expressed in mg/dL.

nitrites and nitrates (NO2

**2.2.3 Statistical analysis** 

**2.3 Results** 

Technology, USA).


systemic level with the oxidative stress and endothelial function parameters, patients were divided into two groups: a group with normal lipid profile (TC<200 mg/dL, LDL<130 mg/dL, TG<150 mg/dL; n=45), and a group with high cardiovascular risk lipid profile (TC>200 mg/dL, LDL>130 mg/dL, TG < or > 150 mg/dL; n = 125).

We firstly collected anthropometric data (body weight, height), clinical parameters, including systolic blood pressure, body mass index (BMI), fasting plasma glucose and the lipid profile in the two groups of our interest.

Both cardiovascular disease risk and healthy control subjects showed not significantly different values of weight and body mass index, but systolic blood pressure, fasting plasma glucose and triglycerides were significantly higher in hyperlipidemic patients. The HDLcholesterol concentrations of the two groups were comparable. The atherogenic index and the TC /HDL-cholesterol ratio were significantly higher in hyperlipidemic group *versus* control (Table 1).

Also, the increased lipid profile group had a significantly higher circulating levels of oxLDL associated with a significant decrease of the plasma nitric oxide metabolic pathway products (NOx) compared to the normolipidemic group (Table 1).


Values are expressed as meansstandard deviation LDL, low-density lipoprotein; HDL-C high-density lipoprotein cholesterol; oxLDL, oxidized low-density lipoprotein; NOx, nitric oxide metabolic pathway products \* p values derived from Student *t* test: significantly different *vs.* control group; \* p < 0.01; \*\* p < 0.001

Table 1. Clinical characteristics and metabolic variables in control and hyperlipidemic subjects.

Oxidized LDL and NO Synthesis as

**Hyperlipidemic Group (n = 125)** 

Total cholesterol/HDL-C

\*

calculated.

NOx (µmol/L) - 0.143

p < 0.05; \*\* p < 0.01; NS, non-significant

Pearson's correlation coefficients (r)

hyperlipidemic subjects (Table 1).

marker TC/HDL ratio (r= -0.365) (Figure 2).

Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly 481

Total cholesterol (mg/dL) 0.390\*\* 0.298\*\* - 0.340\*\* 0.437\*\* Triglycerides (mg/dL) 0.320\*\* 0.293\*\* 0.037 (NS) 0.128 (NS) LDL-cholesterol (mg/dL) 0.377\*\* 0.391\*\* - 0.315\*\* 0.411\*\* HDL-cholesterol (mg/dL) - 0.445\*\* - 0.762\*\* 0.011 (NS) - 0.223\*

ratio 0.578\*\* 0.796\*\* - 0.191\* 0.416\*\* Atherogenic index (Ai) 0.549\*\* 0.637\*\* 0.054 (NS) 0.220\* Uric acid(mg/dL) 0.270\*\* 0.201\* 0.158 (NS) - 0.006 (NS)

LDL, low-density lipoprotein; HDL-C, high-density lipoprotein cholesterol; oxLDL, oxidized low-

To further estimate the extent of oxLDL involvement in endothelial dysfunction, the ratio of oxLDL to HDL-cholesterol and the newly introduced ratio of oxLDL to NOx, were

Significant differences as regards oxLDL/HDL-cholesterol ratio as well as oxLDL/NOx ratio were found out between the study groups; both ratios were higher in the

Moreover, in the group of subjects with cardiovascular risk, oxLDL/NOx ratio correlated significantly with almost each of the traditional parameters of the metabolic profile, namely: glycemia, total cholesterol, LDL-cholesterol and HDL-cholesterol, as well as the Ai and

We explored the metabolic determinants of oxLDL and NOx by performing the statistical multiple correlation test in the whole study population (n=170). Regarding the oxLDL we identified significant (p < 0.01) positive correlations with each studied parameters of the metabolic profile, such as glycemia (r = 0.351), total cholesterol (r = 0.457), LDL-cholesterol (r = 0.456), triglycerides (r = 0.414) and uric acid (r = 0.253). A strong significant negative

In all 170 subjects we pointed out significantly negative correlations (p < 0.01) of NOx levels and lipid profile: total cholesterol (r = - 0.470), LDL-C (r = - 0.451), and the atherogenic risk

cardiovascular risk markers (TC/HDL-C and oxLDL/HDL-C ratios) (Table 3).

association between oxLDL and HDL-C (r= -0.425, p <0.01) was pointed out.

Table 3. Interrelationships between studied markers of lipoxidative stress, endothelial function, and metabolic profile parameters, in hyperlipidemic subjects, determined as

**ratio NOx oxLDL/NOx** 

(NS) 0.245\*\*

(NS) 0.641\*\*

(NS) 0.546\*\*

(NS) - 0.111 (NS) - 0.729\*\*

**ratio** 

**Variables oxLDL oxLDL/HDL-C** 

Glucose (mg/dL) 0.275\*\* 0.329\*\* - 0.096

oxLDL(U/L) 0.895\*\* - 0.143

oxLDL/HDL-C ratio 0.895\*\* - 0.111

oxLDL/NOx ratio 0.641\*\* 0.546\*\* - 0.729\*\*

density lipoprotein; NOx, nitric oxide metabolic pathway products

To establish which variables other than LDL-C and NO were independent determinants of oxLDL and NO, we explored multiple linear regression models with oxLDL, NOx, oxLDL/HDL-C ratio and oxLDL/NOx ratio, as dependent variables, in control, hyperlipidemia, and whole study population. The results are presented in Table 2 and 3 and Figures 3-6.

In multiple regression analysis for estimating the association between the degree of endothelial dysfunction and metabolic parameters, we found different statistically significant correlations within the two study groups. Tables 2 and 3 show the correlations of oxLDL and NOx with serum metabolic variables, atherogenic markers and indices in normal and hyperlipidemic subjects.

In the control group (table 2) circulating oxLDL level positively correlated with glycemia and triglycerides, as well as the total cholesterol/HDL-cholesterol ratio.

In subjects with high cardio-vascular risk (table 3) significant positive correlations between oxLDL and LDL-cholesterol were pointed out. In both study groups oxLDL was significantly negative correlated with HDL-cholesterol, and significantly positive with the atherogenic index (Ai).


LDL, low-density lipoprotein; HDL-C, high-density lipoprotein cholesterol; oxLDL, oxidized lowdensity lipoprotein; NOx, nitric oxide metabolic pathway products

\* p < 0.05; \*\* p < 0.01; NS, non-significant

Table 2. Interrelationships between studied markers of lipoxidative stress, endothelial function, and metabolic profile parameters, in control subjects, determined as Pearson's correlation coefficients (r).

As regards the nitric oxide metabolic pathway products, the statistical analysis of the data pointed out a significant negative correlation, between NOx and the total cholesterol/HDLcholesterol ratio, but only in the hyperlipidemic group (Table 2 and 3).


LDL, low-density lipoprotein; HDL-C, high-density lipoprotein cholesterol; oxLDL, oxidized lowdensity lipoprotein; NOx, nitric oxide metabolic pathway products

\* p < 0.05; \*\* p < 0.01; NS, non-significant

480 Atherogenesis

To establish which variables other than LDL-C and NO were independent determinants of oxLDL and NO, we explored multiple linear regression models with oxLDL, NOx, oxLDL/HDL-C ratio and oxLDL/NOx ratio, as dependent variables, in control, hyperlipidemia, and whole study population. The results are presented in Table 2 and 3 and

In multiple regression analysis for estimating the association between the degree of endothelial dysfunction and metabolic parameters, we found different statistically significant correlations within the two study groups. Tables 2 and 3 show the correlations of oxLDL and NOx with serum metabolic variables, atherogenic markers and indices in

In the control group (table 2) circulating oxLDL level positively correlated with glycemia

In subjects with high cardio-vascular risk (table 3) significant positive correlations between oxLDL and LDL-cholesterol were pointed out. In both study groups oxLDL was significantly negative correlated with HDL-cholesterol, and significantly positive with the

Glucose (mg/dL) 0.351\* 0.273 (NS) 0.164 (NS) - 0.004 (NS)

(mg/dL) 0.257 (NS) - 0.470\*\* - 0.088 (NS) 0.097 (NS) Triglycerides (mg/dL) 0.358\* 0.273 (NS) - 0.259 (NS) 0.414\*\*

(mg/dL) 0.257 (NS) 0.396\*\* - 0.024 (NS) 0.031 (NS)

(mg/dL) - 0.471\*\* - 0.832\*\* 0.081 (NS) - 0.291 (NS)

<sup>C</sup>0.528\*\* 0.537\*\* - 0.075 (NS) 0.252 (NS) Atherogenic index (Ai) 0.502\*\* 0.605\*\* - 0.267 (NS) 0.234\*\* Uric acid(mg/dL) 0.200 (NS) 0.063 (NS) 0.184 (NS) - 0.045 (NS) oxLDL (U/L) 0.850\*\* 0.074 (NS) 0.404\*\* oxLDL/HDL-C ratio 0.850\*\* 0.045 (NS) 0.357\* NOx (µmol/L) 0.074 (NS) 0.045 (NS) - 0.826\*\*

LDL, low-density lipoprotein; HDL-C, high-density lipoprotein cholesterol; oxLDL, oxidized low-

As regards the nitric oxide metabolic pathway products, the statistical analysis of the data pointed out a significant negative correlation, between NOx and the total cholesterol/HDL-

Table 2. Interrelationships between studied markers of lipoxidative stress, endothelial function, and metabolic profile parameters, in control subjects, determined as Pearson's

**ratio NOx oxLDL/NOx** 

**ratio** 

and triglycerides, as well as the total cholesterol/HDL-cholesterol ratio.

**Variables oxLDL oxLDL/HDL-C** 

oxLDL/NOx ratio 0.404\*\* 0.357\* - 0.826\*\*

cholesterol ratio, but only in the hyperlipidemic group (Table 2 and 3).

density lipoprotein; NOx, nitric oxide metabolic pathway products

Figures 3-6.

normal and hyperlipidemic subjects.

atherogenic index (Ai).

**Control Group (n = 45)** 

Total cholesterol

LDL-cholesterol

HDL-cholesterol

\*

Total cholesterol/HDL-

p < 0.05; \*\* p < 0.01; NS, non-significant

correlation coefficients (r).

Table 3. Interrelationships between studied markers of lipoxidative stress, endothelial function, and metabolic profile parameters, in hyperlipidemic subjects, determined as Pearson's correlation coefficients (r)

To further estimate the extent of oxLDL involvement in endothelial dysfunction, the ratio of oxLDL to HDL-cholesterol and the newly introduced ratio of oxLDL to NOx, were calculated.

Significant differences as regards oxLDL/HDL-cholesterol ratio as well as oxLDL/NOx ratio were found out between the study groups; both ratios were higher in the hyperlipidemic subjects (Table 1).

Moreover, in the group of subjects with cardiovascular risk, oxLDL/NOx ratio correlated significantly with almost each of the traditional parameters of the metabolic profile, namely: glycemia, total cholesterol, LDL-cholesterol and HDL-cholesterol, as well as the Ai and cardiovascular risk markers (TC/HDL-C and oxLDL/HDL-C ratios) (Table 3).

We explored the metabolic determinants of oxLDL and NOx by performing the statistical multiple correlation test in the whole study population (n=170). Regarding the oxLDL we identified significant (p < 0.01) positive correlations with each studied parameters of the metabolic profile, such as glycemia (r = 0.351), total cholesterol (r = 0.457), LDL-cholesterol (r = 0.456), triglycerides (r = 0.414) and uric acid (r = 0.253). A strong significant negative association between oxLDL and HDL-C (r= -0.425, p <0.01) was pointed out.

In all 170 subjects we pointed out significantly negative correlations (p < 0.01) of NOx levels and lipid profile: total cholesterol (r = - 0.470), LDL-C (r = - 0.451), and the atherogenic risk marker TC/HDL ratio (r= -0.365) (Figure 2).

Oxidized LDL and NO Synthesis as

study subjects (n=170)

products (NOx) and the circulating oxidized LDL.

**A B** 

**C** 

markers, in 170 elderly patients.

**2.4 Discussion** 

Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly 483

Fig. 4. oxLDL/NOx ratio is directly correlated with total cholesterol/HDL-cholesterol ratio (A), with oxLDL/HDL-cholesterol ratio (B), and with the Atherogenic index (C) in all the

The purpose of this work was to point to the interrelationships of oxLDL and NO as biomarkers of oxidative stress and endothelial function and the metabolic profile in elderly. We explored the literature on molecular mechanisms involved in the biochemical and metabolic links of NO and oxLDL. We investigated the metabolic determinants of oxLDL and NOx in 170 elderly subjects. Our research results focused mainly on the correlations between lipid and lipoprotein parameters as indices of atherogenic risk, and lipoxidative stress and endothelial dysfunction biomarkers, namely the nitric oxide metabolic pathway

Impairment in NO, a common feature in patients with endothelial dysfunction, is considered to predict atherosclerosis and cardiovascular events. On the other hand, elevated levels of oxidized LDL, formed within the arterial wall, are commonly related to the atherogenic profile (Steinberg, 2009; Steinberg & Witzum, 2010). Therefore, in the present study, we evaluated the relationships of oxLDL and NOx as oxidative stress and endothelial dysfunction biomarkers with the metabolic profile and the cardiovascular high-risk profile

Plasma NOx levels were significantly lower in patients with hyperlipidemia, further suggesting that physiologic levels of NO are necessary to maintain the normal, vasodilatatory and noninflammatory phenotype of the vascular wall. A major finding of this study is that NO release levels measured by its metabolic pathway products significantly

Fig. 2. Plasma nitric oxide metabolic pathway products (NOx) is inversely correlated with total cholesterol/HDL-cholesterol ratio in all the study subjects (n=170)

Finally, it is important to underscore the most interesting significant, negative, correlation, identified between oxLDL and NOx (r = - 0.205, p < 0.01; n = 170) in all study population (Figure 3). In the hyperlipidemic group this association was negative but not significant.

The newly introduced ratio oxLDL/NOx was significantly related to the ratio oxLDL/HDL (r = 0.547, p < 0.01, n=170), the atherogenic index (Ai) and also the total cholesterol/HDLratio (r = 0.478, p < 0.01 and r = 0.537, p < 0.01) (Figure 4 - A, B, C).

Fig. 3. Oxidized LDL (oxLDL) levels are inversely associated with plasma nitric oxide metabolic pathway products (NOx) in all the study subjects (n = 170).

Fig. 4. oxLDL/NOx ratio is directly correlated with total cholesterol/HDL-cholesterol ratio (A), with oxLDL/HDL-cholesterol ratio (B), and with the Atherogenic index (C) in all the study subjects (n=170)

#### **2.4 Discussion**

482 Atherogenesis

Fig. 2. Plasma nitric oxide metabolic pathway products (NOx) is inversely correlated with

Finally, it is important to underscore the most interesting significant, negative, correlation, identified between oxLDL and NOx (r = - 0.205, p < 0.01; n = 170) in all study population (Figure 3). In the hyperlipidemic group this association was negative but not

The newly introduced ratio oxLDL/NOx was significantly related to the ratio oxLDL/HDL (r = 0.547, p < 0.01, n=170), the atherogenic index (Ai) and also the total cholesterol/HDL-

Fig. 3. Oxidized LDL (oxLDL) levels are inversely associated with plasma nitric oxide

metabolic pathway products (NOx) in all the study subjects (n = 170).

total cholesterol/HDL-cholesterol ratio in all the study subjects (n=170)

ratio (r = 0.478, p < 0.01 and r = 0.537, p < 0.01) (Figure 4 - A, B, C).

significant.

The purpose of this work was to point to the interrelationships of oxLDL and NO as biomarkers of oxidative stress and endothelial function and the metabolic profile in elderly. We explored the literature on molecular mechanisms involved in the biochemical and metabolic links of NO and oxLDL. We investigated the metabolic determinants of oxLDL and NOx in 170 elderly subjects. Our research results focused mainly on the correlations between lipid and lipoprotein parameters as indices of atherogenic risk, and lipoxidative stress and endothelial dysfunction biomarkers, namely the nitric oxide metabolic pathway products (NOx) and the circulating oxidized LDL.

Impairment in NO, a common feature in patients with endothelial dysfunction, is considered to predict atherosclerosis and cardiovascular events. On the other hand, elevated levels of oxidized LDL, formed within the arterial wall, are commonly related to the atherogenic profile (Steinberg, 2009; Steinberg & Witzum, 2010). Therefore, in the present study, we evaluated the relationships of oxLDL and NOx as oxidative stress and endothelial dysfunction biomarkers with the metabolic profile and the cardiovascular high-risk profile markers, in 170 elderly patients.

Plasma NOx levels were significantly lower in patients with hyperlipidemia, further suggesting that physiologic levels of NO are necessary to maintain the normal, vasodilatatory and noninflammatory phenotype of the vascular wall. A major finding of this study is that NO release levels measured by its metabolic pathway products significantly

Oxidized LDL and NO Synthesis as

**NO bioavailability**

**ENDOTHELIAL DYSFUNCTION**

NO.

species; HO**.**

Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly 485

oxide and endothelial dysfunction. Hyperlipidemia, oxidative stress and LDL oxidation are harmful at multiple steps in atherogenesis, including direct contributions to endothelial functions. As shown in figure 5, hyperlipidemia induces enhanced oxidative stress, superoxide (O2.-) excessive generation and LDL oxidation. Increased O2.- generation as a result of excess mitochondrial lipid oxidation, LDL oxidation and other sources, is critically involved in reduced NO bioactivity and endothelial dysfunction, by direct elimination of

**NO direct elimination**

LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; FFA, free fatty acids; ApoB-100, apolipoprotein B-100; H2O2, hydrogen peroxide; SOD, superoxide dismutase, ROS, reactive oxygen

Fig. 5. Simplified scheme of the interrelationships between oxidative stress, LDL oxidation

There is abundant experimental evidence indicating the role of NO oxidative inactivation as a mediator of endothelial dysfunction and a pre-pathogenic vascular phenotype (Harrison,

their reaction to generate ONOO- occurs three times faster than the O2.- elimination by

, alkoxyl radical; LOO**.**

1997; Bermudez et al., 2008). The NO is the kinetically preferred scavenger for O2

and NO in the hyperlipidemic state leading to endothelial dysfunction.

*Lipoxygenase Myeloperoxidase Fe2+, Cu2+*

**.- oxLDL**

**+ O2 .-**

**decreased NO by increased oxLDL**

> **O2 .- ONOO-LOO. LO. HO.**

**HYPERLIPIDEMIA**

**LDL FFA**

**Oxidative stress**

*Xanthine oxidase NADH/NADPH oxidase*

> **BH4 oxidation**

**"Uncoupled" eNOS**

**SOD**

**ApoB-100**

**NO**

, peroxynitrite; eNOS,

.-, because

**production**

**H2O2**

**O2**

**ROS**

, peroxyl radical; ONOO**-**

**NO.**

**L- Arginine L- Citrulline**

endothelial nitric oxide synthase; BH4, tetrahydrobiopterin.

, hydroxyl radical; LO**.**

**eNOS BH4 Ca2+-calmodulin**

negatively correlated with circulating oxLDL concentrations. Overall, this work pointed out the link between the vascular endothelium vasodilating/vasoconstricting imbalance and the metabolic profile in hyperlipidemic elderly patients.

The evaluations of LDL-cholesterol, high-density lipoprotein-cholesterol, and triglycerides are the traditionally recommended lipid screening tests for coronary heart disease (CHD). Several studies do suggest that total cholesterol/HDL-cholesterol ratio, a major lipid index, is better than the individual total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides parameters (Lemieux et al., 2001). This ratio is easily obtained and one of the most powerful important risk factors for CHD. Both oxLDL and NOx significantly correlated with all markers and calculated atherogenic indices (TC/HDL-C, oxLDL/HDL-C and Ai). Hence, our data support the fact that measurements of the oxLDL and NOx levels at different times may help to monitor the state and severity of endothelial dysfunction.

Based on the multiple correlations analysis in both study groups and all subjects we found that TC, TC/HDL-C, and oxLDL/HDL-C ratios are major determinants of oxLDL and NO. These associations are stronger for the newly introduced oxLDL/NOx ratio. As well, the oxLDL/NOx ratio is strongly correlated with the atherogenic index and more importantly, with the oxLDL/HDL-C ratio, the best lipid biomarker used for discriminating between coronary artery disease patients and healthy control subjects, and also the best blood biomarker that reflects atherosclerotic disease activity in the arterial wall (Huang et al., 2008; Lankin et al., 2011)

Our results are in accordance with literature with regard to the damaging effects of hyperlipidemia, mediated or stimulated by oxidative stress. Numerous studies have supported the role of hyperlipidemia in atherosclerosis, endothelial dysfunction and progression of coronary heart diseases (Wallace et al., 2010; Deanfield et al., 2007; Highashino et al., 2010; Van den Oever et al., 2010). The hypothesised mechanisms for this effect are via hyperlipemia-induced oxidative stress, especially LDL oxidation and subsequent reduced NO bioavailibility. The strong significant association of oxidized LDL with plasma lipid profile (TC, LDL-C, TG), atherogenic risk markers (TC/HDL-c, oxLDL/HDL) and atherogenic index (Ai) found out in the hyperlipidemic group as well as the whole population studied, underscore the validity of the observation that hyperlipemia induces LDL oxidation and oxidative stress. The oxidative stress generates the superoxide radicals (O2 .-), which are scavenged by nitric oxide to form peroxynitrite (ONOO-), a powerful oxidant. The overproduction of O2.- has direct and indirect effects on vascular NO bioavailability. Moreover, O2.- and ONOO. can oxidize tetrahydrobiopterin (BH4), the cofactor necessary for NO production by eNOS enzyme, leading to eNOS uncoupling, and thus to more O2.- generation and reduced NO production. Also, the significant negative correlation found out in this study between oxLDL and NOx shows that the excess of LDL oxidation itself may contribute to reduce NO level. Taken toghether, hyperlipidemia, oxidative stress and LDL oxidation result in reduced NO bioavailability via combinatory effects of direct elimination and decreased production of NO. This NO reduced bioavailability compromises all the antiatherogenic functions of the endothelium. This hypothesised mechanism shown above could be a target for interventions to protect against hyperlipidemia-induced atherogenesis and cardiovascular disease.

Based on the strong interrelationships pointed out in this clinical study and the numerous experimental and clinical research in the field of atherosclerosis we summarize in figure 5 the important relationships among hyperlipidemia, oxidative stress, LDL oxidation, nitric

negatively correlated with circulating oxLDL concentrations. Overall, this work pointed out the link between the vascular endothelium vasodilating/vasoconstricting imbalance and the

The evaluations of LDL-cholesterol, high-density lipoprotein-cholesterol, and triglycerides are the traditionally recommended lipid screening tests for coronary heart disease (CHD). Several studies do suggest that total cholesterol/HDL-cholesterol ratio, a major lipid index, is better than the individual total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides parameters (Lemieux et al., 2001). This ratio is easily obtained and one of the most powerful important risk factors for CHD. Both oxLDL and NOx significantly correlated with all markers and calculated atherogenic indices (TC/HDL-C, oxLDL/HDL-C and Ai). Hence, our data support the fact that measurements of the oxLDL and NOx levels at different times may help to monitor the state and severity of endothelial dysfunction. Based on the multiple correlations analysis in both study groups and all subjects we found that TC, TC/HDL-C, and oxLDL/HDL-C ratios are major determinants of oxLDL and NO. These associations are stronger for the newly introduced oxLDL/NOx ratio. As well, the oxLDL/NOx ratio is strongly correlated with the atherogenic index and more importantly, with the oxLDL/HDL-C ratio, the best lipid biomarker used for discriminating between coronary artery disease patients and healthy control subjects, and also the best blood biomarker that reflects atherosclerotic disease activity in the arterial wall (Huang et al., 2008;

Our results are in accordance with literature with regard to the damaging effects of hyperlipidemia, mediated or stimulated by oxidative stress. Numerous studies have supported the role of hyperlipidemia in atherosclerosis, endothelial dysfunction and progression of coronary heart diseases (Wallace et al., 2010; Deanfield et al., 2007; Highashino et al., 2010; Van den Oever et al., 2010). The hypothesised mechanisms for this effect are via hyperlipemia-induced oxidative stress, especially LDL oxidation and subsequent reduced NO bioavailibility. The strong significant association of oxidized LDL with plasma lipid profile (TC, LDL-C, TG), atherogenic risk markers (TC/HDL-c, oxLDL/HDL) and atherogenic index (Ai) found out in the hyperlipidemic group as well as the whole population studied, underscore the validity of the observation that hyperlipemia induces LDL oxidation and oxidative stress. The oxidative stress generates the superoxide radicals (O2.-), which are scavenged by nitric oxide to form peroxynitrite (ONOO-), a

cofactor necessary for NO production by eNOS enzyme, leading to eNOS uncoupling, and thus to more O2.- generation and reduced NO production. Also, the significant negative correlation found out in this study between oxLDL and NOx shows that the excess of LDL oxidation itself may contribute to reduce NO level. Taken toghether, hyperlipidemia, oxidative stress and LDL oxidation result in reduced NO bioavailability via combinatory effects of direct elimination and decreased production of NO. This NO reduced bioavailability compromises all the antiatherogenic functions of the endothelium. This hypothesised mechanism shown above could be a target for interventions to protect against

Based on the strong interrelationships pointed out in this clinical study and the numerous experimental and clinical research in the field of atherosclerosis we summarize in figure 5 the important relationships among hyperlipidemia, oxidative stress, LDL oxidation, nitric

.- has direct and indirect effects on vascular NO

can oxidize tetrahydrobiopterin (BH4), the

metabolic profile in hyperlipidemic elderly patients.

Lankin et al., 2011)

powerful oxidant. The overproduction of O2

bioavailability. Moreover, O2.- and ONOO.

hyperlipidemia-induced atherogenesis and cardiovascular disease.

oxide and endothelial dysfunction. Hyperlipidemia, oxidative stress and LDL oxidation are harmful at multiple steps in atherogenesis, including direct contributions to endothelial functions. As shown in figure 5, hyperlipidemia induces enhanced oxidative stress, superoxide (O2 .-) excessive generation and LDL oxidation. Increased O2 .- generation as a result of excess mitochondrial lipid oxidation, LDL oxidation and other sources, is critically involved in reduced NO bioactivity and endothelial dysfunction, by direct elimination of NO.

LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; FFA, free fatty acids; ApoB-100, apolipoprotein B-100; H2O2, hydrogen peroxide; SOD, superoxide dismutase, ROS, reactive oxygen species; HO**.** , hydroxyl radical; LO**.** , alkoxyl radical; LOO**.** , peroxyl radical; ONOO**-** , peroxynitrite; eNOS, endothelial nitric oxide synthase; BH4, tetrahydrobiopterin.

Fig. 5. Simplified scheme of the interrelationships between oxidative stress, LDL oxidation and NO in the hyperlipidemic state leading to endothelial dysfunction.

There is abundant experimental evidence indicating the role of NO oxidative inactivation as a mediator of endothelial dysfunction and a pre-pathogenic vascular phenotype (Harrison, 1997; Bermudez et al., 2008). The NO is the kinetically preferred scavenger for O2 .-, because their reaction to generate ONOO- occurs three times faster than the O2.- elimination by

Oxidized LDL and NO Synthesis as

wall without altering eNOS expression (Wang et al., 2011).

human coronary artery endothelial cells (Yu et al., 2011).

NO degradation by hyperlipidemia, oxidative stress and LDL oxidation.

Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly 487

Recent studies demonstrated that oxLDL causes impairment of endothelium-dependent, nitric oxide-mediated vasodilation involving L-arginine deficiency. The oxLDL may reduce L-arginine availability to eNOS for NO production, by up-regulating arginase. The experimental studies indicated that oxLDL increased arginase expression in the vascular

Experimental studies underscore the dual role of oxLDL on endothelial cells causing either proliferation or apoptosis, depending on its concentration and exposure time (Galle et al., 2001). Thus, oxLDL induced proliferation at low (5 to 10 microg/mL) and apoptosis at higher concentrations (50 to 300 microg/mL). Both effects are mediated by O2.- formation via NADPH oxidase as it major source. Thus, oxLDL contributes importantly to vascular cellular turnover through the induction of oxidative stress. More recently, was demonstrated that oxLDL at low concentrations (5 microg/mL) promotes *in vitro* angiogenesis and activate nitric oxide synthase through Pl3K/Akt/eNOS pathway in

On the whole, the decline in nitric oxide bioavailability is caused by the cummulative effects of many factors and processes discussed above: the decreased expression of the endothelial NO synthase, a reduction of substrate or cofactors for eNOS, alterations of cellular signaling, eNOS inhibition by asymmetric demethyl arginine, reduced NO production and accelerated

Taking into account overall the atherogenic properties of oxidized LDL, involved in all stages of atherosclerosis (Steinberg et al., 1989; Steinberg, 2009), and the vasoprotective and antioxidant functions of NO (Bermudez et al., 2008; Yasa & Turkseven, 2005), we introduced for the first time the ratio oxLDL to NOx for quantifying their possible cumulative effect on vascular endothelium. The strong positive associations of this ratio with the atherogenic index and the atherogenic risk markers: TC/HDL and oxLDL/HDL ratios, supported us to propose this newly introduced ratio (oxLDL/NOx) as a potential marker of endothelial dysfunction. The future in depth studies, will take into consideration the association with clinical parameters of vascular endothelial functions using acethylcholine to induce endothelium dependent dilation, quantitative angiography, and high resolution ultrasound

to measure brachial artery diameter, to further support this new candidate marker.

function and predicts CHD independently of the lipid markers.

oxidative stress in the endothelium.

Wu et al., (2006) suggested in a prospective cohort study that circulating oxLDL as an individual parameter, measured with antibody 4E6, was not an independent overall predictor of coronary heart disease (CHD), after adjustment of lipid markers and less predictive in development of CHD than apoB and total cholesterol/HDL-cholesterol ratio (Wu et al., 2006). Therefore, based on the results obtained in our study it is important to examine in future research whether the ratio oxLDL to NOx correlates with endothelial

Data of this study support the relevance of oxLDL and NOx as biomarkers reflecting, at systemic level, the progressive damage at cellular level under the action of prooxidant pathogenic factors. These biomarkers could be valuable in the complex evaluation of

Despite numerous evidences of oxidative processes involved in atherosclerosis and the multiple experimental research on their inhibition by traditional antioxidants, and the success in several animal trials, the human clinical trials using antioxidants have failed (Parthasarathy et al., 2008; da Luz et al., 2006). There were not taken into consideration all the factors, aspects, processes, steps and stages involved in early or advanced atherosclerotic

superoxide dismutase (SOD) (Beckman & Koppenol, 1996; Cai & Harrison, 2000). Both excess generation of reactive oxygen species (ROS) including O2.- and oxidized LDL, and decreased antioxidant defence mechanisms contribute to enhanced degradation of NO. Many studies support the role of the O2.- as an essential element in the decrease of NO bioavailability in oxidative stress conditions. Thus, studies on rabbbits with aortic atherosclerosis, demonstrated a remarkable decrease in endothelium-related relaxation, which was corrected by SOD treatment (Dulak et al., 1997).

Also, peroxynitrite is itself a powerful oxidant which contributes to enhance oxidative stress and turn the balance NO - ROS in the favour of ROS. Both radicals, O2.- and ONOOcan oxidize tetrahydrobiopterin (BH4) leads to eNOS uncoupling, which in turn will produce O2.- instead of NO, and activate this vicious cycle (Fostermann, 2006). Uncoupling eNOS directly leading to decreased NO production. Not only BH4 oxidation, but also decreases in BH4 concentrations may reduce the NO production. Thus, many studies have shown a significant decrease in BH4 activity in various pathological states, such as: hyperlipidemia, hypercholesterolemia, insulin resistance, probably through the oxLDL increase, as well as increased expression in some proinflammatory cytokine (TNFalpha, interleukin-1 beta) (Bowers et al., 2011; Wever et al., 1997; Stroes et al., 1997). Furthermore, clinical and experimental studies have confirmed these mechanisms, showing that acute administration of BH4 improve the endothelial dysfunctions related to hyperlipidemia, atherosclerosis and hypertension (Setoguchi et al., 2001). Also, a decrease in arginine and consequently a lack in eNOS substrate bioavailability leads to a failure in NO synthesis (Bermudez et al, 2005).

Recent *in vitro* studies (Bowers et al., 2011) demonstrated that tetrahydrobiopterin (BH4) could reduce oxLDL-induced O2.- production by NADPH oxidase, increasing NO synthesis in endothelial cells. The superoxide anion production was increased by pretreatment of cells with an inhibitor of BH4 synthesis, and decreased following pretreatment with a BH4 precursor. Thus, BH4 concentrations can modulate the NADPH oxidase-induced imbalance of endothelial NO and O2.- production. BH4 may be critical in combating oxidative stress, restoring proper redox state and reducing risk for cardiovascular disease including atherosclerosis.

Other mechanisms are also involved in the interrelations of LDL oxidation, nitric oxide and endothelial dysfunction. The oxidized LDL may reduce eNOS levels by inhibiting eNOS gene expression (Dulak et al., 1997) and also can displace eNOS from caveolae by binding to endothelial cell CD36 receptors and by depleting caveolae cholesterol content and therefore disrupt eNOS activity (Barbato et al., 2004). These adverse effects of oxLDL are prevented by HDL via binding to scavenger receptor BI (SR-BI), colocalized with eNOS in endothelial caveolae. This occurs through the maintenance of caveolae cholesterol content by cholesterol ester uptake from HDL. Moreover, HDL binding to SR-BI may stimulate eNOS activity in endothelial cells, and enhance endothelium- and NO-dependent relaxation. Thus, lipoproteins have potent effects on eNOS function in caveolae via actions on both membrane cholesterol homeostasis and the level of activation of the enzyme, processes that may be critically involved in the earliest phases of atherogenesis (Rigotti et al., 1997; Uittenbogaraard et al., 2000; Yuhanna et al., 2001; Schaul, 2003). The significant negative correlations between HDL and oxLDL, oxLDL/HDL ratio, atherogenic index and more important oxLDL/NO ratio pointed out in hyperlipidemic group and all subjects, underscore the beneficial effect of HDL on the endothelium.

superoxide dismutase (SOD) (Beckman & Koppenol, 1996; Cai & Harrison, 2000). Both excess generation of reactive oxygen species (ROS) including O2.- and oxidized LDL, and decreased antioxidant defence mechanisms contribute to enhanced degradation of NO. Many studies support the role of the O2.- as an essential element in the decrease of NO bioavailability in oxidative stress conditions. Thus, studies on rabbbits with aortic atherosclerosis, demonstrated a remarkable decrease in endothelium-related relaxation,

Also, peroxynitrite is itself a powerful oxidant which contributes to enhance oxidative stress and turn the balance NO - ROS in the favour of ROS. Both radicals, O2.- and ONOOcan oxidize tetrahydrobiopterin (BH4) leads to eNOS uncoupling, which in turn will produce O2.- instead of NO, and activate this vicious cycle (Fostermann, 2006). Uncoupling eNOS directly leading to decreased NO production. Not only BH4 oxidation, but also decreases in BH4 concentrations may reduce the NO production. Thus, many studies have shown a significant decrease in BH4 activity in various pathological states, such as: hyperlipidemia, hypercholesterolemia, insulin resistance, probably through the oxLDL increase, as well as increased expression in some proinflammatory cytokine (TNFalpha, interleukin-1 beta) (Bowers et al., 2011; Wever et al., 1997; Stroes et al., 1997). Furthermore, clinical and experimental studies have confirmed these mechanisms, showing that acute administration of BH4 improve the endothelial dysfunctions related to hyperlipidemia, atherosclerosis and hypertension (Setoguchi et al., 2001). Also, a decrease in arginine and consequently a lack in eNOS substrate bioavailability leads to a failure in

Recent *in vitro* studies (Bowers et al., 2011) demonstrated that tetrahydrobiopterin (BH4) could reduce oxLDL-induced O2.- production by NADPH oxidase, increasing NO synthesis in endothelial cells. The superoxide anion production was increased by pretreatment of cells with an inhibitor of BH4 synthesis, and decreased following pretreatment with a BH4 precursor. Thus, BH4 concentrations can modulate the NADPH oxidase-induced imbalance of endothelial NO and O2.- production. BH4 may be critical in combating oxidative stress, restoring proper redox state and reducing risk for cardiovascular disease including

Other mechanisms are also involved in the interrelations of LDL oxidation, nitric oxide and endothelial dysfunction. The oxidized LDL may reduce eNOS levels by inhibiting eNOS gene expression (Dulak et al., 1997) and also can displace eNOS from caveolae by binding to endothelial cell CD36 receptors and by depleting caveolae cholesterol content and therefore disrupt eNOS activity (Barbato et al., 2004). These adverse effects of oxLDL are prevented by HDL via binding to scavenger receptor BI (SR-BI), colocalized with eNOS in endothelial caveolae. This occurs through the maintenance of caveolae cholesterol content by cholesterol ester uptake from HDL. Moreover, HDL binding to SR-BI may stimulate eNOS activity in endothelial cells, and enhance endothelium- and NO-dependent relaxation. Thus, lipoproteins have potent effects on eNOS function in caveolae via actions on both membrane cholesterol homeostasis and the level of activation of the enzyme, processes that may be critically involved in the earliest phases of atherogenesis (Rigotti et al., 1997; Uittenbogaraard et al., 2000; Yuhanna et al., 2001; Schaul, 2003). The significant negative correlations between HDL and oxLDL, oxLDL/HDL ratio, atherogenic index and more important oxLDL/NO ratio pointed out in hyperlipidemic group and all subjects,

which was corrected by SOD treatment (Dulak et al., 1997).

underscore the beneficial effect of HDL on the endothelium.

NO synthesis (Bermudez et al, 2005).

atherosclerosis.

Recent studies demonstrated that oxLDL causes impairment of endothelium-dependent, nitric oxide-mediated vasodilation involving L-arginine deficiency. The oxLDL may reduce L-arginine availability to eNOS for NO production, by up-regulating arginase. The experimental studies indicated that oxLDL increased arginase expression in the vascular wall without altering eNOS expression (Wang et al., 2011).

Experimental studies underscore the dual role of oxLDL on endothelial cells causing either proliferation or apoptosis, depending on its concentration and exposure time (Galle et al., 2001). Thus, oxLDL induced proliferation at low (5 to 10 microg/mL) and apoptosis at higher concentrations (50 to 300 microg/mL). Both effects are mediated by O2 .- formation via NADPH oxidase as it major source. Thus, oxLDL contributes importantly to vascular cellular turnover through the induction of oxidative stress. More recently, was demonstrated that oxLDL at low concentrations (5 microg/mL) promotes *in vitro* angiogenesis and activate nitric oxide synthase through Pl3K/Akt/eNOS pathway in human coronary artery endothelial cells (Yu et al., 2011).

On the whole, the decline in nitric oxide bioavailability is caused by the cummulative effects of many factors and processes discussed above: the decreased expression of the endothelial NO synthase, a reduction of substrate or cofactors for eNOS, alterations of cellular signaling, eNOS inhibition by asymmetric demethyl arginine, reduced NO production and accelerated NO degradation by hyperlipidemia, oxidative stress and LDL oxidation.

Taking into account overall the atherogenic properties of oxidized LDL, involved in all stages of atherosclerosis (Steinberg et al., 1989; Steinberg, 2009), and the vasoprotective and antioxidant functions of NO (Bermudez et al., 2008; Yasa & Turkseven, 2005), we introduced for the first time the ratio oxLDL to NOx for quantifying their possible cumulative effect on vascular endothelium. The strong positive associations of this ratio with the atherogenic index and the atherogenic risk markers: TC/HDL and oxLDL/HDL ratios, supported us to propose this newly introduced ratio (oxLDL/NOx) as a potential marker of endothelial dysfunction. The future in depth studies, will take into consideration the association with clinical parameters of vascular endothelial functions using acethylcholine to induce endothelium dependent dilation, quantitative angiography, and high resolution ultrasound to measure brachial artery diameter, to further support this new candidate marker.

Wu et al., (2006) suggested in a prospective cohort study that circulating oxLDL as an individual parameter, measured with antibody 4E6, was not an independent overall predictor of coronary heart disease (CHD), after adjustment of lipid markers and less predictive in development of CHD than apoB and total cholesterol/HDL-cholesterol ratio (Wu et al., 2006). Therefore, based on the results obtained in our study it is important to examine in future research whether the ratio oxLDL to NOx correlates with endothelial function and predicts CHD independently of the lipid markers.

Data of this study support the relevance of oxLDL and NOx as biomarkers reflecting, at systemic level, the progressive damage at cellular level under the action of prooxidant pathogenic factors. These biomarkers could be valuable in the complex evaluation of oxidative stress in the endothelium.

Despite numerous evidences of oxidative processes involved in atherosclerosis and the multiple experimental research on their inhibition by traditional antioxidants, and the success in several animal trials, the human clinical trials using antioxidants have failed (Parthasarathy et al., 2008; da Luz et al., 2006). There were not taken into consideration all the factors, aspects, processes, steps and stages involved in early or advanced atherosclerotic

Oxidized LDL and NO Synthesis as

**4. Acknowledgements** 

the manuscript.

**5. References** 

1188

0194911X

840-844, ISSN 0009-7300

(May 2008), pp. 86-99, ISSN 1573-4021

Biomarkers of Atherogenesis Correlations with Metabolic Profile in Elderly 489

approaches in the prevention and treatment of atherosclerosis based on improving NO

The authors thank Professor Sampath Parthasarathy for comments that greatly improved

The authors are grateful for the support provided by the 7th Framework Program (FP7) European Study "MARK-AGE, European Study to Establish Biomarkers of Human Ageing".

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bioactivity and reducing LDL oxidation may become a challenge for future studies.

lesions, their interrelations, and the most important the pro-oxidant properties and actions of antioxidants in different oxidative process steps and disease stages.

These interrelatioships pointed out in our study could be very important in the management of new effective therapeutic strategies for atherosclerosis and cardiovascular disease. Because oxidative stress, LDL oxidation and endothelial dysfunction centrally contributes to cardiovascular disease, further sustained efforts must be undertaken to translate this knowledge into the characterization and identification of biomarkers that enable preventive or early detection of injuries and allow improved risk stratification by integration into cardiovascular risk stratification models.

#### **2.5 Limitations of our study**

The study population included only elderly and therefore the results may be different in other age-groups subjects, in order to have identified the early onset of hyperlipidemiainduced vascular impairment. Another important limitation was that we did not evaluate the endothelial function using the flow-mediated dilation (FMD) and ultrasound examination of the right brachial artery.

#### **3. Conclusion**

The results of this correlations study pointed out that in hyperlipidemic elderly patients the endothelial NO synthesis could indeed be impaired and associated with a higher oxidative stress exerted on circulating LDL particles. Oxidized LDL has a large range of biological effects that contribute to atherogenesis, but NO also has many biological effects that prevent atherogenesis. In this context, the interrelations pointed out between hyperlipidemia, oxidative stress, LDL oxidation and nitric oxide leading to endothelial dysfunctions, emphasized their implications in molecular mechanisms of endothelial dysfunction.

It is important to distinguish between the effect of oxidized LDL and the effect of a deficiency in the release of NO and to draw a link between these two biomarkers. According to the results obtained in this study, we propose the use of a new marker of endothelial dysfunction, the ratio of oxLDL to NOx, which could be a more accurate estimation of the *in vivo* cumulative implications of oxLDL and NO in atherogenesis. Future studies taking into account the association of this newly introduced marker with other markers of endothelial function will be undertaken to support the marker validity.

The strong interrelations pointed out in our study underscore the molecular mechanisms implicated in endothelial dysfunctions and atherosclerosis presented in this chapter. Future research is needed to translate this knowledge into the identification, characterization and validation of new and known biomarkers of lipoxidative stress-induced endothelial dysfunctions and atherosclerosis, and their integration into cardiovascular risk stratifications models.

These findings suggest the importance of understanding the senescent specific changes occurring in endothelium associated with age-related disease. Such an understanding may not only provide answers regarding mechanisms of disease development, but may also provide biomarkers of endothelium specific ageing.

As perspectives, the nutritional and therapeutic strategies should attempt to correct the lipid profile and lipoxidative stress in order to prevent the amplification of redox and inflammatory phenomena that lead to increased cardiovascular risk. As well, therapeutic approaches in the prevention and treatment of atherosclerosis based on improving NO bioactivity and reducing LDL oxidation may become a challenge for future studies.

#### **4. Acknowledgements**

The authors thank Professor Sampath Parthasarathy for comments that greatly improved the manuscript.

The authors are grateful for the support provided by the 7th Framework Program (FP7) European Study "MARK-AGE, European Study to Establish Biomarkers of Human Ageing".

#### **5. References**

488 Atherogenesis

lesions, their interrelations, and the most important the pro-oxidant properties and actions

These interrelatioships pointed out in our study could be very important in the management of new effective therapeutic strategies for atherosclerosis and cardiovascular disease. Because oxidative stress, LDL oxidation and endothelial dysfunction centrally contributes to cardiovascular disease, further sustained efforts must be undertaken to translate this knowledge into the characterization and identification of biomarkers that enable preventive or early detection of injuries and allow improved risk stratification by integration into

The study population included only elderly and therefore the results may be different in other age-groups subjects, in order to have identified the early onset of hyperlipidemiainduced vascular impairment. Another important limitation was that we did not evaluate the endothelial function using the flow-mediated dilation (FMD) and ultrasound

The results of this correlations study pointed out that in hyperlipidemic elderly patients the endothelial NO synthesis could indeed be impaired and associated with a higher oxidative stress exerted on circulating LDL particles. Oxidized LDL has a large range of biological effects that contribute to atherogenesis, but NO also has many biological effects that prevent atherogenesis. In this context, the interrelations pointed out between hyperlipidemia, oxidative stress, LDL oxidation and nitric oxide leading to endothelial dysfunctions,

It is important to distinguish between the effect of oxidized LDL and the effect of a deficiency in the release of NO and to draw a link between these two biomarkers. According to the results obtained in this study, we propose the use of a new marker of endothelial dysfunction, the ratio of oxLDL to NOx, which could be a more accurate estimation of the *in vivo* cumulative implications of oxLDL and NO in atherogenesis. Future studies taking into account the association of this newly introduced marker with other markers of endothelial

The strong interrelations pointed out in our study underscore the molecular mechanisms implicated in endothelial dysfunctions and atherosclerosis presented in this chapter. Future research is needed to translate this knowledge into the identification, characterization and validation of new and known biomarkers of lipoxidative stress-induced endothelial dysfunctions and atherosclerosis, and their integration into cardiovascular risk

These findings suggest the importance of understanding the senescent specific changes occurring in endothelium associated with age-related disease. Such an understanding may not only provide answers regarding mechanisms of disease development, but may also

As perspectives, the nutritional and therapeutic strategies should attempt to correct the lipid profile and lipoxidative stress in order to prevent the amplification of redox and inflammatory phenomena that lead to increased cardiovascular risk. As well, therapeutic

emphasized their implications in molecular mechanisms of endothelial dysfunction.

function will be undertaken to support the marker validity.

provide biomarkers of endothelium specific ageing.

of antioxidants in different oxidative process steps and disease stages.

cardiovascular risk stratification models.

examination of the right brachial artery.

**2.5 Limitations of our study** 

**3. Conclusion** 

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**23** 

*USA* 

Jerome L. Sullivan

**Are Hemochromatosis Mutations Protective** 

Modest levels of stored iron, far less than conventional iron overload, may promote cardiovascular disease, i.e. sustained iron depletion may be protective [1-6]. This so-called "iron hypothesis" was initially presented to explain for the sex difference in cardiovascular disease and the increase in disease following menopause. The idea, although continually debated for more than 25 years, has achieved standing as a plausible and testable hypothesis

The hypothesis has not yet been definitively tested. A first randomized clinical trial (FeAST) to address aspects of the hypothesis was recently reported [7]. The FeAST trial [7] had significant limitations as a general test of the idea: 1) it was a trial of secondary prevention, and 2) the iron reduction protocol fell far short of achieving full iron depletion. Zacharski et al [7] reported that reducing iron stores significantly improves survival for patients with symptomatic but stable peripheral arterial disease (PAD), if iron reduction begins at a young age. The FeAST trial provides compelling support for a new trial designed to test the

Controversial results from multiple epidemiological studies investigating a variety of atherosclerotic events using all kinds of variable parameters of body iron load have presented a confusing picture of the iron hypothesis [20]. Confusion became complete when it appeared that patients with homozygous hemochromatosis who were afflicted with serious, life long iron overload had no increased atherosclerosis and might even be protected against atherosclerosis. In the debate on the hypothesis, the disease pattern in homozygous hemochromatosis has been intrepreted as perhaps the most persuasive evidence against the hypothesis [21]. This "hemochromatosis paradox" is seen as a anomaly that makes the hypothesis untenable for some investigators. How can normal stored iron levels be bad for the vascular system, when massive amounts of stored iron in genetic iron

**2. Hemochromatosis and atherosclerosis: More to it than iron load alone** 

An early corollary to the iron hypothesis was the proposal that heterozygous hemochromatosis might be a significant risk factor for premature myocardial infarction [22]. This was proposed despite the general impression at the time that homozygous hemochromatosis was not prominently associated with increased atherosclerosis. In the

overload are not associated with increased atherosclerosis?

**1. Introduction** 

[7-18] [19].

original hypothesis.

**Against Iron-Mediated Atherogenesis?** 

*University of Central Florida College of Medicine, Orlando,* 


### **Are Hemochromatosis Mutations Protective Against Iron-Mediated Atherogenesis?**

Jerome L. Sullivan *University of Central Florida College of Medicine, Orlando, USA* 

#### **1. Introduction**

494 Atherogenesis

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Anderson, R.G., Mendelsohn, M.E., Hobbs, H.H. & Shaul, P.W. (2001). High-Density Lipoprotein Binding to Scavenger Receptor-BI Activates Endothelial Nitric Oxide Synthase, *Nature Medicine*, Vol.7, No.7, (July 2001), pp. 853–857, ISSN 1078Modest levels of stored iron, far less than conventional iron overload, may promote cardiovascular disease, i.e. sustained iron depletion may be protective [1-6]. This so-called "iron hypothesis" was initially presented to explain for the sex difference in cardiovascular disease and the increase in disease following menopause. The idea, although continually debated for more than 25 years, has achieved standing as a plausible and testable hypothesis [7-18] [19].

The hypothesis has not yet been definitively tested. A first randomized clinical trial (FeAST) to address aspects of the hypothesis was recently reported [7]. The FeAST trial [7] had significant limitations as a general test of the idea: 1) it was a trial of secondary prevention, and 2) the iron reduction protocol fell far short of achieving full iron depletion. Zacharski et al [7] reported that reducing iron stores significantly improves survival for patients with symptomatic but stable peripheral arterial disease (PAD), if iron reduction begins at a young age. The FeAST trial provides compelling support for a new trial designed to test the original hypothesis.

Controversial results from multiple epidemiological studies investigating a variety of atherosclerotic events using all kinds of variable parameters of body iron load have presented a confusing picture of the iron hypothesis [20]. Confusion became complete when it appeared that patients with homozygous hemochromatosis who were afflicted with serious, life long iron overload had no increased atherosclerosis and might even be protected against atherosclerosis. In the debate on the hypothesis, the disease pattern in homozygous hemochromatosis has been intrepreted as perhaps the most persuasive evidence against the hypothesis [21]. This "hemochromatosis paradox" is seen as a anomaly that makes the hypothesis untenable for some investigators. How can normal stored iron levels be bad for the vascular system, when massive amounts of stored iron in genetic iron overload are not associated with increased atherosclerosis?

#### **2. Hemochromatosis and atherosclerosis: More to it than iron load alone**

An early corollary to the iron hypothesis was the proposal that heterozygous hemochromatosis might be a significant risk factor for premature myocardial infarction [22]. This was proposed despite the general impression at the time that homozygous hemochromatosis was not prominently associated with increased atherosclerosis. In the

Hemochromatosis and Iron-Mediated Atherogenesis 497

*defect in hemochromatosis leading to a constriction. of the macrophage/reticuloendothelial iron pool" [24]. This macrophage defect [41] in hereditary hemochromatosis was suggested as a factor that might "protect homozygotes from foam cell formation and thus, to a degree, gives some specific protection* 

The discovery of hepcidin [42-44] and the details of its influence on iron metabolism [45-49] illuminated patterns of macrophage iron retention and led to a conceptual *volte-face* on the

Hepcidin is the major regulator for the amount of iron retained within macrophages. Production of hepcidin is regulated by iron intake and a number of interrelated factors. Elevated levels, favoring macrophage iron retention, are seen with increased iron intake, infection and inflammation. Iron loading in secondary iron overload in wild type individuals is associated with increased hepcidin expression. Reduced hepcidin levels and iron-poor macrophages accompany iron deficiency, hypoxia, anemia and hereditary hemochromatosis. Hepcidin binds to the iron exporter protein ferroportin, leading to the internalization, and intracellular degradation of ferroportin. Loss of the iron exporter function of ferroportin from macrophages leads to intracellular retention of iron and to reduced serum iron levels. In intestinal epithelial cells, hepcidin-induced loss of ferroportin

Remarkably, the most extreme reductions in hepcidin level are associated with the opposite extremes of total body iron load, i.e. in iron deficiency anemia and in homozygous hemochromatosis [50]. Loss of hepcidin expression can be produced by mutations in hepcidin, hemojuvelin, *TFR2*, and *HFE* [51]. Mutations at these sites leads to hereditary iron overload. In this discussion, the term "hemochromatosis" indicates hereditary iron overload associated with one of the mutations causing lower hepcidin expression. The homozygous *HFE* C282Y mutation is the most common cause of hereditary iron overload and is

The very low hepcidin levels seen in homozygous hemochromatosis are associated with systemic iron loading because reduced hepcidin levels permit unregulated ferroportinmediated transfer of iron from intestinal epithelial cells into the systemic iron pool. The more extreme the degree of hepcidin deficiency, the more severe the level of parenchymal iron load, but also the more extreme the macrophage iron retention deficit. These patterns offer a potential resolution of the paradox of the proposed protection by iron depletion in wild type subjects against cardiovascular disease despite of the lack of increased atherosclerosis in genetic iron overload [4;6]. Hepcidin may act as an iron-dependent risk factor for atherosclerosis by causing iron loading of plaque macrophages with promotion of foam cell formation. According to this proposal, hepcidin amplifies the plaque iron loading effects of an increased iron load as iron itself upregulates hepcidin concentration. At the other end of the iron status spectrum, iron deficiency downregulates hepcidin and promotes removal of iron from plaque macrophages. In hemochromatosis, the associated hepcidin deficiency is hypothesized to reduce progressive iron accumulation within arterial walls and foam cell formation. Hemochromatosis patients may thus enjoy a specific protection against plaque progression in proportion to the severity of hepcidin deficiency. Hepcidin deficiency would not protect these patients from direct iron-mediated injury to heart muscle from parenchymal iron accumulation in myocardial tissue. The corollary hypothesis that identifies hepcidin as a risk factor for atherogenesis [4] may explain the conundrum of decreased atherosclerosis in the face of massive iron loading and provide additional

justification for the contention that the macrophage has a key role in atherogenesis.

*against atherosclerosis*," [24] with a partial protective effect in heterozygotes.

possibility of diminished atherosclerosis in homozygotes [4;6].

results in reduced iron internalization into the systemic circulation.

associated with lower liver expression of hepcidin mRNA [51].

absence of definitive data, this was not seen as necessarily incompatible with the iron hypothesis [22-24]. An impact on cardiovascular event rates in hemochromatosis was not excluded based on available data. In addition, even without promotion of atherosclerosis by genetic iron overload, relevant issues that continue to be unresolved include roles of hemochromatosis mutation-associated iron overload in myocardial reperfusion injury [2;24- 26] and endothelial dysfunction [27;28]. Future investigations are needed, as long term exposure to non-transferrin bound iron (NTBI) in genetic iron overload may contribute to life-long progression of atherosclerosis as it promotes monocyte-endothelium interaction and inflammatory pathways.

Mutational effects other than promotion of an increase in total body iron were not considered in the 1990 hypothesis relating heterozygosity to early onset of myocardial infarction [22]. The idea that total body iron load was the only factor that might influence cardiovascular disease expression in hemochromatosis was restated as recently as 2007 in a JAMA editorial on the status of the iron hypothesis by Hu [8]:

"*The 1996 discovery of HFE gene mutations responsible for most cases of hereditary hemochromatosis [[29]] has led to the use of genetic markers of iron stores (ie, heterozygosity for the C282Y mutation in the HFE gene as a marker of lifelong moderate iron overload) in epidemiologic studies. In contrast to biomarkers, genetic markers of iron overload can be measured exactly and are not influenced by such factors as inflammation, recent blood loss, diet, and use of medications (eg, aspirin)*."

The corollary hypothesis that heterozygosity might be associated with myocardial infarction [22] led to a number of investigations, especially after the identification of the diseasecausing mutation in most cases of hemochromatosis in 1996 [29]. Early findings appeared to support some increase in cardiovascular events among heterozygotes [23;30;31]. However, these studies taken together with subsequent investigations [32-36] do not support an increase in myocardial infarction, stroke or atherosclerosis in patients who are heterozygous for hemochromatosis. In fact, the body of relevant work, including some older studies [37;38] does not exclude protection against atherosclerosis in hemochromatosis. In an autopsy series that examined coronary artery disease in heavily iron overloaded individuals, Miller and Hutchins [37] reported an odds ratio of coronary artery disease with iron overload of 0.18. This is suggestive of a significant protective effect in patients presumptively homozygous for hemochromatosis who comprised 80% of the autopsy cases reviewed by Miller and Hutchins [37]. Could some poorly understood feature of homozygous hemochromatosis confound the relationship between iron load and atherosclerosis?

#### **3. Hepcidin and a resolution of the hemochromatosis paradox**

An iron loading mutation is not just "a marker of lifelong moderate iron overload" as indicated by Hu [8]. Hemochromatosis mutations also radically alter the distribution of body iron [39]. Iron-poor Kupffer cells adjacent to iron-loaded hepatocytes are a classic finding in hereditary hemochromatosis [39]. Another classic finding in homozygotes is a relative scarcity of coronary artery iron deposition despite extensive iron deposits in myocardial tissue [39;40].

In 1998, Moura et al [41] reported that monocytes from hereditary hemochromatosis patients released twice as much iron in the low molecular weight form as normal human monocytes after erythrocyte phagocytosis. Thus, even before the discovery and understanding of the iron regulatory hormone, hepcidin [42-44], there was an understanding of "*a macrophage* 

absence of definitive data, this was not seen as necessarily incompatible with the iron hypothesis [22-24]. An impact on cardiovascular event rates in hemochromatosis was not excluded based on available data. In addition, even without promotion of atherosclerosis by genetic iron overload, relevant issues that continue to be unresolved include roles of hemochromatosis mutation-associated iron overload in myocardial reperfusion injury [2;24- 26] and endothelial dysfunction [27;28]. Future investigations are needed, as long term exposure to non-transferrin bound iron (NTBI) in genetic iron overload may contribute to life-long progression of atherosclerosis as it promotes monocyte-endothelium interaction

Mutational effects other than promotion of an increase in total body iron were not considered in the 1990 hypothesis relating heterozygosity to early onset of myocardial infarction [22]. The idea that total body iron load was the only factor that might influence cardiovascular disease expression in hemochromatosis was restated as recently as 2007 in a

"*The 1996 discovery of HFE gene mutations responsible for most cases of hereditary hemochromatosis [[29]] has led to the use of genetic markers of iron stores (ie, heterozygosity for the C282Y mutation in the HFE gene as a marker of lifelong moderate iron overload) in epidemiologic studies. In contrast to biomarkers, genetic markers of iron overload can be measured exactly and are not influenced by* 

The corollary hypothesis that heterozygosity might be associated with myocardial infarction [22] led to a number of investigations, especially after the identification of the diseasecausing mutation in most cases of hemochromatosis in 1996 [29]. Early findings appeared to support some increase in cardiovascular events among heterozygotes [23;30;31]. However, these studies taken together with subsequent investigations [32-36] do not support an increase in myocardial infarction, stroke or atherosclerosis in patients who are heterozygous for hemochromatosis. In fact, the body of relevant work, including some older studies [37;38] does not exclude protection against atherosclerosis in hemochromatosis. In an autopsy series that examined coronary artery disease in heavily iron overloaded individuals, Miller and Hutchins [37] reported an odds ratio of coronary artery disease with iron overload of 0.18. This is suggestive of a significant protective effect in patients presumptively homozygous for hemochromatosis who comprised 80% of the autopsy cases reviewed by Miller and Hutchins [37]. Could some poorly understood feature of homozygous hemochromatosis confound the relationship between iron load and

*such factors as inflammation, recent blood loss, diet, and use of medications (eg, aspirin)*."

**3. Hepcidin and a resolution of the hemochromatosis paradox** 

An iron loading mutation is not just "a marker of lifelong moderate iron overload" as indicated by Hu [8]. Hemochromatosis mutations also radically alter the distribution of body iron [39]. Iron-poor Kupffer cells adjacent to iron-loaded hepatocytes are a classic finding in hereditary hemochromatosis [39]. Another classic finding in homozygotes is a relative scarcity of coronary artery iron deposition despite extensive iron deposits in

In 1998, Moura et al [41] reported that monocytes from hereditary hemochromatosis patients released twice as much iron in the low molecular weight form as normal human monocytes after erythrocyte phagocytosis. Thus, even before the discovery and understanding of the iron regulatory hormone, hepcidin [42-44], there was an understanding of "*a macrophage* 

JAMA editorial on the status of the iron hypothesis by Hu [8]:

and inflammatory pathways.

atherosclerosis?

myocardial tissue [39;40].

*defect in hemochromatosis leading to a constriction. of the macrophage/reticuloendothelial iron pool" [24]. This macrophage defect [41] in hereditary hemochromatosis was suggested as a factor that might "protect homozygotes from foam cell formation and thus, to a degree, gives some specific protection against atherosclerosis*," [24] with a partial protective effect in heterozygotes.

The discovery of hepcidin [42-44] and the details of its influence on iron metabolism [45-49] illuminated patterns of macrophage iron retention and led to a conceptual *volte-face* on the possibility of diminished atherosclerosis in homozygotes [4;6].

Hepcidin is the major regulator for the amount of iron retained within macrophages. Production of hepcidin is regulated by iron intake and a number of interrelated factors. Elevated levels, favoring macrophage iron retention, are seen with increased iron intake, infection and inflammation. Iron loading in secondary iron overload in wild type individuals is associated with increased hepcidin expression. Reduced hepcidin levels and iron-poor macrophages accompany iron deficiency, hypoxia, anemia and hereditary hemochromatosis. Hepcidin binds to the iron exporter protein ferroportin, leading to the internalization, and intracellular degradation of ferroportin. Loss of the iron exporter function of ferroportin from macrophages leads to intracellular retention of iron and to reduced serum iron levels. In intestinal epithelial cells, hepcidin-induced loss of ferroportin results in reduced iron internalization into the systemic circulation.

Remarkably, the most extreme reductions in hepcidin level are associated with the opposite extremes of total body iron load, i.e. in iron deficiency anemia and in homozygous hemochromatosis [50]. Loss of hepcidin expression can be produced by mutations in hepcidin, hemojuvelin, *TFR2*, and *HFE* [51]. Mutations at these sites leads to hereditary iron overload. In this discussion, the term "hemochromatosis" indicates hereditary iron overload associated with one of the mutations causing lower hepcidin expression. The homozygous *HFE* C282Y mutation is the most common cause of hereditary iron overload and is associated with lower liver expression of hepcidin mRNA [51].

The very low hepcidin levels seen in homozygous hemochromatosis are associated with systemic iron loading because reduced hepcidin levels permit unregulated ferroportinmediated transfer of iron from intestinal epithelial cells into the systemic iron pool. The more extreme the degree of hepcidin deficiency, the more severe the level of parenchymal iron load, but also the more extreme the macrophage iron retention deficit. These patterns offer a potential resolution of the paradox of the proposed protection by iron depletion in wild type subjects against cardiovascular disease despite of the lack of increased atherosclerosis in genetic iron overload [4;6]. Hepcidin may act as an iron-dependent risk factor for atherosclerosis by causing iron loading of plaque macrophages with promotion of foam cell formation. According to this proposal, hepcidin amplifies the plaque iron loading effects of an increased iron load as iron itself upregulates hepcidin concentration. At the other end of the iron status spectrum, iron deficiency downregulates hepcidin and promotes removal of iron from plaque macrophages. In hemochromatosis, the associated hepcidin deficiency is hypothesized to reduce progressive iron accumulation within arterial walls and foam cell formation. Hemochromatosis patients may thus enjoy a specific protection against plaque progression in proportion to the severity of hepcidin deficiency. Hepcidin deficiency would not protect these patients from direct iron-mediated injury to heart muscle from parenchymal iron accumulation in myocardial tissue. The corollary hypothesis that identifies hepcidin as a risk factor for atherogenesis [4] may explain the conundrum of decreased atherosclerosis in the face of massive iron loading and provide additional justification for the contention that the macrophage has a key role in atherogenesis.

Hemochromatosis and Iron-Mediated Atherogenesis 499

Of special significance in the present discussion, the reduced translation of cytokine mRNAs of the mutant macrophages could be reproduced in wild-type cells by reducing the intracellular iron concentration with chelation. Atherosclerotic plaque macrophages in patients with hemochromatosis mutations associated with diminished hepcidin may display similar attenuated inflammatory responses such as those from *Hfe* -/- mice [56], and thereby

Iron plays a role in vascular disease in other cell types than the macrophage, e.g. endothelial cells [3;9;14;18;57-59] and vascular smooth muscle cells [60-62]. Patients with hemochromatosis have endothelial dysfunction that is improved by iron reduction therapy [63]. This suggests that iron overload itself rather than mutational effects of iron overload genes influences endothelial function. Proliferaton of vascular smooth muscle cells [60-62] also requires iron. How hemochromatosis mutations might modifies iron-mediated

**7. Serum cholesterol level, hemochromatosis, macrophage iron loss, and** 

Adams et al [64] reported that hemochromatosis patients homzygous for C282Y have diminished serum cholesterol and low-density lipoprotein (LDL) levels. Systemically lower cholesterol and LDL could represent an additional mechanism by which hemochromatosis patients are relatively protected from atherosclerosis. This could be associated with the iron retention deficit in mutant macrophages. A role for macrophage iron metabolism in regulation of cellular lipid level has been proposed [65]. As noted above, the most extreme reductions in hepcidin level are seen at the opposite extremes of total body iron load, i.e. in both iron deficiency anemia and in homozygous hemochromatosis. Consistent with a hepcidin level similar to that in hemochromatosis, iron deficiency is also associated with lower systemic levels of serum cholesterol and LDL [12;66;67]. Future studies are needed to determine if lower macrophage iron level in iron deficiency or inherited iron overload

**8. Mutational protection against atherogenesis: Epidemiological implications**  The literature on the role of iron in cardiovascular disease in the general population is contradictory and inconsistent, as has often been noted [8]. There have been misconceptions regarding the hypothesis leading to inadequate study designs [20;68]. Another key limitation of previous studies that has not been addressed is the possibility of a protective effect of hemochromatosis mutations against iron-mediated atherogenesis. If hemochromatosis mutations confer protection against atherogenesis, previous epidemiological studies of iron and atherosclerosis may be critically flawed. The highest serum ferritin levels in population groups whose hemochromatosis gene status has not been ascertained will select a disproportionate share of subjects who are heterozygous or homozygous for hemochromatosis. These high serum ferritin individuals may have less disease because of mutational protection against atherosclerosis and may confound

underlying associations of iron load and atherosclerosis in normal subjects.

**6. Iron, hemochromatosis and other cell types in vascular disease** 

atherogenic processes in these cell types will require additional studies.

a diminished tendency to form atherosclerotic foam cells.

**cardiovascular disease** 

negatively regulates systemic cholesterol level.

Previous studies, especially the work of Miller and Hutchins [37] and Pirart and Barbier [38], raised the possibility of a protective effect of hereditary hemochromatosis against atherosclerosis. An unknown "*facteur constitutionnel*" [38] linked to hemochromatosis that enhances resistance to vascular lesions was proposed. A mechanistic hypothesis to explain the findings [37;38] was not proposed as the studies were done prior to identification of either the principal iron overloading genotypes or the iron regulatory hormone hepcidin. More recent evidence supporting the hypothesis that hemochromatosis-associated hepcidin deficiency is protective against atherosclerosis has been reported [52]. Valenti et al [52] studied vascular disease, iron status, hepcidin levels and *HFE* mutations in 506 consecutive patients with nonalcholic fatty liver disease (NAFLD). None were homozygous for hereditary hemochromatosis. Serum ferritin was associated with common carotid intimamedia thickness (CC-IMT) (p = 0.048) and with prevalence of atherosclerotic carotid plaques (p = 0.0004), except in patients whose heterozygous *HFE* mutations lower hepcidin levels. Hyperferritinemia was associated with vascular damage only in patients with wild type *HFE* genotypes (p<0.0001). Hepcidin was elevated in those without such an *HFE* mutation and was found to be an independent predictor of the presence of carotid atherosclerosis.

#### **4. Iron, hepcidin, inflammation and vascular disease**

Inflammation accelerates atherogenesis [53]. The mechanism may involve iron- and hepcidin-mediated mechanisms [4;6]. Hepcidin is upregulated by interleukin-6 (IL-6), a cytokine induced by inflammatory processes. IL-6 has also been found to be a cardiovascular disease risk factor [54]. An important end result of any process that induces IL-6 is increased deposition of iron within reticuloendothelial cells, including atherosclerotic plaque macrophages, because of hepcidin upregulation. Continued inflammation-mediated hepcidin synthesis maintains iron in storage sites even in the face of a low hematocrit as in the anemia of inflammation (i.e. the "anemia of chronic disorders").

Hepatic hepcidin may be normally upregulated in inflammation even in hemochromatosis homozygotes who usually have markedly low hepcidin levels [55]. The effects of inflammatory processes in hemochromatosis patients on possible redistribution of iron from parenchymal cells to the reticuloendothelial compartment, including arterial plaque macrophages, are not currently known. Interactions between mutational effects and inflammation-induced effects on hepcidin level may result in complex epidemiological patterns in studies of cardiovascular disease expression in hemochromatosis patients.

#### **5. Blunted inflammatory responses in macrophages in hemochromatosis or induced iron depletion**

A recent study of macrophages in the *Hfe* knockout (*Hfe* -/-) mouse [56] is pertinent to the present discussion of iron, inflammation and atherosclerosis. Wang et al [56] found attenuated inflammatory responses in a mouse model of human hemochromatosis and reduced translation of cytokine mRNAs in *Hfe* -/- macrophages in response to Salmonella and LPS exposure. Intramacrophage iron levels were decreased in the *Hfe* -/- mice in association with upregulation of macrophage iron exporter ferroportin (FPN). Salmonellaand LPS-induced inflammatory responses were diminished in the *Hfe* knockout animals. Less severe enterocolitis was observed in vivo and reduced macrophage TNF- and IL-6 secretion was observed in vitro.

Previous studies, especially the work of Miller and Hutchins [37] and Pirart and Barbier [38], raised the possibility of a protective effect of hereditary hemochromatosis against atherosclerosis. An unknown "*facteur constitutionnel*" [38] linked to hemochromatosis that enhances resistance to vascular lesions was proposed. A mechanistic hypothesis to explain the findings [37;38] was not proposed as the studies were done prior to identification of either the principal iron overloading genotypes or the iron regulatory hormone hepcidin. More recent evidence supporting the hypothesis that hemochromatosis-associated hepcidin deficiency is protective against atherosclerosis has been reported [52]. Valenti et al [52] studied vascular disease, iron status, hepcidin levels and *HFE* mutations in 506 consecutive patients with nonalcholic fatty liver disease (NAFLD). None were homozygous for hereditary hemochromatosis. Serum ferritin was associated with common carotid intimamedia thickness (CC-IMT) (p = 0.048) and with prevalence of atherosclerotic carotid plaques (p = 0.0004), except in patients whose heterozygous *HFE* mutations lower hepcidin levels. Hyperferritinemia was associated with vascular damage only in patients with wild type *HFE* genotypes (p<0.0001). Hepcidin was elevated in those without such an *HFE* mutation and was found to be an independent predictor of the presence of carotid atherosclerosis.

Inflammation accelerates atherogenesis [53]. The mechanism may involve iron- and hepcidin-mediated mechanisms [4;6]. Hepcidin is upregulated by interleukin-6 (IL-6), a cytokine induced by inflammatory processes. IL-6 has also been found to be a cardiovascular disease risk factor [54]. An important end result of any process that induces IL-6 is increased deposition of iron within reticuloendothelial cells, including atherosclerotic plaque macrophages, because of hepcidin upregulation. Continued inflammation-mediated hepcidin synthesis maintains iron in storage sites even in the face of a low hematocrit as in

Hepatic hepcidin may be normally upregulated in inflammation even in hemochromatosis homozygotes who usually have markedly low hepcidin levels [55]. The effects of inflammatory processes in hemochromatosis patients on possible redistribution of iron from parenchymal cells to the reticuloendothelial compartment, including arterial plaque macrophages, are not currently known. Interactions between mutational effects and inflammation-induced effects on hepcidin level may result in complex epidemiological patterns in studies of cardiovascular disease expression in hemochromatosis patients.

**5. Blunted inflammatory responses in macrophages in hemochromatosis or** 

A recent study of macrophages in the *Hfe* knockout (*Hfe* -/-) mouse [56] is pertinent to the present discussion of iron, inflammation and atherosclerosis. Wang et al [56] found attenuated inflammatory responses in a mouse model of human hemochromatosis and reduced translation of cytokine mRNAs in *Hfe* -/- macrophages in response to Salmonella and LPS exposure. Intramacrophage iron levels were decreased in the *Hfe* -/- mice in association with upregulation of macrophage iron exporter ferroportin (FPN). Salmonellaand LPS-induced inflammatory responses were diminished in the *Hfe* knockout animals. Less severe enterocolitis was observed in vivo and reduced macrophage TNF- and IL-6

**4. Iron, hepcidin, inflammation and vascular disease** 

the anemia of inflammation (i.e. the "anemia of chronic disorders").

**induced iron depletion** 

secretion was observed in vitro.

Of special significance in the present discussion, the reduced translation of cytokine mRNAs of the mutant macrophages could be reproduced in wild-type cells by reducing the intracellular iron concentration with chelation. Atherosclerotic plaque macrophages in patients with hemochromatosis mutations associated with diminished hepcidin may display similar attenuated inflammatory responses such as those from *Hfe* -/- mice [56], and thereby a diminished tendency to form atherosclerotic foam cells.

#### **6. Iron, hemochromatosis and other cell types in vascular disease**

Iron plays a role in vascular disease in other cell types than the macrophage, e.g. endothelial cells [3;9;14;18;57-59] and vascular smooth muscle cells [60-62]. Patients with hemochromatosis have endothelial dysfunction that is improved by iron reduction therapy [63]. This suggests that iron overload itself rather than mutational effects of iron overload genes influences endothelial function. Proliferaton of vascular smooth muscle cells [60-62] also requires iron. How hemochromatosis mutations might modifies iron-mediated atherogenic processes in these cell types will require additional studies.

#### **7. Serum cholesterol level, hemochromatosis, macrophage iron loss, and cardiovascular disease**

Adams et al [64] reported that hemochromatosis patients homzygous for C282Y have diminished serum cholesterol and low-density lipoprotein (LDL) levels. Systemically lower cholesterol and LDL could represent an additional mechanism by which hemochromatosis patients are relatively protected from atherosclerosis. This could be associated with the iron retention deficit in mutant macrophages. A role for macrophage iron metabolism in regulation of cellular lipid level has been proposed [65]. As noted above, the most extreme reductions in hepcidin level are seen at the opposite extremes of total body iron load, i.e. in both iron deficiency anemia and in homozygous hemochromatosis. Consistent with a hepcidin level similar to that in hemochromatosis, iron deficiency is also associated with lower systemic levels of serum cholesterol and LDL [12;66;67]. Future studies are needed to determine if lower macrophage iron level in iron deficiency or inherited iron overload negatively regulates systemic cholesterol level.

#### **8. Mutational protection against atherogenesis: Epidemiological implications**

The literature on the role of iron in cardiovascular disease in the general population is contradictory and inconsistent, as has often been noted [8]. There have been misconceptions regarding the hypothesis leading to inadequate study designs [20;68]. Another key limitation of previous studies that has not been addressed is the possibility of a protective effect of hemochromatosis mutations against iron-mediated atherogenesis. If hemochromatosis mutations confer protection against atherogenesis, previous epidemiological studies of iron and atherosclerosis may be critically flawed. The highest serum ferritin levels in population groups whose hemochromatosis gene status has not been ascertained will select a disproportionate share of subjects who are heterozygous or homozygous for hemochromatosis. These high serum ferritin individuals may have less disease because of mutational protection against atherosclerosis and may confound underlying associations of iron load and atherosclerosis in normal subjects.

Hemochromatosis and Iron-Mediated Atherogenesis 501

A role for iron in foam cell formation and lesion progression has been implicated by numerous observations and experiments [4-6;75-83]. Recent work shows that iron can be mobilized out of atherosclerotic plaque by manipulation of body iron status, and that this process may be associated with reduction in lesion size. Animal experiments suggest that systemic lowering of stored iron levels reduces intralesional iron content and also the size of atherosclerotic plaques [70;84]. It is well known that iron-deficient erythropoiesis can mobilize and relocate almost all stored iron in the body to maturing erythroid precursors. In iron deficiency, mobilization is facilitated by extreme downregulation of hepcidin. Key questions in future human studies include the following: What duration and degree of iron reduction therapy is needed for restoring iron levels in atherosclerotic vessel segments to the much lower level seen in healthy vascular tissue? How much reduction in the level of hepcidin is required to facilitate the relocation of stored iron from intralesional macrophages to erythroid precursors? And, is it possible in normal subjects to inhibit the formation of atherosclerotic foam cells by rendering their macrophages as iron poor as in those with

[1] Sullivan JL. Iron and the sex difference in heart disease risk. Lancet 1981; 1(8233):1293-

[2] Sullivan JL. The iron paradigm of ischemic heart disease. Am Heart J 1989; 117(5):1177-

[3] Sullivan JL. Stored Iron and Vascular Reactivity. Arterioscler Thromb Vasc Biol 2005;

[4] Sullivan J. Macrophage Iron, Hepcidin, and Atherosclerotic Plaque Stability. Exp Biol

[5] Li W, XU LH, Forssell C, Sullivan JL, YUAN XM. Overexpression of Transferrin

[6] Sullivan JL. Iron in arterial plaque: A modifiable risk factor for atherosclerosis. Biochimica et Biophysica Acta (BBA) - General Subjects 2009; 1790(7):718-723. [7] Zacharski LR, Chow BK, Howes PS, Shamayeva G, Baron JA, Dalman RL, Malenka DJ,

[8] Hu FB. The Iron-Heart Hypothesis: Search for the Ironclad Evidence. JAMA 2007;

[9] Zheng H, Cable R, Spencer B, Votto N, Katz SD. Iron stores and vascular function in voluntary blood donors. Arterioscler Thromb Vasc Biol 2005; 25(8):1577-1583. [10] Salonen JT, Nyyssonen K, Korpela H, Tuomilehto J, Seppanen R, Salonen R. High

Carotid Plaques. Proc Soc Exp Biol Med 2008; 233(7):818-826.

Finnish men [see comments]. Circulation 1992; 86(3):803-811.

Receptor and Ferritin Related to Clinical Symptoms and Destabilization of Human

Ozaki CK, Lavori PW. Reduction of Iron Stores and Cardiovascular Outcomes in Patients With Peripheral Arterial Disease: A Randomized Controlled Trial. JAMA

stored iron levels are associated with excess risk of myocardial infarction in eastern

hemochromatosis mutations?

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**11. Conflict of interest disclosures** 

#### **9. Penetrance and testing the hepcidin hypothesis**

This problem of clinical penetrance of the hemochromatosis mutations needs to be considered in the design of a study to test the hepcidin hypothesis. There is undoubtedy a variable impact of genotype on hepcidin expression. Genotype of subjects in a study to test the hypothesis shouldbe determined; however, testing the hypothesis would not rely directly on showing an association of genotype with disease. The hypothesis suggests that protection against atherogenesis is inversely proportional to hepcidin expression. In an epidemiological study, the hypothesis suggests that, among those with any one of a number of iron overloading genotypes, protection against atherogenesis would be seen in proportion to the degree of life long hepcidin downregulation.

It would be inappropriate to simply look at a group of all subjects with hepcidin expression below some prespecified level. It would be necessary to exclude the iron deficient subjects from a group defined by such a criterion, as iron deficiency is associated with quite low hepcidin levels. A future interventional study of the effect of long term iron deficiency-induced reduction in hepcidin expression on atherogenesis would be of interest.

#### **10. Conclusions and future directions**

The hypothesis that iron depletion protects against atherosclerosis may apply even in hemochromatosis homozygotes because of the mutational effect of selective iron depletion of the macrophage, a key cell type in atherogenesis. In homozygotes, a sea of tissue iron deposition surrounds islands of iron depleted cells of the reticuloendothelial system. Low hepcidin expression is a mutational feature of hemochromatosis and also of systemic iron deficiency that may protect against iron-mediated atherogenesis in both conditions. What is known at present about disease patterns in genetic iron overload is compatible with the hypothesis that iron depletion protects against atherosclerosis. Hereditary hemochromatosis may be a special case of selective cellular iron depletion that inhibits atherogenesis.

More detailed investigations are needed on hepcidin as a risk factor for atherosclerosis including more studies of atherosclerotic disease in patients with hemochromatosis mutations. Work is also needed on the effects of the inflammatory response on iron metabolism, especially the impact of inflammatory processes on hepcidin and macrophage iron in patients with hemochromatosis mutations.

It would be of interest to replicate the low hepcidin levels of those with hemochromatosis mutations in normal subjects and to assess the effects of low hepcidin levels on atherogenesis. A well established and safe method that would have the effect of reducing hepcidin production in normal subjects is induced iron depletion. Long-term modest reduction in storage iron can be achieved in patients with established vascular disease and is associated with decreased cancer mortality [69] and, among younger participants, decreased cardiovascular mortality [7].

In humans with intact hepcidin responses, atherosclerotic plaque has a substantially higher iron concentration than that in healthy arterial wall [15]. Increased lesional iron is also seen in cholesterol fed animals. In a series of studies with rabbits fed a 1% cholesterol diet, Watt and colleagues [70-74] used nuclear microscopy to show a 7-fold increase in iron concentration within newly formed atherosclerotic lesions compared to healthy arteries. Iron accumulation was seen at the onset of lesion formation.

A role for iron in foam cell formation and lesion progression has been implicated by numerous observations and experiments [4-6;75-83]. Recent work shows that iron can be mobilized out of atherosclerotic plaque by manipulation of body iron status, and that this process may be associated with reduction in lesion size. Animal experiments suggest that systemic lowering of stored iron levels reduces intralesional iron content and also the size of atherosclerotic plaques [70;84]. It is well known that iron-deficient erythropoiesis can mobilize and relocate almost all stored iron in the body to maturing erythroid precursors. In iron deficiency, mobilization is facilitated by extreme downregulation of hepcidin. Key questions in future human studies include the following: What duration and degree of iron reduction therapy is needed for restoring iron levels in atherosclerotic vessel segments to the much lower level seen in healthy vascular tissue? How much reduction in the level of hepcidin is required to facilitate the relocation of stored iron from intralesional macrophages to erythroid precursors? And, is it possible in normal subjects to inhibit the formation of atherosclerotic foam cells by rendering their macrophages as iron poor as in those with hemochromatosis mutations?

#### **11. Conflict of interest disclosures**

None.

500 Atherogenesis

This problem of clinical penetrance of the hemochromatosis mutations needs to be considered in the design of a study to test the hepcidin hypothesis. There is undoubtedy a variable impact of genotype on hepcidin expression. Genotype of subjects in a study to test the hypothesis shouldbe determined; however, testing the hypothesis would not rely directly on showing an association of genotype with disease. The hypothesis suggests that protection against atherogenesis is inversely proportional to hepcidin expression. In an epidemiological study, the hypothesis suggests that, among those with any one of a number of iron overloading genotypes, protection against atherogenesis would be seen in

It would be inappropriate to simply look at a group of all subjects with hepcidin expression below some prespecified level. It would be necessary to exclude the iron deficient subjects from a group defined by such a criterion, as iron deficiency is associated with quite low hepcidin levels. A future interventional study of the effect of long term iron deficiency-induced reduction in hepcidin expression on atherogenesis would be of

The hypothesis that iron depletion protects against atherosclerosis may apply even in hemochromatosis homozygotes because of the mutational effect of selective iron depletion of the macrophage, a key cell type in atherogenesis. In homozygotes, a sea of tissue iron deposition surrounds islands of iron depleted cells of the reticuloendothelial system. Low hepcidin expression is a mutational feature of hemochromatosis and also of systemic iron deficiency that may protect against iron-mediated atherogenesis in both conditions. What is known at present about disease patterns in genetic iron overload is compatible with the hypothesis that iron depletion protects against atherosclerosis. Hereditary hemochromatosis

More detailed investigations are needed on hepcidin as a risk factor for atherosclerosis including more studies of atherosclerotic disease in patients with hemochromatosis mutations. Work is also needed on the effects of the inflammatory response on iron metabolism, especially the impact of inflammatory processes on hepcidin and macrophage

It would be of interest to replicate the low hepcidin levels of those with hemochromatosis mutations in normal subjects and to assess the effects of low hepcidin levels on atherogenesis. A well established and safe method that would have the effect of reducing hepcidin production in normal subjects is induced iron depletion. Long-term modest reduction in storage iron can be achieved in patients with established vascular disease and is associated with decreased cancer mortality [69] and, among younger participants,

In humans with intact hepcidin responses, atherosclerotic plaque has a substantially higher iron concentration than that in healthy arterial wall [15]. Increased lesional iron is also seen in cholesterol fed animals. In a series of studies with rabbits fed a 1% cholesterol diet, Watt and colleagues [70-74] used nuclear microscopy to show a 7-fold increase in iron concentration within newly formed atherosclerotic lesions compared to healthy arteries.

may be a special case of selective cellular iron depletion that inhibits atherogenesis.

**9. Penetrance and testing the hepcidin hypothesis** 

proportion to the degree of life long hepcidin downregulation.

**10. Conclusions and future directions** 

iron in patients with hemochromatosis mutations.

Iron accumulation was seen at the onset of lesion formation.

decreased cardiovascular mortality [7].

interest.

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**24** 

*Croatia* 

**Atherosclerosis** 

*Merkur University Hospital, Zagreb* 

*Biochemistry, University of Zagreb, Zagreb,* 

**Paraoxonase Polymorphisms and Platelet** 

**Activating Factor Acetylhydrolase Activity** 

*3Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and* 

Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries (Lusis, JA. 2000). Investigations into the genetics of atherosclerosis, along with biochemical approaches, have greatly advanced today knowledge of the mechanisms of this complex multifactorial disease (Lusis et al., 2004a, 2004b; Lusis &Weiss , 2010). According to the oxidation hypothesis, oxidative stress is a key mechanism through which atherosclerosis as a chronic inflammatory disease develops. It is mediated by reactive oxygen species that alter the fundamental properties of cholesterol, cholesterol esters, and phospholipids on lipoproteins, as well as other proteins, to make them dysfunctional, immunogenic, and pro-atherogenic (Tsimikas et. al., 2009). Oxidative stress can be enhanced by non-enzymatic pathways, such as by copper and iron cations, as well as by enzymatic pathways, such as by lipoxygenases, myeloperoxidase, and NADPH oxidase. These pro-oxidant pathways are balanced by anti-oxidant mechanisms, such as anti-oxidant vitamins (alpha-tocopherol and carotenoids) present within lipoproteins, and anti-oxidant enzymes, such as superoxide dismutase and glutathione peroxidase. Many of these enzymes and products of oxidation can be measured in the circulation, including oxidized low-density lipoprotein, oxidized phospholipids, isoprostanes, and myeloperoxidase, and have been shown to predict the presence of cardiovascular disease

Human serum paraoxonase [(PON1); aryldialkylphosphatase (EC 3.1.8.1)] is associated with high density lipoprotein particles (HDL) responsible in part for the ability of HDL to prevent lipid peroxidation. The decreased serum paraoxonase (PON1) activity in patients with atherosclerosis disease may cause decreased HDL antioxidant capacity and therefore significantly influence the risk of the development of atherosclerosis (Aviram, M. 2004;

(CVD) and incident cardiovascular events (Tsimikas et al., 2007, 2009).

**1. Introduction** 

**as a Genetic Risk Factors in Cerebral** 

Zlata Flegar-Meštrić1, Mirjana Mariana Kardum Paro1, Sonja Perkov1, Vinko Vidjak2 and Marija Grdić Rajković<sup>3</sup>

> *1Institute of Clinical Chemistry and Laboratory Medicine, 2Clinical Department for Diagnostic and Clinical Radiology,*

decreases lesion iron concentrations in the cholesterol-fed rabbit. Free Radic Biol Med 2005; 38(9):1206-1211.


### **Paraoxonase Polymorphisms and Platelet Activating Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis**

Zlata Flegar-Meštrić1, Mirjana Mariana Kardum Paro1, Sonja Perkov1, Vinko Vidjak2 and Marija Grdić Rajković<sup>3</sup> *1Institute of Clinical Chemistry and Laboratory Medicine, 2Clinical Department for Diagnostic and Clinical Radiology, Merkur University Hospital, Zagreb 3Department of Medical Biochemistry and Hematology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia* 

#### **1. Introduction**

506 Atherogenesis

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associated macrophage protein 1 in atherosclerotic lesions may be associated with

YUAN XM. Cytocidal effects of atheromatous plaque components: the death zone

Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries (Lusis, JA. 2000). Investigations into the genetics of atherosclerosis, along with biochemical approaches, have greatly advanced today knowledge of the mechanisms of this complex multifactorial disease (Lusis et al., 2004a, 2004b; Lusis &Weiss , 2010). According to the oxidation hypothesis, oxidative stress is a key mechanism through which atherosclerosis as a chronic inflammatory disease develops. It is mediated by reactive oxygen species that alter the fundamental properties of cholesterol, cholesterol esters, and phospholipids on lipoproteins, as well as other proteins, to make them dysfunctional, immunogenic, and pro-atherogenic (Tsimikas et. al., 2009). Oxidative stress can be enhanced by non-enzymatic pathways, such as by copper and iron cations, as well as by enzymatic pathways, such as by lipoxygenases, myeloperoxidase, and NADPH oxidase. These pro-oxidant pathways are balanced by anti-oxidant mechanisms, such as anti-oxidant vitamins (alpha-tocopherol and carotenoids) present within lipoproteins, and anti-oxidant enzymes, such as superoxide dismutase and glutathione peroxidase. Many of these enzymes and products of oxidation can be measured in the circulation, including oxidized low-density lipoprotein, oxidized phospholipids, isoprostanes, and myeloperoxidase, and have been shown to predict the presence of cardiovascular disease (CVD) and incident cardiovascular events (Tsimikas et al., 2007, 2009).

Human serum paraoxonase [(PON1); aryldialkylphosphatase (EC 3.1.8.1)] is associated with high density lipoprotein particles (HDL) responsible in part for the ability of HDL to prevent lipid peroxidation. The decreased serum paraoxonase (PON1) activity in patients with atherosclerosis disease may cause decreased HDL antioxidant capacity and therefore significantly influence the risk of the development of atherosclerosis (Aviram, M. 2004;

Paraoxonase Polymorphisms and Platelet Activating

AJ. 2009).

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 509

As it was mentioned earlier, PON2 is a ubiquitously expressed intracellular protein with a relative molecular mass of approximately 44 kDa (Ng et al., 2005; Li et al., 2003). PON2 has antioxidant properties, lowers the intracellular oxidative stress and prevents the cellmediated oxidation of LDL (Ng at al., 2005; Li et al., 2003). In the *pon2* gene two common polymorphisms were identified. Alanine or glycine could be at the position 148 (A148G), and serine or cysteine could be at the position 311 (S311C). S311C polymorphism has been related with eg. coronary artery disease, ischemic stroke in patients with type 2 diabetes mellitus, Alzheimer's disease and reduced bone mass in postmenopausal women (Ng at al., 2005; Li et al., 2003). The mechanisms by which PON2 exerts its atheroprotective effects remain to be clarified. Large-scale epidemiologic studies are needed to further examine the relationship between PON2 genetic polymorphisms and risk for CVD (Shih, DM. Lusius,

Human PON3 is a '40-kDa protein primarily synthesized in the liver with biological activity similar to PON1. PON3 is a secreted protein associated with HDL in the plasma and can participate in the prevention of LDL oxidation. The PON3 protein may play a role, distinct from that of PON1, in the lipoprotein metabolism of the kidney. These characteristics link PON3 with a group of enzymes, such as PON1, platelet-activating factor–acetylhydrolase, and lecithin-cholesterol acyltransferase, which together may contribute to the antiatherogenic properties of HDL, but the role of PON3 in atherosclerosis needs further

Another lipoprotein-associated enzyme, the platelet-activating factor acetylhydrolase (PAF-AH), also referred to as lipoprotein-associated phospholipase A2 (Lp-PLA2), is an enzyme (EC 3.1.1.47) recently described as a potentially useful plasma biomarker associated with cardiovascular disease (Srinivasan, B. Bahson, BJ. 2010; Koenig et al., 2004; Yamada et al., 2000; Karasawa, K. 2006; Mallat et al., 2010). The biological role of Lp-PLA2 (PAF-AH) has been controversial, with contradictory antiatherogenic and proatherogenic functions. The antiatherogenic properties of Lp-PLA2 were first suggested because plasma PAF-AH might play an anti-inflammatory role in human diseases by preventing the accumulation of PAF (1- O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and PAF-like oxidized phospholipids (Karasawa, K. 2006; Mallat et al., 2010; Mitsios et al., 2006). PAF is a biologically active phospholipid involved in diverse pathologies such as inflammation and atherosclerosis. PAF can activate various cell types including platelets. In the presence of PAF, platelets aggregate and degranulate, releasing biologically potent agents. PAF is hydrolyzed and converted to lysoPAF by the catalytic reaction of PAF-AH (Mitsios et al., 2006). The atherogenic role of Lp-PLA2 comes from the observation that this enzyme can also produce lysophosphatidylcholine and oxidatively modified nonesterified fatty acids which could promote the pathogenesis of atherosclerosis (Karasawa, K. 2006; Mallat et al., 2010). Lysophosphatidylcholine is an important chemoattractant for macrophages and T cells, it induces migration of vascular smooth muscle cells, affects endothelial function, and increases the expression of adhesion

Phospholipases A2 (PLA2s) comprise distinct sets of enzymes with different localizations: the intracellular (cytosolic) enzymes that are Ca2+ dependent (cPLA2), Ca2+ independent (iPLA2), or specific for PAF (intracellular PAF acetylhydrolase) and extracellular (plasma) enzymes, either associated with lipoproteins (Lp-PLA2) or secreted PLA2s (sPLA2) (Mallat et al., 2010). Extracellular (plasma) PAF-AH shares 41% sequence identity with intracellular (cytosolic) Type II PAFAH, whereas both enzymes show less structural similarity to Type I PAF-AH (Karasawa, K. 2006; Mitsios et al., 2006). Secreted PLA2s (sPLA2) represent a

investigation (Reddy et al., 2001; Getz, GS. Resardon, CA. 2004).

molecules and cytokines (Garza et al., 2007; Tsimikas et al., 2009).

Nieminen et al., 2006; Shih DM. & Lusis AJ. 2009). The enormous between-individual biological variability in serum PON1 activity seems to be regulated mainly by genetic determinants. The paraoxonase gene family includes *pon1*, *pon2* and *pon3* genes which produce three enzyme paraoxonase 1 (PON1), paraoxonase 2 (PON2) and paraoxonase 3 (PON3). These genes are located on the long arm of chromosome 7 and they are structurally similar. They share about 70% of identity in nucleotide sequences and about 60% of identity in amino acid sequences. PON1 mRNA is expressed in the liver, and PON3 mRNA is expressed primarily in the liver but also in the kidneys. On the other hand PON2 mRNA is ubiquitously expressed in different kinds of tissues like kidneys, liver, lungs, small intestine, placenta, spleen, stomach and testicles and in the cells of the artery wall (including endothelial cell, smooth muscle cell and macrophages) (Draganov, DI. La Du, BN. 2004; Ng et al., 2005). PON1 is a 354 amino acid long glycosylated protein and has an apparent mass of 43-47 kDa. The enzyme is synthesized in the liver and is secreted into plasma. In the plasma, PON1 is mainly bounded to high density lipoproteins (HDL) but also small amount of this enzyme was detected in very low-density lipoprotein (VLDL), and postprandial chylomicrons. PON1 has hydrophobic signal sequence on the N-terminal region, from which only the initiator methionine residue is removed, and this region is for the association of PON1 with HDL (Draganov, DI. La Du, BN. 2004; Fuhrman et al., 2005.) PON1 possesses organophosphatase, arylesterase and lactonase activities and hydrolyzes different kinds of substrates (like paraoxon, chlorpyrifos oxon, diazoxon, sarin, soman, phenylacetate, tiophenylacetate homogentisic acid lactone, dihydrocoumarin, γ-butyrolactone and homocysteine thiolactone) (Draganov, DI. La Du, BN. 2004; Ng et al., 2005). PON1 is also well known to possess antioxidative and antiatherogenic activity, to protect HDL and lowdensity lipoprotein (LDL) from oxidation, and to destroy biologically active oxidized lipids on lipoproteins and in arterial cells (Draganov, DI. La Du, BN. 2004; Aviram, M. 2004). More than 160 polymorphisms of *pon1* gene are known, and some of them have been recognized to affect PON1 concentration and activity (Deakin, SP. James, RW. 2004; Costa et al., 2005). Two polymorphisms in the coding region of *pon1* gene result in the substitution of amino acid glutamine with arginine at the position 192 (Q192R polymorphism, the exchange of codon CAA to CGA in exon 6) and in the substitution of amino acid leucine to methionine at the position 55 (L55M polymorphism, the exchange of codon TTG to ATG in exon 3) (Adkins et al., 1993). Q192 and R192 alloenzymes have a different affinity and catalytic activity towards numerous substrates, the R192 alloenzyme hydrolyzes paraoxon six times faster than Q192 alloenzyme while Q192 alloenzyme hydrolyzes sarin, soman and diazoxon faster than R192 alloenzyme (Deakin, SP. James, RW. 2004). These two alloenzymes are also different in their ability to protect LDL from oxidation *in vitro*, Q192 alloenzyme is more efficient than R192 alloenzyme (Deakin, SP. James, RW. 2004; Mackness et al., 1999). L55M polymorphism affects PON1 mRNA levels, concentration and enzyme activity. M55 alloenzyme is associated with a lower level of PON1 mRNA, concentration and activity (Deakin, SP. James, RW. 2004). These two alloenzymes are also different in protection of LDL against oxidation, where M55 alloenzyme shows to be more protective (Mackness et al., 1999). In the promoter region of *pon1* gene at least five polymorphisms were detected and - 108C>T polymorphism is one of them. This polymorphism affects *pon1* gene expression, and enzyme concentration and activity. It is believed that -108C>T polymorphism is the main contributor to serum PON1 variation (accounting for 23-24% of total variation), while other polymorphisms in *pon1* promoter region made little or no difference to serum PON1 levels (Deakin, SP. James, RW. 2004; Leviev, I. James, RW. 2000; Suehiro et al., 2000).

Nieminen et al., 2006; Shih DM. & Lusis AJ. 2009). The enormous between-individual biological variability in serum PON1 activity seems to be regulated mainly by genetic determinants. The paraoxonase gene family includes *pon1*, *pon2* and *pon3* genes which produce three enzyme paraoxonase 1 (PON1), paraoxonase 2 (PON2) and paraoxonase 3 (PON3). These genes are located on the long arm of chromosome 7 and they are structurally similar. They share about 70% of identity in nucleotide sequences and about 60% of identity in amino acid sequences. PON1 mRNA is expressed in the liver, and PON3 mRNA is expressed primarily in the liver but also in the kidneys. On the other hand PON2 mRNA is ubiquitously expressed in different kinds of tissues like kidneys, liver, lungs, small intestine, placenta, spleen, stomach and testicles and in the cells of the artery wall (including endothelial cell, smooth muscle cell and macrophages) (Draganov, DI. La Du, BN. 2004; Ng et al., 2005). PON1 is a 354 amino acid long glycosylated protein and has an apparent mass of 43-47 kDa. The enzyme is synthesized in the liver and is secreted into plasma. In the plasma, PON1 is mainly bounded to high density lipoproteins (HDL) but also small amount of this enzyme was detected in very low-density lipoprotein (VLDL), and postprandial chylomicrons. PON1 has hydrophobic signal sequence on the N-terminal region, from which only the initiator methionine residue is removed, and this region is for the association of PON1 with HDL (Draganov, DI. La Du, BN. 2004; Fuhrman et al., 2005.) PON1 possesses organophosphatase, arylesterase and lactonase activities and hydrolyzes different kinds of substrates (like paraoxon, chlorpyrifos oxon, diazoxon, sarin, soman, phenylacetate, tiophenylacetate homogentisic acid lactone, dihydrocoumarin, γ-butyrolactone and homocysteine thiolactone) (Draganov, DI. La Du, BN. 2004; Ng et al., 2005). PON1 is also well known to possess antioxidative and antiatherogenic activity, to protect HDL and lowdensity lipoprotein (LDL) from oxidation, and to destroy biologically active oxidized lipids on lipoproteins and in arterial cells (Draganov, DI. La Du, BN. 2004; Aviram, M. 2004). More than 160 polymorphisms of *pon1* gene are known, and some of them have been recognized to affect PON1 concentration and activity (Deakin, SP. James, RW. 2004; Costa et al., 2005). Two polymorphisms in the coding region of *pon1* gene result in the substitution of amino acid glutamine with arginine at the position 192 (Q192R polymorphism, the exchange of codon CAA to CGA in exon 6) and in the substitution of amino acid leucine to methionine at the position 55 (L55M polymorphism, the exchange of codon TTG to ATG in exon 3) (Adkins et al., 1993). Q192 and R192 alloenzymes have a different affinity and catalytic activity towards numerous substrates, the R192 alloenzyme hydrolyzes paraoxon six times faster than Q192 alloenzyme while Q192 alloenzyme hydrolyzes sarin, soman and diazoxon faster than R192 alloenzyme (Deakin, SP. James, RW. 2004). These two alloenzymes are also different in their ability to protect LDL from oxidation *in vitro*, Q192 alloenzyme is more efficient than R192 alloenzyme (Deakin, SP. James, RW. 2004; Mackness et al., 1999). L55M polymorphism affects PON1 mRNA levels, concentration and enzyme activity. M55 alloenzyme is associated with a lower level of PON1 mRNA, concentration and activity (Deakin, SP. James, RW. 2004). These two alloenzymes are also different in protection of LDL against oxidation, where M55 alloenzyme shows to be more protective (Mackness et al., 1999). In the promoter region of *pon1* gene at least five polymorphisms were detected and - 108C>T polymorphism is one of them. This polymorphism affects *pon1* gene expression, and enzyme concentration and activity. It is believed that -108C>T polymorphism is the main contributor to serum PON1 variation (accounting for 23-24% of total variation), while other polymorphisms in *pon1* promoter region made little or no difference to serum PON1 levels

(Deakin, SP. James, RW. 2004; Leviev, I. James, RW. 2000; Suehiro et al., 2000).

As it was mentioned earlier, PON2 is a ubiquitously expressed intracellular protein with a relative molecular mass of approximately 44 kDa (Ng et al., 2005; Li et al., 2003). PON2 has antioxidant properties, lowers the intracellular oxidative stress and prevents the cellmediated oxidation of LDL (Ng at al., 2005; Li et al., 2003). In the *pon2* gene two common polymorphisms were identified. Alanine or glycine could be at the position 148 (A148G), and serine or cysteine could be at the position 311 (S311C). S311C polymorphism has been related with eg. coronary artery disease, ischemic stroke in patients with type 2 diabetes mellitus, Alzheimer's disease and reduced bone mass in postmenopausal women (Ng at al., 2005; Li et al., 2003). The mechanisms by which PON2 exerts its atheroprotective effects remain to be clarified. Large-scale epidemiologic studies are needed to further examine the relationship between PON2 genetic polymorphisms and risk for CVD (Shih, DM. Lusius, AJ. 2009).

Human PON3 is a '40-kDa protein primarily synthesized in the liver with biological activity similar to PON1. PON3 is a secreted protein associated with HDL in the plasma and can participate in the prevention of LDL oxidation. The PON3 protein may play a role, distinct from that of PON1, in the lipoprotein metabolism of the kidney. These characteristics link PON3 with a group of enzymes, such as PON1, platelet-activating factor–acetylhydrolase, and lecithin-cholesterol acyltransferase, which together may contribute to the antiatherogenic properties of HDL, but the role of PON3 in atherosclerosis needs further investigation (Reddy et al., 2001; Getz, GS. Resardon, CA. 2004).

Another lipoprotein-associated enzyme, the platelet-activating factor acetylhydrolase (PAF-AH), also referred to as lipoprotein-associated phospholipase A2 (Lp-PLA2), is an enzyme (EC 3.1.1.47) recently described as a potentially useful plasma biomarker associated with cardiovascular disease (Srinivasan, B. Bahson, BJ. 2010; Koenig et al., 2004; Yamada et al., 2000; Karasawa, K. 2006; Mallat et al., 2010). The biological role of Lp-PLA2 (PAF-AH) has been controversial, with contradictory antiatherogenic and proatherogenic functions. The antiatherogenic properties of Lp-PLA2 were first suggested because plasma PAF-AH might play an anti-inflammatory role in human diseases by preventing the accumulation of PAF (1- O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and PAF-like oxidized phospholipids (Karasawa, K. 2006; Mallat et al., 2010; Mitsios et al., 2006). PAF is a biologically active phospholipid involved in diverse pathologies such as inflammation and atherosclerosis. PAF can activate various cell types including platelets. In the presence of PAF, platelets aggregate and degranulate, releasing biologically potent agents. PAF is hydrolyzed and converted to lysoPAF by the catalytic reaction of PAF-AH (Mitsios et al., 2006). The atherogenic role of Lp-PLA2 comes from the observation that this enzyme can also produce lysophosphatidylcholine and oxidatively modified nonesterified fatty acids which could promote the pathogenesis of atherosclerosis (Karasawa, K. 2006; Mallat et al., 2010). Lysophosphatidylcholine is an important chemoattractant for macrophages and T cells, it induces migration of vascular smooth muscle cells, affects endothelial function, and increases the expression of adhesion molecules and cytokines (Garza et al., 2007; Tsimikas et al., 2009).

Phospholipases A2 (PLA2s) comprise distinct sets of enzymes with different localizations: the intracellular (cytosolic) enzymes that are Ca2+ dependent (cPLA2), Ca2+ independent (iPLA2), or specific for PAF (intracellular PAF acetylhydrolase) and extracellular (plasma) enzymes, either associated with lipoproteins (Lp-PLA2) or secreted PLA2s (sPLA2) (Mallat et al., 2010). Extracellular (plasma) PAF-AH shares 41% sequence identity with intracellular (cytosolic) Type II PAFAH, whereas both enzymes show less structural similarity to Type I PAF-AH (Karasawa, K. 2006; Mitsios et al., 2006). Secreted PLA2s (sPLA2) represent a

Paraoxonase Polymorphisms and Platelet Activating

Zagreb, Croatia.

**2.2 Samples** 

**2.3 Methods** 

lumen of carotid arteries in three patients were found.

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 511

was angiographically determined. In this group, intracranial stenosis less than 50% of the

The control no-stenosis group consisted of 90 patients, 46 female, median age 60 years (range, 44-76 years) and 44 male, median age 63 years (range, 46-82 years) with suspected cerebrovascular symptoms, but with normal Doppler examination of the carotid arteries. Vertigo, headache and transitory vision problems were indications for Doppler examination for 72 patients (80%). Twelve out of 90 (13.3%) patients had had nonischemic cerebrovascular insult a few months or years priorly with new symptoms like headache, suspected motor deficit or vertigo. The remaining six patients (6.7%) had the same symptoms combined with the carotid bruit. All of them had normal appearance and normal hemodynamic results at Doppler examination of carotid arteries. The third group of patients, with Doppler established carotid stenosis between 1-49% of cerebral arteries, was not included in the present investigation. They were proceeded to other non-invasive

All Doppler and DSA procedures were performed at the Institute for Diagnostic and Interventional Radiology of the Merkur University Hospital. Doppler examinations were performed at the center of excellence with more than 3,000 examinations per year. DSA was

Smokers were defined as those reporting daily smoking. Obesity was defined in terms of the

The patients with the BMI 25 were considered overweight. Written informed consent was

This study was approved by the Ethics Committee of the Merkur University Hospital,

Blood samples were collected by venopuncture after overnight fasting and under controlled pre-analytical conditions. Serum was prepared 30 min after blood collection into vacutainer tubes (Becton Dickinson) without additives by centrifugation at 3000 rpm for 15 minutes. Blood collected in EDTA-coated tubes was used for determination of *pon1 and pon2*  genotypes while sera were analyzed for triacylglycerol, total cholesterol, LDL and HDL-

**2.3.1 Serum triacylglycerol, total cholesterol, LDL and HDL cholesterol assays** 

Serum triacylglycerol and total cholesterol were measured by enzymatic PAP- method. HDLcholesterol was measured with direct method based on selective inhibition of the non-HDL fractions by means of polyanions. A homogeneous assay for the selective measurement of LDLcholesterol in serum was used. All measurements were performed on fresh sera on the day of blood collection using standard commercial kits (Olympus Diagnostic GmbH, Hamburg, Germany) on the Olympus AU 600 analyzer (Olympus Mishima Co., Ltd., Shizuoka, Japan).

PON1 paraoxonase activity was assessed by using paraoxon as the substrate in the presence of NaCl (NaCl stimulated activity) (Juretić et al., 2006). The assay was performed on Olympus AU 600 biochemical analyzer (Olympus Mishima Co., Ltd., Shizuoka, Japan) at

performed by the interventional radiologists skilled in neurovascular interventions.

carotid investigations like MR angiography or multislice CT angiography.

patient's body mass index (BMI) calculated as weight in kg/height in m2 .

cholesterol concentrations and PON1 and PAF-AH activities.

**2.3.2 Paraoxonase activity measurement** 

obtained from all subjects according to the guidelines of our Ethics Committee.

diverse family of structurally related, disulfide-rich calcium-dependent secreted enzymes that hydrolyze the sn-2 position of glycerophospholipids generating potent lipid mediators: lysophospholipids and free fatty acids, including the precursor of eicosanoids, arachidonic acid. Extracellular levels of secreted PLA2s are increased in both plasma and inflammatory fluids in various inflammatory diseases ( Karabina et al., 2010; Mallat et al., 2010 ).

The extracellular (plasma) enzyme Lp-PLA2 is a single polypeptide that originates mostly from cells of the hematopoietic lineage, primarily from monocytes/macrophages (Karabina et al., 2010; Stafforini, DM. 2009). Lp-PLA2 (PAF-AH) exhibits unique substrate specificity toward PAF and oxidized phospholipids. In human plasma, PAF-AH activity is associated mainly with the apolipoprotein B (apoB)-containing lipoproteins and primarily with low-density lipoprotein (LDL). A small proportion of the circulating enzyme activity is also associated with high density lipoprotein and lipoprotein(a), an atherogenic lipoprotein particle that appears to be a preferential carrier of oxidized phospholipids in human plasma (Mallat et al., 2010; Wolfert et al., 2004; Karasawa, K. 2006). In plasma, approximately 80% of Lp-PLA2 is attached to low-density lipoproteins (LDLs), and the remaining 20% is linked to high-density lipoproteins (HDLs) and lipoprotein (a) (Garza et al., 2007). HDL protects LDL from oxidation and HDL-associated PAF-AH might be involved in this effect together with other HDLassociated enzymes, including PON1 and lecithin-cholesterol acyltransferase (LCAT) . Dyslipidemia-induced decrease in the ratio of HDL-associated PAF-AH to the plasma PAF-AH levels might thus lead to the promotion of atherosclerosis (Karasawa, K. 2006 ; Garza et al., 2007). Many studies appeared on the role of lipoprotein–associated PLA2 and secreted PLA2s in atherosclerosis at the level of biology and epidemiology. It is still unclear whether these PLA2s act as true biological effectors of cardiovascular diseases in humans and whether they have proven utility as biomarkers of disease severity (Mallat et al., 2010).

We explored relations between serum PON1 and PAF-AH activities as well as the distribution of polymorphisms of *pon1* and *pon2* genes and cerebral atherosclerosis in wellcharacterized groups of patients with angiografically assessed severe stenosis of cerebral arteries and matched control no-stenosis group.

#### **2. Patients and methods**

#### **2.1 Patients**

The study comprised 119 patients, 35 women and 84 men with symptoms of cerebrovascular insufficiency and stenosis of carotid artery more than 50% of the lumen. Among them, 87 (73.1%) had transitory ischemic attacks, 19 (16.0%) had suffered a cerebrovascular insult with motor deficit 5-9 months previously, and 13 patients (10.9%) had headache and vertigo with carotid bruit. All patients were examined by neurologists and referred to Doppler examination. At the Doppler examination, all of them had stenosis of one or both carotid arteries more than 50% of the arterial lumen and were preceded to digital subtraction angiography (DSA) and possible endovascular carotid PTA/stent treatment. Based on the angiographic findings, for the purpose of present investigation they were divided in two groups. The first group consisted of 73 patients, 25 female, median age 67 years (range, 41-79 years) and 48 male, median age 65 years (range, 46-83 years) with a moderate degree of carotid extra cranial stenosis between 50% and 69% of the arterial lumen. In this group there was no intracranial stenosis of cerebral arteries. The second group consisted of 46 patients, 10 female, median age 67 years (range, 46-78 years) and 36 male, median age 68 years (range, 54-78 years) in whom stenosis between 70-99% or obliteration of the carotid artery was angiographically determined. In this group, intracranial stenosis less than 50% of the lumen of carotid arteries in three patients were found.

The control no-stenosis group consisted of 90 patients, 46 female, median age 60 years (range, 44-76 years) and 44 male, median age 63 years (range, 46-82 years) with suspected cerebrovascular symptoms, but with normal Doppler examination of the carotid arteries. Vertigo, headache and transitory vision problems were indications for Doppler examination for 72 patients (80%). Twelve out of 90 (13.3%) patients had had nonischemic cerebrovascular insult a few months or years priorly with new symptoms like headache, suspected motor deficit or vertigo. The remaining six patients (6.7%) had the same symptoms combined with the carotid bruit. All of them had normal appearance and normal hemodynamic results at Doppler examination of carotid arteries. The third group of patients, with Doppler established carotid stenosis between 1-49% of cerebral arteries, was not included in the present investigation. They were proceeded to other non-invasive carotid investigations like MR angiography or multislice CT angiography.

All Doppler and DSA procedures were performed at the Institute for Diagnostic and Interventional Radiology of the Merkur University Hospital. Doppler examinations were performed at the center of excellence with more than 3,000 examinations per year. DSA was performed by the interventional radiologists skilled in neurovascular interventions.

Smokers were defined as those reporting daily smoking. Obesity was defined in terms of the patient's body mass index (BMI) calculated as weight in kg/height in m2 .

The patients with the BMI 25 were considered overweight. Written informed consent was obtained from all subjects according to the guidelines of our Ethics Committee.

This study was approved by the Ethics Committee of the Merkur University Hospital, Zagreb, Croatia.

#### **2.2 Samples**

510 Atherogenesis

diverse family of structurally related, disulfide-rich calcium-dependent secreted enzymes that hydrolyze the sn-2 position of glycerophospholipids generating potent lipid mediators: lysophospholipids and free fatty acids, including the precursor of eicosanoids, arachidonic acid. Extracellular levels of secreted PLA2s are increased in both plasma and inflammatory

The extracellular (plasma) enzyme Lp-PLA2 is a single polypeptide that originates mostly from cells of the hematopoietic lineage, primarily from monocytes/macrophages (Karabina et al., 2010; Stafforini, DM. 2009). Lp-PLA2 (PAF-AH) exhibits unique substrate specificity toward PAF and oxidized phospholipids. In human plasma, PAF-AH activity is associated mainly with the apolipoprotein B (apoB)-containing lipoproteins and primarily with low-density lipoprotein (LDL). A small proportion of the circulating enzyme activity is also associated with high density lipoprotein and lipoprotein(a), an atherogenic lipoprotein particle that appears to be a preferential carrier of oxidized phospholipids in human plasma (Mallat et al., 2010; Wolfert et al., 2004; Karasawa, K. 2006). In plasma, approximately 80% of Lp-PLA2 is attached to low-density lipoproteins (LDLs), and the remaining 20% is linked to high-density lipoproteins (HDLs) and lipoprotein (a) (Garza et al., 2007). HDL protects LDL from oxidation and HDL-associated PAF-AH might be involved in this effect together with other HDLassociated enzymes, including PON1 and lecithin-cholesterol acyltransferase (LCAT) . Dyslipidemia-induced decrease in the ratio of HDL-associated PAF-AH to the plasma PAF-AH levels might thus lead to the promotion of atherosclerosis (Karasawa, K. 2006 ; Garza et al., 2007). Many studies appeared on the role of lipoprotein–associated PLA2 and secreted PLA2s in atherosclerosis at the level of biology and epidemiology. It is still unclear whether these PLA2s act as true biological effectors of cardiovascular diseases in humans and whether they

We explored relations between serum PON1 and PAF-AH activities as well as the distribution of polymorphisms of *pon1* and *pon2* genes and cerebral atherosclerosis in wellcharacterized groups of patients with angiografically assessed severe stenosis of cerebral

The study comprised 119 patients, 35 women and 84 men with symptoms of cerebrovascular insufficiency and stenosis of carotid artery more than 50% of the lumen. Among them, 87 (73.1%) had transitory ischemic attacks, 19 (16.0%) had suffered a cerebrovascular insult with motor deficit 5-9 months previously, and 13 patients (10.9%) had headache and vertigo with carotid bruit. All patients were examined by neurologists and referred to Doppler examination. At the Doppler examination, all of them had stenosis of one or both carotid arteries more than 50% of the arterial lumen and were preceded to digital subtraction angiography (DSA) and possible endovascular carotid PTA/stent treatment. Based on the angiographic findings, for the purpose of present investigation they were divided in two groups. The first group consisted of 73 patients, 25 female, median age 67 years (range, 41-79 years) and 48 male, median age 65 years (range, 46-83 years) with a moderate degree of carotid extra cranial stenosis between 50% and 69% of the arterial lumen. In this group there was no intracranial stenosis of cerebral arteries. The second group consisted of 46 patients, 10 female, median age 67 years (range, 46-78 years) and 36 male, median age 68 years (range, 54-78 years) in whom stenosis between 70-99% or obliteration of the carotid artery

fluids in various inflammatory diseases ( Karabina et al., 2010; Mallat et al., 2010 ).

have proven utility as biomarkers of disease severity (Mallat et al., 2010).

arteries and matched control no-stenosis group.

**2. Patients and methods** 

**2.1 Patients** 

Blood samples were collected by venopuncture after overnight fasting and under controlled pre-analytical conditions. Serum was prepared 30 min after blood collection into vacutainer tubes (Becton Dickinson) without additives by centrifugation at 3000 rpm for 15 minutes. Blood collected in EDTA-coated tubes was used for determination of *pon1 and pon2*  genotypes while sera were analyzed for triacylglycerol, total cholesterol, LDL and HDLcholesterol concentrations and PON1 and PAF-AH activities.

#### **2.3 Methods**

#### **2.3.1 Serum triacylglycerol, total cholesterol, LDL and HDL cholesterol assays**

Serum triacylglycerol and total cholesterol were measured by enzymatic PAP- method. HDLcholesterol was measured with direct method based on selective inhibition of the non-HDL fractions by means of polyanions. A homogeneous assay for the selective measurement of LDLcholesterol in serum was used. All measurements were performed on fresh sera on the day of blood collection using standard commercial kits (Olympus Diagnostic GmbH, Hamburg, Germany) on the Olympus AU 600 analyzer (Olympus Mishima Co., Ltd., Shizuoka, Japan).

#### **2.3.2 Paraoxonase activity measurement**

PON1 paraoxonase activity was assessed by using paraoxon as the substrate in the presence of NaCl (NaCl stimulated activity) (Juretić et al., 2006). The assay was performed on Olympus AU 600 biochemical analyzer (Olympus Mishima Co., Ltd., Shizuoka, Japan) at

Paraoxonase Polymorphisms and Platelet Activating

morphism Primer °C

Poly-

*pon1*

*pon1*

*pon1* - 108C>T

*pon*2

1CT: 5′

**2.3.4 PAF-AH activity assay** 

frozen at -80°C until the day of analysis.

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 513

Q192R 1QR: 5′ TATTGTTGCTGTGGGACCTGAG 3′ 60 BspPI 238 bp Q allele:

L55M 1LM: 5′ CCTGCAATAATATGAAACAACCTG 3′ 63 Hin1II 172 bp L allele:

S311C 1SC: 5′ ACATGCATGTACGGTGGTCTTATA 3′ 55 DdeI 265 bp S allele:

Table 1. Conditions for PCR-RFLP method. The lower case base "a" in *pon1* -108CNT 1CT primer indicates a mismatch, introducing a restriction site for restriction enzyme BsrBI.

fragments of 142, 123, 75 and 67 bp were detected in genotype SC.

Determination of *pon2* S311C polymorphism by PCR-RFLP procedure using DdeI restriction enzyme was carried out as follows. The exchange of the nucleotide C with G results in substitution of codon TCT to TGT (exon 9 of *pon2* gene), and with substitution of serine to cystein at position 311 (S311C, SNP ID rs7493). S and C alleles have a restriction site for DdeI restriction enzyme but the presence of codon TCT in S allele introduces an additional restriction site for this enzyme. The amplified fragment of 265 bp was digested in two fragments (142 and 123 bp) in both S and C allele. In the case of S allele 142 bp fragment is additionally digested in two fragments (75 and 67 bp). Fragments of 123, 75 and 67 bp were detected in genotype SS, fragments of 142 and 123 bp were detected in genotype CC, and

Platelet-activating factor acetylhydrolase (PAF-AH) activity was measured in plain serum with the new automated spectrophotometric assay (Azwell Inc., Auto PAF-AH, Osaka, Japan) at 37C (Kosaka et al., 2000). In the first phase, 2µL of serum was added to 240 µL of 200 mmol/L HEPES (*N*-2-hydroxyethylpiperazine–*N´*-2-ethanesulfonic acid) buffer (Reagent 1), pH 7.6 and pre-incubated at 37ºC for 5 min. The reaction was started by adding 80 µL of 20 mmol/L citric acid monohydrate buffer, pH 4.5 containing 90 mmol/L 1 myristoyl-2-(4-nitrophenylsuccinyl)phosphatidylcholine (Reagent 2). The liberation of *p*nitrophenol was monitored at 405 and 505 nm at 1 and 3 min after the addition of Reagent 2 using the automatic biochemical analyzer OlympusAU600 (Olympus Mishima Co., Ltd., Shizuoka, Japan). Enzyme activities are expressed in international units per liter of serum and standardized against concentration of LDL-cholesterol. Serum samples were kept

2QR: 5′ CCTGAGAATCTGAGTAAATCCACT 3′R allele:

2LM: 5′ TGAAAGACTTAAACTGCCAGTC 3′M allele:

AGCTAGCTGCCGACCCGGCGGGGAGGaG 3′ 68 BsrBI 240 bp C allele:

2CT: 5′ GGCTGCAGCCCTCACCACAACCC 3′T allele:

2SC: 5′ AGCAATTCATAGAAAATTAATTGTTA 3′C allele:

Restrictio n enzyme

PCR fragme nt

RFLP fragments

238 bp

172 bp

175+63 bp

103+69 bp

212+28 bp

123+75+67 bp

142+123 bp

240 bp

37°C, as previously described, with a minor modification (Grdić et al., 2008). Briefly, 15 μL of serum was added to 300 μL of reaction mixture containing 2.5 mmol/L paraoxon of ~90% purity, 2.2 mmol/L CaCl2 and 1.0 mol/L NaCl in 0.1 mol/L Tris– HCl buffer, pH 8.0. The release of p-nitrophenol from paraoxon was measured at 410/480 nm (ε410/480=17900 L/ mol cm) and the enzyme activity is expressed in international units per 1 L of serum and standardized against concentration of HDL-cholesterol . Serum samples were kept frozen at -80°C until the day of analysis.

#### **2.3.3 Paraoxonase polymorphisms determinations**

Polymorphisms of *pon1* and *pon2* genes were determined by the polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (PCR-RFLP) (Table 1). The PCR reaction was performed in a Gene Amp PCR System 2720 (Applied Biosystems) PCR machine. *Pon1* gene polymorphisms (Q192R, L55M and -108C>T) were determined by the method described by Campo et al., (Campo et al., 2004). with some modifications concerning the sequence of 1CT primer, annealing temperature and restriction enzyme for -108C>T polymorphism (Grdić et al., 2008; Grdić Rajković et al., 2011).

*Pon2* gene polymorphism (S311C) was determined by the method described by Sanghera et al. (Sangera et al., 1998) with a few modifications including the sequence of 2SC primer and annealing temperature (Grdić et al., 2011). Briefly, the amplification mixture (total volume 25 μL) for each *pon1* gene polymorphism and for *pon2* gene polymorphism contained 250 ng of genomic DNA, 0.4 μmol/L of each primer, 0.2 mmol/L of each dNTP, 2mmol/LMgCl2, 0.5 units of PlatinumTaqDNA Polymerase and 2.5 μL of reaction buffer (200mmol/L Tris– HCl, pH 8.4 and 500mmol/L KCl). PCR reaction was carried out using the following procedure: the first step of predenaturation at 95 °C for 12 min, 35 cycles of amplification (30 seconds at 94 °C followed by 30 seconds at specific primers annealing temperature and 60 seconds at 72 °C), and the last cycle of final extension at 72 °C for 7 min. PCR was attenuated by lowering the temperature to 4 °C for at least 6 min. The primers, annealing temperatures and lengths of PCR fragments are given in Table 1. Endonuclease mixture for each polymorphism explored in this study (total volume 15 μL) contained 9 μL of amplified fragment, an appropriate buffer for each restriction enzyme and 4 units of BspPI (for *pon1* Q192R), 5 units of Hin1II (for *pon1* L55M), 3 units of BsrBI (for *pon1* -108C>T) and 3 units of DdeI (for *pon2* S311C). For separation of restriction products electrophoresis on 4% agarose gel in TAE buffer (0.04 mol/L Tris–HCl, 5 mmol/L Na-acetate, 0.04 mmol/L EDTA, pH 7.9) and stained with ethidium bromide (final concentration was 0.5 μg/mL) were used. The length of RFLP fragments is given in Table 1.

Determination of *pon1* Q192R, *pon1* L55M and *pon1* -108C>T polymorphisms by the PCR-RFLP procedure using specific restriction enzymes were described in details previously (Grdić et al., 2008, 2011). Briefly, for *pon1* Q192R polymorphism undigested fragment (238 bp) was detected in genotype QQ, digested fragments (175 and 63 bp) were detected in genotype RR, and both digested and undigested fragments (238, 175 and 63 bp) were detected in genotype QR. For *pon1* L55M polymorphism undigested fragment (172 bp) was detected in genotype LL, digested fragments (103 and 69 bp) were detected in genotype MM, and digested and undigested fragments (172, 103 and 69 bp) were detected in genotype LM. For *pon1*-108C>T polymorphism undigested fragment (240 bp) was detected in genotype TT, digested fragment (212 bp) was detected in genotype CC, and both undigested and digested fragments (240 and 212 bp) were detected in genotype CT .


Table 1. Conditions for PCR-RFLP method. The lower case base "a" in *pon1* -108CNT 1CT primer indicates a mismatch, introducing a restriction site for restriction enzyme BsrBI.

Determination of *pon2* S311C polymorphism by PCR-RFLP procedure using DdeI restriction enzyme was carried out as follows. The exchange of the nucleotide C with G results in substitution of codon TCT to TGT (exon 9 of *pon2* gene), and with substitution of serine to cystein at position 311 (S311C, SNP ID rs7493). S and C alleles have a restriction site for DdeI restriction enzyme but the presence of codon TCT in S allele introduces an additional restriction site for this enzyme. The amplified fragment of 265 bp was digested in two fragments (142 and 123 bp) in both S and C allele. In the case of S allele 142 bp fragment is additionally digested in two fragments (75 and 67 bp). Fragments of 123, 75 and 67 bp were detected in genotype SS, fragments of 142 and 123 bp were detected in genotype CC, and fragments of 142, 123, 75 and 67 bp were detected in genotype SC.

#### **2.3.4 PAF-AH activity assay**

512 Atherogenesis

37°C, as previously described, with a minor modification (Grdić et al., 2008). Briefly, 15 μL of serum was added to 300 μL of reaction mixture containing 2.5 mmol/L paraoxon of ~90% purity, 2.2 mmol/L CaCl2 and 1.0 mol/L NaCl in 0.1 mol/L Tris– HCl buffer, pH 8.0. The release of p-nitrophenol from paraoxon was measured at 410/480 nm (ε410/480=17900 L/ mol cm) and the enzyme activity is expressed in international units per 1 L of serum and standardized against concentration of HDL-cholesterol . Serum samples were kept frozen at

Polymorphisms of *pon1* and *pon2* genes were determined by the polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis (PCR-RFLP) (Table 1). The PCR reaction was performed in a Gene Amp PCR System 2720 (Applied Biosystems) PCR machine. *Pon1* gene polymorphisms (Q192R, L55M and -108C>T) were determined by the method described by Campo et al., (Campo et al., 2004). with some modifications concerning the sequence of 1CT primer, annealing temperature and restriction enzyme for

*Pon2* gene polymorphism (S311C) was determined by the method described by Sanghera et al. (Sangera et al., 1998) with a few modifications including the sequence of 2SC primer and annealing temperature (Grdić et al., 2011). Briefly, the amplification mixture (total volume 25 μL) for each *pon1* gene polymorphism and for *pon2* gene polymorphism contained 250 ng of genomic DNA, 0.4 μmol/L of each primer, 0.2 mmol/L of each dNTP, 2mmol/LMgCl2, 0.5 units of PlatinumTaqDNA Polymerase and 2.5 μL of reaction buffer (200mmol/L Tris– HCl, pH 8.4 and 500mmol/L KCl). PCR reaction was carried out using the following procedure: the first step of predenaturation at 95 °C for 12 min, 35 cycles of amplification (30 seconds at 94 °C followed by 30 seconds at specific primers annealing temperature and 60 seconds at 72 °C), and the last cycle of final extension at 72 °C for 7 min. PCR was attenuated by lowering the temperature to 4 °C for at least 6 min. The primers, annealing temperatures and lengths of PCR fragments are given in Table 1. Endonuclease mixture for each polymorphism explored in this study (total volume 15 μL) contained 9 μL of amplified fragment, an appropriate buffer for each restriction enzyme and 4 units of BspPI (for *pon1* Q192R), 5 units of Hin1II (for *pon1* L55M), 3 units of BsrBI (for *pon1* -108C>T) and 3 units of DdeI (for *pon2* S311C). For separation of restriction products electrophoresis on 4% agarose gel in TAE buffer (0.04 mol/L Tris–HCl, 5 mmol/L Na-acetate, 0.04 mmol/L EDTA, pH 7.9) and stained with ethidium bromide (final concentration was 0.5 μg/mL) were used. The

Determination of *pon1* Q192R, *pon1* L55M and *pon1* -108C>T polymorphisms by the PCR-RFLP procedure using specific restriction enzymes were described in details previously (Grdić et al., 2008, 2011). Briefly, for *pon1* Q192R polymorphism undigested fragment (238 bp) was detected in genotype QQ, digested fragments (175 and 63 bp) were detected in genotype RR, and both digested and undigested fragments (238, 175 and 63 bp) were detected in genotype QR. For *pon1* L55M polymorphism undigested fragment (172 bp) was detected in genotype LL, digested fragments (103 and 69 bp) were detected in genotype MM, and digested and undigested fragments (172, 103 and 69 bp) were detected in genotype LM. For *pon1*-108C>T polymorphism undigested fragment (240 bp) was detected in genotype TT, digested fragment (212 bp) was detected in genotype CC, and both undigested

and digested fragments (240 and 212 bp) were detected in genotype CT .


**2.3.3 Paraoxonase polymorphisms determinations** 

length of RFLP fragments is given in Table 1.


Platelet-activating factor acetylhydrolase (PAF-AH) activity was measured in plain serum with the new automated spectrophotometric assay (Azwell Inc., Auto PAF-AH, Osaka, Japan) at 37C (Kosaka et al., 2000). In the first phase, 2µL of serum was added to 240 µL of 200 mmol/L HEPES (*N*-2-hydroxyethylpiperazine–*N´*-2-ethanesulfonic acid) buffer (Reagent 1), pH 7.6 and pre-incubated at 37ºC for 5 min. The reaction was started by adding 80 µL of 20 mmol/L citric acid monohydrate buffer, pH 4.5 containing 90 mmol/L 1 myristoyl-2-(4-nitrophenylsuccinyl)phosphatidylcholine (Reagent 2). The liberation of *p*nitrophenol was monitored at 405 and 505 nm at 1 and 3 min after the addition of Reagent 2 using the automatic biochemical analyzer OlympusAU600 (Olympus Mishima Co., Ltd., Shizuoka, Japan). Enzyme activities are expressed in international units per liter of serum and standardized against concentration of LDL-cholesterol. Serum samples were kept frozen at -80°C until the day of analysis.

Paraoxonase Polymorphisms and Platelet Activating

**Control nostenosis group (N=90)** 

(44-82)

26.3 (20.2 – 35.7)

6.3 (4.2 – 8.4)

1.39 (0.34 – 4.14)

1.6 (1.0 – 3.1)

3.9 (2.6 – 5.9)

Whitney test; p <0.05 was considered as statistically significant.

(tryacylglicerol, total cholesterol, HDL cholesterol, LDL cholesterol) (Table 4).

**3.3 Paraoxonase activity measurement** 

kg/m2 , indicating overweight.

Age (years) <sup>61</sup>

(Table 2).

**Parameter** 

(kg/m2 )

Body mass index

Total cholesterol (mmol/L)

Triacylglicerol (mmol/L)

HDL- cholesterol (mmol/L)

LDL- cholesterol (mmol/L)

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 515

subgroups (Yates corrected χ² =0.003, p=0.338 in the group with <70% of stenosis; Yates corrected χ² =0.023, p=0.638 in the group with >70% of stenosis) or smoking habits and cerebrovascular stenosis subgroups (Yates corrected χ² =0.001, p=0.478 in the group with <70% of stenosis; Yates corrected χ² =0.012, p=0.962 in the group with >70% of stenosis). The proportion of daily smokers in the group of patients with <70% of stenosis was 33.3% and 32.6% in the group of patients with >70% of stenosis versus 25.8 % in control no-stenosis group. The mean values of the body mass index in all groups examined were more than 25

**3.2 Serum triacylglycerol, total cholesterol, LDL and HDL cholesterol concentrations**  Comparing the results obtained for the traditional risk factors (triacylglycerol, total cholesterol, HDL-cholesterol, LDL-cholesterol) between the groups of patients with cerebrovascular stenosis and control no-stenosis group using the Mann-Whitney univariate statistic method, significant differences were found for all serum lipid parameters (p<0.05)

> **<70% of stenosis (N=73)**

> > 66

25.7

5.4

1.75

1.3

3.6

Basal and stimulated PON1 activities differ significantly between patients group with stenosis and the control no-stenosis group, and HDL standardized basal and stimulated PON1 activity did not show statistical difference. Kolmogorov –Smirnov test for normal distribution reject normality for all examined data (Table 3) . There were no statistically significant relationships between basal and stimulated PON1 activity and examined lipid and lipoprotein parameters

Table 2. Demographic and biochemical parameters for control no-stenosis group and patients with <70% and >70% of cerebrovascular stenosis. Results are given as medians, with ranges in parentheses. p values: significance level for difference between the group of patients with cerebrovascular stenosis and the control no-stenosis group tested by Mann-

**Patients with cerebrovascular stenosis** 

**>70% of stenosis (N=46)** 

(46 - 83) 0.160

(19.0 – 35.1) 0.944

(3.5 - 9.6) 0.000

(0.71 – 5.09) 0.026

(0.8 – 1.8) 0.000

(1.2 – 8.4) 0.001

*P*

*P*

(41 - 83) 0.068 <sup>68</sup>

(19.1 – 34.1) 0.143 26.5

(3.4 – 11.5) 0.001 5.7

(0.43 – 8.18) 0.003 1.66

(0.7 – 2.3) 0.000 1.1

(1.8 – 6.3) 0.021 3.5

#### **2.3.5 Quality control of measurements**

The Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital has been accredited to ISO 15189, Medical laboratories - Particular requirements for quality and competence since 2007 (ISO 15189, 2008). Analytical methods for measurement of serum triacylglycerol, total cholesterol, LDL and HDL-cholesterol concentrations as well as for paraoxonase polymorphisms determinations used in this study have been accredited according to this norm (Flegar- Meštrić et al., 2010a). Traceability of analytical methods is achieved through a manufacturer's reference materials (calibrators) or reference methods for enzyme activities. Analyzer–based calibrations are routinely performed for compensation of systematic effects. Estimates of within-laboratory precision are provided by internal quality control data using commercial control sera (Olympus Diagnostic) for triacylglycerol, total cholesterol, LDL and HDL-cholesterol concentrations and pool serum samples for the paraoxonase and PAF-AH activities. Trueness estimates are based on the long-term results of external quality assessment (EQA) obtained by the participation of the Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital in the National External Quality Assessment Scheme organized by the Croatian Society of Medical Biochemists and international EQA schemes for general and special medical biochemistry organized by Labquality - WHO Collaborating Centre for Education and Training in Laboratory Quality Assurance, FIN-00520 Helsinki, Finland (Flegar-Meštrić, Z. et al., 2010b). According to the requirements of the international standard ISO 15189, interlaboratory comparisons were performed for the paraoxonase polymorphisms determinations between the Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital and Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia. Estimation of measurement uncertainties is done on the basis of the "Guide to the Expression of Uncertainty in Measurement" (GUM, 2005). The uncertainty components that we use are uncertainties related to calibrator, within-laboratory precision and trueness estimates based on the results of external quality assessment (EQA). The expanded measurement uncertainties (k=2) obtained for triacylglycerol, total cholesterol, LDL and HDL-cholesterol concentrations and pool serum samples for the paraoxonase and PAF-AH activities in the normal concentration range were 4.8, 4.0, 8.0, 11.1, 4.2 and 3.8%, respectively.

#### **2.4 Statistical analysis**

The Mann-Whitney U-test was applied to evaluate the differences between the groups, with p< 0.05 considered statistically significant. The correlations between serum PAF-AH activity and concentrations of total and LDL cholesterol were estimated using Pearson's correlation. Chi-square test was used for comparisons of allele and genotype proportions. MedCalc statistical program (MedCalc Software Version 8.1.0.0, 2005 Frank Schoonjans for Windows, available at the website:www.medcalc.be/) was used.

#### **3. Results**

#### **3.1 Patients**

The results of the Mann-Whitney U-test showed that, according to the demographic and lifestyle characteristics (age, body mass index), the control no-stenosis group matched the groups of patients with different degrees of cerebrovascular stenosis (Table 2). The chisquared test showed no significant differences between sex and cerebrovascular stenosis

The Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital has been accredited to ISO 15189, Medical laboratories - Particular requirements for quality and competence since 2007 (ISO 15189, 2008). Analytical methods for measurement of serum triacylglycerol, total cholesterol, LDL and HDL-cholesterol concentrations as well as for paraoxonase polymorphisms determinations used in this study have been accredited according to this norm (Flegar- Meštrić et al., 2010a). Traceability of analytical methods is achieved through a manufacturer's reference materials (calibrators) or reference methods for enzyme activities. Analyzer–based calibrations are routinely performed for compensation of systematic effects. Estimates of within-laboratory precision are provided by internal quality control data using commercial control sera (Olympus Diagnostic) for triacylglycerol, total cholesterol, LDL and HDL-cholesterol concentrations and pool serum samples for the paraoxonase and PAF-AH activities. Trueness estimates are based on the long-term results of external quality assessment (EQA) obtained by the participation of the Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital in the National External Quality Assessment Scheme organized by the Croatian Society of Medical Biochemists and international EQA schemes for general and special medical biochemistry organized by Labquality - WHO Collaborating Centre for Education and Training in Laboratory Quality Assurance, FIN-00520 Helsinki, Finland (Flegar-Meštrić, Z. et al., 2010b). According to the requirements of the international standard ISO 15189, interlaboratory comparisons were performed for the paraoxonase polymorphisms determinations between the Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital and Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia. Estimation of measurement uncertainties is done on the basis of the "Guide to the Expression of Uncertainty in Measurement" (GUM, 2005). The uncertainty components that we use are uncertainties related to calibrator, within-laboratory precision and trueness estimates based on the results of external quality assessment (EQA). The expanded measurement uncertainties (k=2) obtained for triacylglycerol, total cholesterol, LDL and HDL-cholesterol concentrations and pool serum samples for the paraoxonase and PAF-AH activities in the normal concentration range were

The Mann-Whitney U-test was applied to evaluate the differences between the groups, with p< 0.05 considered statistically significant. The correlations between serum PAF-AH activity and concentrations of total and LDL cholesterol were estimated using Pearson's correlation. Chi-square test was used for comparisons of allele and genotype proportions. MedCalc statistical program (MedCalc Software Version 8.1.0.0, 2005 Frank Schoonjans for Windows,

The results of the Mann-Whitney U-test showed that, according to the demographic and lifestyle characteristics (age, body mass index), the control no-stenosis group matched the groups of patients with different degrees of cerebrovascular stenosis (Table 2). The chisquared test showed no significant differences between sex and cerebrovascular stenosis

**2.3.5 Quality control of measurements** 

4.8, 4.0, 8.0, 11.1, 4.2 and 3.8%, respectively.

available at the website:www.medcalc.be/) was used.

**2.4 Statistical analysis** 

**3. Results 3.1 Patients**  subgroups (Yates corrected χ² =0.003, p=0.338 in the group with <70% of stenosis; Yates corrected χ² =0.023, p=0.638 in the group with >70% of stenosis) or smoking habits and cerebrovascular stenosis subgroups (Yates corrected χ² =0.001, p=0.478 in the group with <70% of stenosis; Yates corrected χ² =0.012, p=0.962 in the group with >70% of stenosis). The proportion of daily smokers in the group of patients with <70% of stenosis was 33.3% and 32.6% in the group of patients with >70% of stenosis versus 25.8 % in control no-stenosis group. The mean values of the body mass index in all groups examined were more than 25 kg/m2 , indicating overweight.

**3.2 Serum triacylglycerol, total cholesterol, LDL and HDL cholesterol concentrations**  Comparing the results obtained for the traditional risk factors (triacylglycerol, total cholesterol, HDL-cholesterol, LDL-cholesterol) between the groups of patients with cerebrovascular stenosis and control no-stenosis group using the Mann-Whitney univariate statistic method, significant differences were found for all serum lipid parameters (p<0.05) (Table 2).


Table 2. Demographic and biochemical parameters for control no-stenosis group and patients with <70% and >70% of cerebrovascular stenosis. Results are given as medians, with ranges in parentheses. p values: significance level for difference between the group of patients with cerebrovascular stenosis and the control no-stenosis group tested by Mann-Whitney test; p <0.05 was considered as statistically significant.

#### **3.3 Paraoxonase activity measurement**

Basal and stimulated PON1 activities differ significantly between patients group with stenosis and the control no-stenosis group, and HDL standardized basal and stimulated PON1 activity did not show statistical difference. Kolmogorov –Smirnov test for normal distribution reject normality for all examined data (Table 3) . There were no statistically significant relationships between basal and stimulated PON1 activity and examined lipid and lipoprotein parameters (tryacylglicerol, total cholesterol, HDL cholesterol, LDL cholesterol) (Table 4).

Paraoxonase Polymorphisms and Platelet Activating

**Control no-stenosis group (N=81)** 

LM 41 51 25 35 MM 7 9 14 20

QR 39 48 32 45 RR 4 5 6 8

CT 47 58 32 45 TT 12 15 21 30

CS 37 46 24 34 CC 0 0 2 3

(%) of individuals having a certain genotype; checked by Chi-square test.

**Genotype** 

*pon1* L55M

*pon1* Q192R

*pon1* -108C>T

*pon2* S311C

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 517

**n % n %**

LL 33 40 32 45 *P = 0,910*

QQ 38 47 33 47 *P = 0,995*

CC 22 27 18 25 *P = 0,912*

SS 44 54 45 63 *P = 0,981*

Table 5. Genotype frequencies of *pon1* and *pon2* polymorphisms in control no-stenosis group and patients with cerebrovascular stenosis. Data are shown as number (n) and percentage

Fig. 1. Determination of L55M *pon1* gene polymorphism by the PCR-RFLP procedure using

*Hin*1II restriction enzyme. Lines 1- 4 LL, line 5 MM, and line 6 LM genotype.

**Patients with cerebrovascular stenosis (N=71)** 

**p** 


Table 3. Serum paraoxonase (PON1) activity and HDL standardized paraoxonase activity in control no-stenosis group and patients with cerebrovascular stenosis.Abbreviation: IQR, Interquartile range; p values: significance level for difference between the group of patients with cerebrovascular stenosis and the control no-stenosis group tested by Mann-Whitney test; p <0.05 was considered as statistically significant.


Table 4. Relationships between paraoxonase activity and serum lipids and lipoproteins levels. p <0.05 was considered as statistically significant.

#### **3.4 Paraoxonase polymorphisms determinations**

Genotype frequencies of *pon1* and *pon2* polymorphisms found in the group of patients with angiografically assessed stenosis of cerebral arteries vs. control no-stenosis group are presented in Table 5 and Figures 1-4. Observed and expected genotype frequencies of all examined *pon1* and *pon2* genes polymorphisms were in Hardy-Weinberg equilibrium. There were no statistically significant differences between genotype frequencies of *pon1* and *pon2* (Table 5) as well as for the alleles frequencies in patients group vs. control no-stenosis group (p>0,05) (Table 6).


Table 5. Genotype frequencies of *pon1* and *pon2* polymorphisms in control no-stenosis group and patients with cerebrovascular stenosis. Data are shown as number (n) and percentage (%) of individuals having a certain genotype; checked by Chi-square test.

Fig. 1. Determination of L55M *pon1* gene polymorphism by the PCR-RFLP procedure using *Hin*1II restriction enzyme. Lines 1- 4 LL, line 5 MM, and line 6 LM genotype.

516 Atherogenesis

**Control no-stenosis group (N=90)** 

Basal PON1 activity (U/L) 187 (137) 103 (180) 0.0056

(U/L) 379 (326) 213 (339) 0.0079

basal PON1 activity (U/mmol) 110 (125) 93 (142) 0.9390

Table 3. Serum paraoxonase (PON1) activity and HDL standardized paraoxonase activity in control no-stenosis group and patients with cerebrovascular stenosis.Abbreviation: IQR, Interquartile range; p values: significance level for difference between the group of patients with cerebrovascular stenosis and the control no-stenosis group tested by Mann-Whitney

Tryacylglicerol 0.1638 0.1229 0.0754 0.4211 Total cholesterol 0.0105 0.9219 0.1534 0.1003 HDL cholesterol 0.1278 0.2301 0.1146 0.2205 LDL cholesterol -0.3067 0.3182 0.1201 0.1992

Tryacylglicerol 0.1587 0.1358 0.0691 0.4606 Total cholesterol -0.0140 0.8956 0.1589 0.0844 HDL cholesterol 0.1283 0.2281 0.1154 0.2173 LDL cholesterol -0.1262 0.2359 0.1306 0.1623 Table 4. Relationships between paraoxonase activity and serum lipids and lipoproteins

Genotype frequencies of *pon1* and *pon2* polymorphisms found in the group of patients with angiografically assessed stenosis of cerebral arteries vs. control no-stenosis group are presented in Table 5 and Figures 1-4. Observed and expected genotype frequencies of all examined *pon1* and *pon2* genes polymorphisms were in Hardy-Weinberg equilibrium. There were no statistically significant differences between genotype frequencies of *pon1* and *pon2* (Table 5) as well as for the alleles frequencies in patients group vs. control no-stenosis group

Median (IQR) Median (IQR)

**Correlation coefficient** 

**Basal PON1 activity r p r p** 

**NaCl stimulated PON1 activity** 

**Patients with cerebrovascular** 

228 (238) 189 (310) 0.9605

**Patients with cerebrovascular stenosis (N=119)** 

**stenosis (N=119) <sup>p</sup>**

**Paraoxonase (unit)** 

NaCl –stimulated PON1 activity

NaCl –stimulated PON1 activity

test; p <0.05 was considered as statistically significant.

levels. p <0.05 was considered as statistically significant.

**3.4 Paraoxonase polymorphisms determinations** 

(p>0,05) (Table 6).

**Control no-stenosis group (N=90)** 

HDL standardized

HDL standardized

(U/mmol)

Paraoxonase Polymorphisms and Platelet Activating

genotype.

**Allele** 

*pon1* L55M

*pon1* Q192R

*pon1* -108C>T

*pon2* S311C

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 519

Fig. 4. Determination of S311C *pon2* gene polymorphism by the PCR-RFLP procedure using *Dde*I restriction enzyme. Lines 1- 2 SS, line 3 CS, line 4 SS, line 5 CS, and line 6 CC

**n % n %** 

L 107 62 89 63 *P = 0,9744*

Q 115 66 139 70 *P = 0,8477*

C 91 56 95 48 *P = 0,1246*

S 125 77 160 80 *P = 0,5980*

Table 6. Allele frequencies of *pon1* and *pon2* polymorphisms in control no-stenosis group

and patients with cerebrovascular stenosis. Data are shown as number (n) and percentage (%) of individuals having a certain allele; checked by Chi-square test.

**Patients with** 

**cerebrovascular stenosis <sup>p</sup>**

**Control no-stenosis group** 

M 65 38 53 37

R 47 34 61 30

T 71 44 105 52

C 37 23 40 20

Fig. 2. Determination of Q192R *pon1* gene polymorphism by the PCR-RFLP procedure using *Bsp*PI restriction enzyme. Line 1 RR, line 2 QQ, line 3 QR, line 4 RR, line 5 QQ, and line 6 QR genotype.

Fig. 3. Determination of -108C>T *pon1* gene polymorphism by the PCR-RFLP procedure using *Bsr*BI restriction enzyme. Line 1 CT, line 2 CC, lines 3, 4 CT, line 5 TT and line 6 CT genotype.

Fig. 2. Determination of Q192R *pon1* gene polymorphism by the PCR-RFLP procedure using *Bsp*PI restriction enzyme. Line 1 RR, line 2 QQ, line 3 QR, line 4 RR, line 5 QQ, and line 6 QR

Fig. 3. Determination of -108C>T *pon1* gene polymorphism by the PCR-RFLP procedure using *Bsr*BI restriction enzyme. Line 1 CT, line 2 CC, lines 3, 4 CT, line 5 TT and line 6 CT

genotype.

genotype.

Fig. 4. Determination of S311C *pon2* gene polymorphism by the PCR-RFLP procedure using *Dde*I restriction enzyme. Lines 1- 2 SS, line 3 CS, line 4 SS, line 5 CS, and line 6 CC genotype.


Table 6. Allele frequencies of *pon1* and *pon2* polymorphisms in control no-stenosis group and patients with cerebrovascular stenosis. Data are shown as number (n) and percentage (%) of individuals having a certain allele; checked by Chi-square test.

Paraoxonase Polymorphisms and Platelet Activating

variation in PON1 activity (Gupta et al., 2009).

(Flegar-Meštrić et al., 2007; Vrhovski-Hebrang et al., 2002).

dyslipidemia to the risk of developing future stenosis of cerebral arteries.

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 521

as the possible risk factors that contribute to the increased risk of cerebrovascular stenosis

It has been reported that raised levels of atherogenic lipoproteins are a prerequisite for most forms of atherosclerotic disease (Mallat et al., 2010; Tsimikas et al., 2009; Lusis, JA. 2000). In our study, the median values obtained in the groups of patients with different degrees of cerebrovascular stenosis were for total cholesterol, LDL-cholesterol and tryacilglycerols higher and for HDL-cholesterol lower than the recommended values for prevention of atherosclerotic disease (De Backer et al., 2004), indicating a possible contribution of

Today, the aim of cardiovascular risk prevention is to determine atherosclerotic disease activity and shift the present focus from identification of stenosis, which is a focal disease, to identification of patients with inflamed and rupture-prone plaque (Karabina et al., 2010). Numerous biomarkers have been proposed to better discern the vulnerability of plaque rupture, pathogenesis, or cardiovascular risk. Epidemiologic, genetic, and biochemical studies support an antiatherogenic role for paraoxonase (PON) 1. The two other members of the PON gene family, namely, PON2 and PON3, have also been reported to possess antioxidant properties and may exhibit antiatherogenic capacities as well (Shih, DM Lusis, AJ. 2009). Previous studies have demonstrated that PON1 expression is down regulated by oxidative stress. In contrast, more recent studies have shown that PON2 expression is up regulated in response to oxidative stress-inducing agents, while PON3 expression remains unchanged (Ng et al., 2005). Although PON1 activity is determined genetically, various factors, such as diet, lifestyle and environmental factors, can influence PON1 activity (Ng et al., 2005; Gupta et al., 2009). Between individuals, there is an approximately 10- to 40-fold

Only a few studies have examined the relationship between PON1 activity and angiographically proven cardiovascular disease (Graner et al., 2006; Mackness et al., 2001). Our results indicated that basal and stimulated PON1 activities were significantly decreased in patients group with angiographically proven cerebrovascular stenosis (>50%) versus control no-stenosis group (p<0.05), and there were no statistically significant relationships between basal and stimulated PON1 activity and examined lipid parameters (total cholesterol, LDL-cholesterol, HDL-cholesterol and tryacilglycerols), p>0.05. Those results are in line with previous studies, indicating that PON1 activities toward paraoxon are lower in subjects with cardiovascular disease than in control subjects regardless of the PON1 genotype. This would suggest that the quality of the PON1 enzyme is a more important

Polymorphisms in *pon1* and *pon2* genes (L55M and Q192R in *pon1*, and S311C in *pon2*) have been reported to be associated with the risk for the development of atherosclerosis as well as polymorphism in *pon1* promoter region (-108C>T) (Pasdar et al., 2006; Granér et al., 2006). Paraoxonase-1 has several genetic polymorphisms that modify its activity and mass concentration. Hypothesized differences in the ability of the polymorphic forms to protect oxidation of LDL have led to numerous studies attempting to determine the relationship between *PON1* polymorphisms and cardiovascular disease. The results of meta-analysis of 88 studies on 4 *PON* polymorphisms [Q192R, L55M, and T(−107)C in the *PON1* and the S311C in the *PON2*] suggested an overall weak association between the R192 polymorphism and CHD risk. Despite these limitations, this meta-analysis suggests that Q192R polymorphisms may increase the risk of CHD, but no significant effect for L55M, T(−107)C

factor in cardiovascular disease than the PON1 gene (Mackness et al., 2001).

#### **3.5 PAF-AH activity assay**

The values of PAF-AH activity did not differ significantly between control no-stenosis group and group of patients with cerebrovascular stenosis (Table 7) while LDL standardized PAF-AH activity (U/mmol) showed significant difference. The PAF-AH activity showed significant relationship with total and LDL cholesterol in both groups studied (Table 8).


Table 7. Serum Platelet-activating factor acetylhydrolase (PAF-AH) activity in groups studied. p values: significance level for difference between the group of patients with cerebrovascular stenosis and the control no-stenosis group tested by Mann-Whitney test; p <0.05 was considered as statistically significant.


Table 8. Relationships between platelet-activating factor acetylhydrolase (PAF-AH) activity and serum lipids and lipoproteins levels. p <0.05 was considered as statistically significant.

#### **4. Discussion**

Atherosclerosis, a disease of large arteries, is the primary cause of heart disease and stroke (Lusis, JA. 2000). Epidemiological studies over the past 50 years have revealed various risk factors for atherosclerosis and cardiovascular disease, which can be grouped into factors with an important genetic component and those that are largely environmental (Gupta et al., 2009; Lusis, JA. 2000). The results of our study indicated that significant changes associated with cerebrovascular stenosis could be the result of the environmental factors and demographic characteristics of the examined population, which is in accordance with previous studies that have investigated the atherosclerosis and the severity and extent of cardiovascular disease (Mallat et al., 2010; Costa et al., 2005; Granér et al., 2006). All groups examined in our study were characterized by a high frequency of cigarette smoking and overweight, which is consistent with the results of a previous large cross-sectional epidemiological study of Croatian population (Turek et al., 2001), and could be considered

The values of PAF-AH activity did not differ significantly between control no-stenosis group and group of patients with cerebrovascular stenosis (Table 7) while LDL standardized PAF-AH activity (U/mmol) showed significant difference. The PAF-AH activity showed significant relationship with total and LDL cholesterol in both groups studied (Table 8).

(U/L) 405 (134) 414 (171) 0.769

Table 7. Serum Platelet-activating factor acetylhydrolase (PAF-AH) activity in groups studied. p values: significance level for difference between the group of patients with cerebrovascular stenosis and the control no-stenosis group tested by Mann-Whitney test; p

Tryacylglicerol 0.353 0.0006 0.153 0.1018 Total cholesterol 0.417 <0.0001 0.591 <0.0001 HDL cholesterol -0.360 0.0005 -0.006 0.9495 LDL cholesterol 0.459 <0.0001 0.5879 <0.0001

Table 8. Relationships between platelet-activating factor acetylhydrolase (PAF-AH) activity and serum lipids and lipoproteins levels. p <0.05 was considered as statistically significant.

Atherosclerosis, a disease of large arteries, is the primary cause of heart disease and stroke (Lusis, JA. 2000). Epidemiological studies over the past 50 years have revealed various risk factors for atherosclerosis and cardiovascular disease, which can be grouped into factors with an important genetic component and those that are largely environmental (Gupta et al., 2009; Lusis, JA. 2000). The results of our study indicated that significant changes associated with cerebrovascular stenosis could be the result of the environmental factors and demographic characteristics of the examined population, which is in accordance with previous studies that have investigated the atherosclerosis and the severity and extent of cardiovascular disease (Mallat et al., 2010; Costa et al., 2005; Granér et al., 2006). All groups examined in our study were characterized by a high frequency of cigarette smoking and overweight, which is consistent with the results of a previous large cross-sectional epidemiological study of Croatian population (Turek et al., 2001), and could be considered

**Patients with cerebrovascular stenosis** 

**Median (IQR) Median (IQR) p** 

**Correlation coefficient Control no-stenosis group Patients with cerebrovascular** 

**r p r p** 

**stenosis** 

99 (30) 119 (41) <0.0001

**Control no-stenosis group** 

**3.5 PAF-AH activity assay** 

PAF-AH activity

LDL standardized PAF-AH activity (U/mmol)

**4. Discussion** 

<0.05 was considered as statistically significant.

as the possible risk factors that contribute to the increased risk of cerebrovascular stenosis (Flegar-Meštrić et al., 2007; Vrhovski-Hebrang et al., 2002).

It has been reported that raised levels of atherogenic lipoproteins are a prerequisite for most forms of atherosclerotic disease (Mallat et al., 2010; Tsimikas et al., 2009; Lusis, JA. 2000).

In our study, the median values obtained in the groups of patients with different degrees of cerebrovascular stenosis were for total cholesterol, LDL-cholesterol and tryacilglycerols higher and for HDL-cholesterol lower than the recommended values for prevention of atherosclerotic disease (De Backer et al., 2004), indicating a possible contribution of dyslipidemia to the risk of developing future stenosis of cerebral arteries.

Today, the aim of cardiovascular risk prevention is to determine atherosclerotic disease activity and shift the present focus from identification of stenosis, which is a focal disease, to identification of patients with inflamed and rupture-prone plaque (Karabina et al., 2010). Numerous biomarkers have been proposed to better discern the vulnerability of plaque rupture, pathogenesis, or cardiovascular risk. Epidemiologic, genetic, and biochemical studies support an antiatherogenic role for paraoxonase (PON) 1. The two other members of the PON gene family, namely, PON2 and PON3, have also been reported to possess antioxidant properties and may exhibit antiatherogenic capacities as well (Shih, DM Lusis, AJ. 2009). Previous studies have demonstrated that PON1 expression is down regulated by oxidative stress. In contrast, more recent studies have shown that PON2 expression is up regulated in response to oxidative stress-inducing agents, while PON3 expression remains unchanged (Ng et al., 2005). Although PON1 activity is determined genetically, various factors, such as diet, lifestyle and environmental factors, can influence PON1 activity (Ng et al., 2005; Gupta et al., 2009). Between individuals, there is an approximately 10- to 40-fold variation in PON1 activity (Gupta et al., 2009).

Only a few studies have examined the relationship between PON1 activity and angiographically proven cardiovascular disease (Graner et al., 2006; Mackness et al., 2001). Our results indicated that basal and stimulated PON1 activities were significantly decreased in patients group with angiographically proven cerebrovascular stenosis (>50%) versus control no-stenosis group (p<0.05), and there were no statistically significant relationships between basal and stimulated PON1 activity and examined lipid parameters (total cholesterol, LDL-cholesterol, HDL-cholesterol and tryacilglycerols), p>0.05. Those results are in line with previous studies, indicating that PON1 activities toward paraoxon are lower in subjects with cardiovascular disease than in control subjects regardless of the PON1 genotype. This would suggest that the quality of the PON1 enzyme is a more important factor in cardiovascular disease than the PON1 gene (Mackness et al., 2001).

Polymorphisms in *pon1* and *pon2* genes (L55M and Q192R in *pon1*, and S311C in *pon2*) have been reported to be associated with the risk for the development of atherosclerosis as well as polymorphism in *pon1* promoter region (-108C>T) (Pasdar et al., 2006; Granér et al., 2006).

Paraoxonase-1 has several genetic polymorphisms that modify its activity and mass concentration. Hypothesized differences in the ability of the polymorphic forms to protect oxidation of LDL have led to numerous studies attempting to determine the relationship between *PON1* polymorphisms and cardiovascular disease. The results of meta-analysis of 88 studies on 4 *PON* polymorphisms [Q192R, L55M, and T(−107)C in the *PON1* and the S311C in the *PON2*] suggested an overall weak association between the R192 polymorphism and CHD risk. Despite these limitations, this meta-analysis suggests that Q192R polymorphisms may increase the risk of CHD, but no significant effect for L55M, T(−107)C

Paraoxonase Polymorphisms and Platelet Activating

indicator of cerebrovascular stenosis.

Republic of Croatia (No. 044-0061245-0551).

No.3, pp. 598-608, ISSN

517 , ISSN

0006-2952

2004), pp. 1301-1303, ISSN 0891-5849

**6. Acknowledgement** 

**7. References** 

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 523

significant relationships between PON1 activity and lipid parameters (total cholesterol, LDL-cholesterol, HDL-cholesterol and tryacilglycerols), p>0.05. According to the results obtained, we assume that decreased PON1 activities in patients with cerebrovascular stenosis may cause a decreased HDL antioxidant capacity and therefore contribute to the increased risk of the development of cerebrovascular atherosclerosis. However, there were no significant differences in genotype or allele frequencies of *pon1* and *pon2* genes between patients with stenosis of cerebral arteries and no-stenosis control group, indicating that changes in paraoxonase activity are determined by both genetic and environmental factors. Our results show the most significant linear relationship between PAF-AH activity and total cholesterol and LDL-cholesterol ( p<0.001) in the control no-stenosis group, as well as in the group of patients with cerebrovascular stenosis. The median serum PAF-AH activity did not differ significantly between the patients with cerebrovascular stenosis and control nostenosis group (p>0,05), while LDL standardized PAF-AH activity showed significant difference between both examined groups (p<0.0001). According to our results, the LDLstandardized PAF-AH activity could be used as an additional discriminating biochemical

This work was supported by a grant of the Ministry of Science, Education and Sports of the

Adkins, S.; Gan, KN.; Mody, M. & La Du, BN. (1993). Molecular basis for the polymorphic

Aviram M. (2004). Introduction to the serial review on paraoxonases, oxidative stress, and

Ballantyne, C., Cushman, M., Psaty, B., et al. (2007). Collaborative meta-analysis of

Costa, LC.; Vitalone, A.; Cole, TB.& Furlong, CE. (2005). Modulation of paraoxonase (PON1)

Deakin, SP.& James, RW. (2004). Genetic and environmental factors modulating serum

*Science,* Vol.107, No. 5, (November 2004), pp. 435-47, ISSN 0143-5221

*Rehabilitation*, Vol. 14, No.1, (February 2007), pp. 3–11, ISSN 1741-8267 Carlquist, JF.; Muhlestein, JB. & Anderson, JL. (2007). Lipoprotein-associated phospholipase

forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes. *American Journal of Human Genetics*, Vol.52,

cardiovascular diseases. *Free Radical Biology and Medicine,* Vol.37, No.9, (November

individual participant data from observational studies of Lp-PLA2 and cardiovascular diseases. *European Journal of Cardiovascular Prevention &* 

A2: a new biomarker for cardiovascular risk assessment and potential therapeutic target. *Expert Review of Molecular Diagnostics,* Vol.7, No.5, *(*September 2007), pp. 511-

activity. *Biochemical Pharmacoloy*, Vol.69, No.4, (February 2005), pp. 541-550, ISSN

concentrations and activities of the antioxidant enzyme paraoxonase–1. *Clinical* 

and S311C polymorphisms (Wang et al., 2011). Additionally, it has been reported that no significant genotypic or allelic frequency differences between stroke cases and controls for any of the structural polymorphisms of the *PON* genes tested were found (Pasdar et al., 2006).

In our study, there were no significant differences in genotype or allele frequencies of *pon1* and *pon2* genes between patients with stenosis of cerebral arteries and controls, indicating that there is no relationship between examined polymorphisms and reduced paraoxonase activity in patients group with angiographically proven cerebrovascular stenosis.

The platelet-activating factor acetylhydrolase (PAF-AH) or lipoprotein-associated phospholipase A2 (Lp-PLA2) is among the multiple biomarkers that have been associated with an increased CHD risk (Karabina et al., 2010; Garza et al., 2007; Tsimikas et al., 2009; Reddy et al., 2009; Wolfert et al., 2004). A recent meta-analysis of 14 prospective epidemiologic studies involving more than 20,000 patients established a high relative risk for cardiovascular events with high Lp-PLA2.( Garza et. al., 2007; Ballantyne et al., 2007). The LDL-associated PAF-AH activity increases in parallel with the severity of hypercholesterolemia, thus one of the major factors that determines plasma levels of PAF-AH is the rate of removal of LDL from the circulation (Karabina et al., 2010; Tsimikas et al., 2009). In our study, the PAF-AH activity shows the most significant linear relationship with total cholesterol and LDL cholesterol in the control no-stenosis group and the group of patients with cerebrovascular stenosis. It has been reported that increased Lp-PLA2 activity is significantly related to incident cardiovascular disease (cardiovascular death, myocardial infarction, stroke, and transient ischemic attack) (Tsimikas et al., 2009; Mallat et al., 2010). In our study, the median serum PAF-AH activity did not differ significantly between patients with cerebrovascular stenosis and control no-stenosis group (median values 414 U/L versus 405 U/L, p>0,05), which is consistent with results of our previous study (Flegar-Meštrić et al., 2003), while LDL standardized PAF-AH activity a showed significant difference between the patients with cerebrovascular stenosis and control group (median values 119 U/mmol versus 99 U/mmol, p<0,0001).

Previous studies show that Lp-PLA2 is a unique inflammatory biomarker that plays a critical role in the development of atherosclerosis and may be involved in the causal pathway of plaque inflammation and plaque rupture (Munzel, T. Gori, T. 2009; Cariquist et al., 2007). The association of Lp-PLA2 with cardiovascular risk among different population studies independent of classical risk factors makes the premise even stronger that Lp-PLA2 is involved in progression of atherosclerosis to advanced rupture-prone unstable plaques (Reddy et al., 2009) . As Lp-PLA2 is produced by macrophages and foam cells of atherosclerotic plaques that are numerous in unstable plaque, the differentiation between stable versus unstable plaque could be established by the presence of elevated Lp-PLA2 (Reddy et al., 2009; Munzel, T. Gori, T. 2009; Hiramoto et al., 1997; Zalewski, A. Macphee, C. 2005). However, the clinical utility of Lp-PLA2 activity for prediction of cardiovascular risk has to be explored in future studies.

#### **5. Conclusion**

The results of the present study show that basal and stimulated PON1 activities were significantly decreased in the patients group with cerebrovascular stenosis (group of patients with symptoms of cerebrovascular insufficiency and stenosis of carotid artery more than 50% of the lumen) versus control no-stenosis group (p<0.05). There were no statistically significant relationships between PON1 activity and lipid parameters (total cholesterol, LDL-cholesterol, HDL-cholesterol and tryacilglycerols), p>0.05. According to the results obtained, we assume that decreased PON1 activities in patients with cerebrovascular stenosis may cause a decreased HDL antioxidant capacity and therefore contribute to the increased risk of the development of cerebrovascular atherosclerosis. However, there were no significant differences in genotype or allele frequencies of *pon1* and *pon2* genes between patients with stenosis of cerebral arteries and no-stenosis control group, indicating that changes in paraoxonase activity are determined by both genetic and environmental factors. Our results show the most significant linear relationship between PAF-AH activity and total cholesterol and LDL-cholesterol ( p<0.001) in the control no-stenosis group, as well as in the group of patients with cerebrovascular stenosis. The median serum PAF-AH activity did not differ significantly between the patients with cerebrovascular stenosis and control nostenosis group (p>0,05), while LDL standardized PAF-AH activity showed significant difference between both examined groups (p<0.0001). According to our results, the LDLstandardized PAF-AH activity could be used as an additional discriminating biochemical indicator of cerebrovascular stenosis.

#### **6. Acknowledgement**

This work was supported by a grant of the Ministry of Science, Education and Sports of the Republic of Croatia (No. 044-0061245-0551).

#### **7. References**

522 Atherogenesis

and S311C polymorphisms (Wang et al., 2011). Additionally, it has been reported that no significant genotypic or allelic frequency differences between stroke cases and controls for any of the structural polymorphisms of the *PON* genes tested were found (Pasdar et al.,

In our study, there were no significant differences in genotype or allele frequencies of *pon1* and *pon2* genes between patients with stenosis of cerebral arteries and controls, indicating that there is no relationship between examined polymorphisms and reduced paraoxonase

The platelet-activating factor acetylhydrolase (PAF-AH) or lipoprotein-associated phospholipase A2 (Lp-PLA2) is among the multiple biomarkers that have been associated with an increased CHD risk (Karabina et al., 2010; Garza et al., 2007; Tsimikas et al., 2009; Reddy et al., 2009; Wolfert et al., 2004). A recent meta-analysis of 14 prospective epidemiologic studies involving more than 20,000 patients established a high relative risk for cardiovascular events with high Lp-PLA2.( Garza et. al., 2007; Ballantyne et al., 2007). The LDL-associated PAF-AH activity increases in parallel with the severity of hypercholesterolemia, thus one of the major factors that determines plasma levels of PAF-AH is the rate of removal of LDL from the circulation (Karabina et al., 2010; Tsimikas et al., 2009). In our study, the PAF-AH activity shows the most significant linear relationship with total cholesterol and LDL cholesterol in the control no-stenosis group and the group of patients with cerebrovascular stenosis. It has been reported that increased Lp-PLA2 activity is significantly related to incident cardiovascular disease (cardiovascular death, myocardial infarction, stroke, and transient ischemic attack) (Tsimikas et al., 2009; Mallat et al., 2010). In our study, the median serum PAF-AH activity did not differ significantly between patients with cerebrovascular stenosis and control no-stenosis group (median values 414 U/L versus 405 U/L, p>0,05), which is consistent with results of our previous study (Flegar-Meštrić et al., 2003), while LDL standardized PAF-AH activity a showed significant difference between the patients with cerebrovascular stenosis and control group (median values 119 U/mmol

Previous studies show that Lp-PLA2 is a unique inflammatory biomarker that plays a critical role in the development of atherosclerosis and may be involved in the causal pathway of plaque inflammation and plaque rupture (Munzel, T. Gori, T. 2009; Cariquist et al., 2007). The association of Lp-PLA2 with cardiovascular risk among different population studies independent of classical risk factors makes the premise even stronger that Lp-PLA2 is involved in progression of atherosclerosis to advanced rupture-prone unstable plaques (Reddy et al., 2009) . As Lp-PLA2 is produced by macrophages and foam cells of atherosclerotic plaques that are numerous in unstable plaque, the differentiation between stable versus unstable plaque could be established by the presence of elevated Lp-PLA2 (Reddy et al., 2009; Munzel, T. Gori, T. 2009; Hiramoto et al., 1997; Zalewski, A. Macphee, C. 2005). However, the clinical utility of Lp-PLA2 activity for prediction of

The results of the present study show that basal and stimulated PON1 activities were significantly decreased in the patients group with cerebrovascular stenosis (group of patients with symptoms of cerebrovascular insufficiency and stenosis of carotid artery more than 50% of the lumen) versus control no-stenosis group (p<0.05). There were no statistically

activity in patients group with angiographically proven cerebrovascular stenosis.

2006).

versus 99 U/mmol, p<0,0001).

**5. Conclusion** 

cardiovascular risk has to be explored in future studies.


Paraoxonase Polymorphisms and Platelet Activating

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**25** 

*USA* 

**G Protein-Coupled Receptor Dependent NF-κB** 

Over the past decade we have witnessed an explosion of information regarding the molecular mechanisms underlying atherogenesis. While at one time atherosclerosis was viewed as a passive process of lipid deposition within muscular arteries, resulting in progressive luminal stenosis, we now understand that the process is much more complex. In particular, there is a growing appreciation for the role of both adaptive and innate immunity in atherogenesis, and for the contribution of other, non-traditional inflammatory stimuli. Indeed, atherogenesis is now understood primarily as an inflammatory disorder and much of the therapeutic focus has turned to devising approaches for reducing systemic levels of pro-inflammatory mediators and/or preventing these mediators from altering the biochemistry and physiology of the cells that make up the vessel wall. The inflammatory component of atherogenesis is particularly important from a clinical standpoint since it appears that atherosclerotic lesions characterized by on-going inflammation are those that are most unstable and susceptible to rupture, possibly leading to luminal thrombosis and

In this chapter, we provide a brief overview of the mechanisms underlying atherogenesis, highlighting known pro-inflammatory influences. We then focus on activation of the NF-B family of transcription factors as a major molecular mediator of inflammation and summarize recent work that has provided new insights into how a diverse set of G proteincoupled receptors (GPCRs) may use a common mechanism to communicate NF-B activation in cells native to the vessel wall, particularly endothelial cells. These discoveries may provide novel avenues for therapeutic intervention as we refine our approach to

Atherosclerosis is a chronic, progressive process through which lipid deposition, extracellular matrix production, immune cell infiltration, and smooth muscle cell proliferation all conspire to produce arterial obstruction and to disrupt normal arterial vasoreactivity (Hansson, 2005). Atherosclerosis and its related diseases account for nearly a third of all deaths, making it the

**1. Introduction** 

acute myocardial infarction.

treating patients at risk for atherosclerosis.

**2. Basic concepts and mechanisms in atherogenesis** 

**Signaling in Atherogenesis** 

Linda M. McAllister-Lucas2 and Peter C. Lucas1

*1Department of Pathology, University of Michigan Medical School, 2Department of Pediatrics, University of Michigan Medical School,* 

Phillip C. Delekta1, Robert L. Panek1,


### **G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis**

Phillip C. Delekta1, Robert L. Panek1, Linda M. McAllister-Lucas2 and Peter C. Lucas1 *1Department of Pathology, University of Michigan Medical School, 2Department of Pediatrics, University of Michigan Medical School, USA* 

#### **1. Introduction**

528 Atherogenesis

Yamada, Y.; Yoshida, H.; Ichihara, S.; Imaizumi, T.; Satoh, K. & Yokota, M. (2000).

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931, ISSN 1049-8834

Correlations between plasma platelet-activating factor acetylhydrolase (PAF-AH) activity and PAF-AH genotype, age, and atherosclerosis in a Japanese population.

Atherosclerosis Biology, Epidemiology, and Possible Therapeutic Target. *Arteriosclerosis, Thrombosis and Vascular Biology*, Vol.25, No.5, (May 2005), pp. 923-

> Over the past decade we have witnessed an explosion of information regarding the molecular mechanisms underlying atherogenesis. While at one time atherosclerosis was viewed as a passive process of lipid deposition within muscular arteries, resulting in progressive luminal stenosis, we now understand that the process is much more complex. In particular, there is a growing appreciation for the role of both adaptive and innate immunity in atherogenesis, and for the contribution of other, non-traditional inflammatory stimuli. Indeed, atherogenesis is now understood primarily as an inflammatory disorder and much of the therapeutic focus has turned to devising approaches for reducing systemic levels of pro-inflammatory mediators and/or preventing these mediators from altering the biochemistry and physiology of the cells that make up the vessel wall. The inflammatory component of atherogenesis is particularly important from a clinical standpoint since it appears that atherosclerotic lesions characterized by on-going inflammation are those that are most unstable and susceptible to rupture, possibly leading to luminal thrombosis and acute myocardial infarction.

> In this chapter, we provide a brief overview of the mechanisms underlying atherogenesis, highlighting known pro-inflammatory influences. We then focus on activation of the NF-B family of transcription factors as a major molecular mediator of inflammation and summarize recent work that has provided new insights into how a diverse set of G proteincoupled receptors (GPCRs) may use a common mechanism to communicate NF-B activation in cells native to the vessel wall, particularly endothelial cells. These discoveries may provide novel avenues for therapeutic intervention as we refine our approach to treating patients at risk for atherosclerosis.

#### **2. Basic concepts and mechanisms in atherogenesis**

Atherosclerosis is a chronic, progressive process through which lipid deposition, extracellular matrix production, immune cell infiltration, and smooth muscle cell proliferation all conspire to produce arterial obstruction and to disrupt normal arterial vasoreactivity (Hansson, 2005). Atherosclerosis and its related diseases account for nearly a third of all deaths, making it the

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 531

The fatty streak is a waxy yellow deposit in the subendothelial space that represents the first grossly visual evidence of atherogenesis (Packard and Libby, 2008). The fatty streak is formed by the accumulation of lipid and lipid-laden macrophages, also known as foam cells, which are recruited during endothelial dysfunction to the intimal space. While these streaks typically develop into more advanced lesions, they do have the potential to involute and resolve, so that at least at this stage, the process of atherogenesis is a reversible one. Lipid residing in the intima, particularly when in an oxidized form (Ox-LDL), can act to further endothelial dysfunction, initiating a vicious cycle that perpetuates fatty streak formation and can lead to more advanced lesions (Packard and Libby, 2008). This occurs in part through activation of scavenger receptors on the basolateral surface of endothelial cells, including the lectin-like oxidized LDL receptor-1 (LOX-1) (Mitra et al., 2011). Among other effects, LOX-1 activation upregulates VCAM-1 and ICAM-1 leading to further monocyte recruitment, upregulates the receptor for Angiotensin II (AGTR1), and increases release of reactive oxygen species (ROS) which cause further oxidation of LDL particles (Mitra et al.,

As the plaque progresses, there is further expansion of the intimal space with lipid and macrophages. Other leukocytes, including lymphocytes and mast cells, begin to accumulate and play key regulatory roles (Hansson, 2005; Packard and Libby, 2008). In this stage, vascular smooth muscle cells (VSMCs) begin to proliferate and some migrate into the superficial intima, leaving their usual position in the media. This occurs in response to increasing concentrations of growth factors released from endothelial cells and inflammatory cells in the developing lesion. The VSMCs in turn contribute to plaque size through their proliferation and through production of extracellular matrix proteins (collagen, elastin, proteoglycans). However, it is these matrix proteins and VSMCs that together form a protective fibrous cap separating the inflammatory core of the plaque from the endothelial layer (Libby et al., 2011). Thus, the integrity of the fibrous cap is essential for maintaining a stable lesion. Also during this stage, the vessel undergoes compensatory remodelling in an effort to maintain luminal patency, although there is invariably a progressive stenosis (Rader and Daugherty, 2008). Part of the remodelling process includes the ingrowth of a neovascular network, extending from the vasa vasorum of the outer adventitial layer of the vessel into the central portion of plaque (Libby et al., 2011) (Fig. 1). While this neovascularization serves a stabilizing function by providing for adequate blood supply to the plaque, and preventing cellular hypoxia in this region, these newly formed vessels are also leaky, delicate, and prone to rupture. In this way, neovascularization represents a double-edged sword, simultaneously promoting and risking lesion stability.

With increasing cycles of lipid deposition and inflammation, the plaque becomes progressively unstable and prone to rupture due to a multitude of factors. The lipid core may become necrotic, leading to release of cytotoxic substances and cellular debris. Hemorrhage of the lipid core microvasculature may occur, leading to intra-lesional thrombosis and production of pro-inflammatory molecules including thrombin. These

effects attract more leukocytes to further weaken the plaque (Libby et al., 2011).

**2.2 Fatty streak** 

2011).

**2.3 Intermediate lesion** 

**2.4 Advanced/vulnerable lesion** 

most common cause of disease-related death in the world (Hansson, 2005; Murray and Lopez, 1997). Although the process is gradual, often taking decades to proceed to a life-threatening stage, it is useful to think of atherogenesis as a series of distinct stages (Fig. 1). We will review these only briefly, as they are discussed in more detail elsewhere in this book.

Fig. 1. The role of the endothelium through the stages of plaque formation. It should be noted that while endothelial dysfunction represents the first stage of plaque formation, it continues through all other stages as well. See text for a detailed description of each stage.

#### **2.1 Endothelial dysfunction**

The earliest recognizable stage in the development of atherosclerosis is characterized by changes in the cellular physiology of endothelial cells, referred to as endothelial dysfunction (Sitia et al., 2010). Endothelial cells form a single cell-thick, selectively permeable barrier, separating circulating blood components from the vessel wall. Aside from their barrier function, these cells also influence overall vessel function, in particular by regulating levels of nitric oxide (NO) which influences vascular contractility and tone (Jin and Loscalzo, 2010). Endothelial dysfunction ensues when these cells are exposed to injurious stimuli, resulting in a disruption in their ability to maintain a proper barrier and to promote vascular relaxation. As will be discussed, many features of endothelial dysfunction can be linked to the stimulation of signal transduction pathways culminating in NF-B activation (de Winther et al., 2005). In particular, NF-B activation induces expression of chemokines such as monocyte chemotactic factor (MCP-1), and adhesion molecules such as vascular and intercellular adhesion molecules (VCAM-1 and ICAM-1), which serve to recruit circulating monocytes and facilitate their process of transmigration through the endothelial barrier into the subendothelial space (de Winther et al., 2005) (Fig. 1). In addition, NF-B activation plays a role in reorganizing tight and adherens junctions, which represent the glue connecting one endothelial cell to the next (Aveleira et al., 2010). Alteration in tight junctions can then influence the permeability of the endothelial layer to serum proteins and lipids. Finally, NF-B activation has a complex role in controlling various aspects of NO production, and vice versa (Csiszar et al., 2008; Farmer and Kennedy, 2009; Laroux et al., 2001). As such, factors that act on endothelial cells to induce NF-B represent important players in the initiation of atherogenesis.

#### **2.2 Fatty streak**

530 Atherogenesis

most common cause of disease-related death in the world (Hansson, 2005; Murray and Lopez, 1997). Although the process is gradual, often taking decades to proceed to a life-threatening stage, it is useful to think of atherogenesis as a series of distinct stages (Fig. 1). We will review

Fig. 1. The role of the endothelium through the stages of plaque formation. It should be noted that while endothelial dysfunction represents the first stage of plaque formation, it continues through all other stages as well. See text for a detailed description of each stage.

The earliest recognizable stage in the development of atherosclerosis is characterized by changes in the cellular physiology of endothelial cells, referred to as endothelial dysfunction (Sitia et al., 2010). Endothelial cells form a single cell-thick, selectively permeable barrier, separating circulating blood components from the vessel wall. Aside from their barrier function, these cells also influence overall vessel function, in particular by regulating levels of nitric oxide (NO) which influences vascular contractility and tone (Jin and Loscalzo, 2010). Endothelial dysfunction ensues when these cells are exposed to injurious stimuli, resulting in a disruption in their ability to maintain a proper barrier and to promote vascular relaxation. As will be discussed, many features of endothelial dysfunction can be linked to the stimulation of signal transduction pathways culminating in NF-B activation (de Winther et al., 2005). In particular, NF-B activation induces expression of chemokines such as monocyte chemotactic factor (MCP-1), and adhesion molecules such as vascular and intercellular adhesion molecules (VCAM-1 and ICAM-1), which serve to recruit circulating monocytes and facilitate their process of transmigration through the endothelial barrier into the subendothelial space (de Winther et al., 2005) (Fig. 1). In addition, NF-B activation plays a role in reorganizing tight and adherens junctions, which represent the glue connecting one endothelial cell to the next (Aveleira et al., 2010). Alteration in tight junctions can then influence the permeability of the endothelial layer to serum proteins and lipids. Finally, NF-B activation has a complex role in controlling various aspects of NO production, and vice versa (Csiszar et al., 2008; Farmer and Kennedy, 2009; Laroux et al., 2001). As such, factors that act on endothelial cells to induce NF-B represent important

**2.1 Endothelial dysfunction** 

players in the initiation of atherogenesis.

these only briefly, as they are discussed in more detail elsewhere in this book.

The fatty streak is a waxy yellow deposit in the subendothelial space that represents the first grossly visual evidence of atherogenesis (Packard and Libby, 2008). The fatty streak is formed by the accumulation of lipid and lipid-laden macrophages, also known as foam cells, which are recruited during endothelial dysfunction to the intimal space. While these streaks typically develop into more advanced lesions, they do have the potential to involute and resolve, so that at least at this stage, the process of atherogenesis is a reversible one. Lipid residing in the intima, particularly when in an oxidized form (Ox-LDL), can act to further endothelial dysfunction, initiating a vicious cycle that perpetuates fatty streak formation and can lead to more advanced lesions (Packard and Libby, 2008). This occurs in part through activation of scavenger receptors on the basolateral surface of endothelial cells, including the lectin-like oxidized LDL receptor-1 (LOX-1) (Mitra et al., 2011). Among other effects, LOX-1 activation upregulates VCAM-1 and ICAM-1 leading to further monocyte recruitment, upregulates the receptor for Angiotensin II (AGTR1), and increases release of reactive oxygen species (ROS) which cause further oxidation of LDL particles (Mitra et al., 2011).

#### **2.3 Intermediate lesion**

As the plaque progresses, there is further expansion of the intimal space with lipid and macrophages. Other leukocytes, including lymphocytes and mast cells, begin to accumulate and play key regulatory roles (Hansson, 2005; Packard and Libby, 2008). In this stage, vascular smooth muscle cells (VSMCs) begin to proliferate and some migrate into the superficial intima, leaving their usual position in the media. This occurs in response to increasing concentrations of growth factors released from endothelial cells and inflammatory cells in the developing lesion. The VSMCs in turn contribute to plaque size through their proliferation and through production of extracellular matrix proteins (collagen, elastin, proteoglycans). However, it is these matrix proteins and VSMCs that together form a protective fibrous cap separating the inflammatory core of the plaque from the endothelial layer (Libby et al., 2011). Thus, the integrity of the fibrous cap is essential for maintaining a stable lesion. Also during this stage, the vessel undergoes compensatory remodelling in an effort to maintain luminal patency, although there is invariably a progressive stenosis (Rader and Daugherty, 2008). Part of the remodelling process includes the ingrowth of a neovascular network, extending from the vasa vasorum of the outer adventitial layer of the vessel into the central portion of plaque (Libby et al., 2011) (Fig. 1). While this neovascularization serves a stabilizing function by providing for adequate blood supply to the plaque, and preventing cellular hypoxia in this region, these newly formed vessels are also leaky, delicate, and prone to rupture. In this way, neovascularization represents a double-edged sword, simultaneously promoting and risking lesion stability.

#### **2.4 Advanced/vulnerable lesion**

With increasing cycles of lipid deposition and inflammation, the plaque becomes progressively unstable and prone to rupture due to a multitude of factors. The lipid core may become necrotic, leading to release of cytotoxic substances and cellular debris. Hemorrhage of the lipid core microvasculature may occur, leading to intra-lesional thrombosis and production of pro-inflammatory molecules including thrombin. These effects attract more leukocytes to further weaken the plaque (Libby et al., 2011).

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 533

AGTR1 is expressed on both endothelial cells and VSMCs (Brasier et al., 2002). In addition, all the components of a local renin-angiotensin system (RAS) exist within the vasculature, so that Ang II can be locally produced and act in an autocrine or paracrine fashion, supplementing the effects of systemically circulating Ang II (Sata and Fukuda, 2010). Although Ang II influences numerous aspects of endothelial cell physiology, at least four categories of genes are induced that contribute to atherogenesis (Fig. 2). These include **1)** genes whose products promote recruitment and activation of monocytes and other inflammatory cells (eg, chemokines, cytokines, and adhesion molecules such as MCP-1, IL-6, IL-8, ICAM-1, VCAM-1, and E-selectin), **2)** genes whose products destabilize plaque and promote both proliferation and migration of underlying VSMCs (eg, MMP-9, PAI-1, and IGF-1R), **3)** genes whose products mediate endothelial dysfunction, particularly in the presence of oxidized LDL (eg, LOX-1), and **4)** genes encoding secondary cytokines that can feed back to ECs and VSMCs, further enhancing the pro-inflammatory milieu (eg, TNFα, Il-1β) (Fig. 2) (Brasier et al., 2002; de Winther et al., 2005; Pober and Sessa, 2007). Importantly, activation of LOX-1 sets in motion a destructive feed-forward cycle, whereby it enhances the expression of both AGTR1 and angiotensin converting enzyme (ACE) (Li et al., 2000; Li et al., 2003). This in turn results in enhanced local production of Ang II (Fig. 2). As will be discussed, all the above are NF-B regulated genes, highlighting NF-B activation as a key

pro-atherogenic signaling event.

Fig. 2. Pleiotropic effects of Ang II on vascular pathophysiology.

(Fig. 2) (Eguchi et al., 1998; Ohtsu et al., 2006; Saito and Berk, 2001).

Ang II, either made locally within the vessel wall or present following diffusion from the vessel lumen, can also act on VSMCs. Here, AGTR1 activation results in many of the same pro-inflammatory responses that are seen in endothelial cells. Additionally, MAPK pathways are activated, partly through transactivation of EGF receptors, thereby promoting the hypertrophy and hyperplasia of VSMCs that is characteristic of atherosclerotic lesions

Ultimately, it is the integrity of the surface endothelial lining of the plaque that represents the greatest clinical concern. Any damage to these endothelial cells may then lead to exposure of the extracellular matrix to circulating blood. This includes the exposure of tissue factor (TF), which triggers the coagulation cascade and can lead to life-threatening local thrombosis. Damage to the endothelium may occur via processes occurring in the vessel lumen, for example as a consequence of shear stresses induced by hypertension and plasma turbulence. Alternatively, damage may come from below, as a consequence of inflammation in the plaque. Ongoing inflammation within the lipid core weakens the fibrous cap, in part because inflammatory cells release proteases that degrade the extracellular matrix. For example, macrophages release matrix metalloproteinase 9 (MMP-9), which alone is sufficient to induce rupture of advanced lesions in mice (Gough et al., 2006). T lymphocytes uniquely contribute to plaque instability via the production of IFN- which then downregulates VSMC matrix production (Packard and Libby, 2008). Thus, lesions that are most susceptible to rupture are those that have been weakened over time by the action of matrix-degrading proteases and have a paucity of VSMCs to provide a protective barrier separating the lipid core from surface endothelial cells (Libby et al., 2011).

#### **3. Specific GPCR agonists as contributors to vascular inflammation**

The GPCR family represents the largest family of cell surface receptors, and includes over 800 known members (Dorsam and Gutkind, 2007; Lappano and Maggiolini, 2011). GPCRs are structurally defined by an extracellular N-terminal tail, seven trans-membrane domains linked together by 6 alternating intracellular and extracellular loops, and a C-terminal tail (Strader et al., 1994). Members of this receptor family respond to a diverse array of ligands including peptides, amines, glycoproteins and enzymes. The receptors relay extracellular signals by activating multiple intracellular signaling pathways which include those for ERK, Akt, JNK, p38MAPK, STAT, and NF-B activation, to name only a few (Dorsam and Gutkind, 2007; Lappano and Maggiolini, 2011). Several GPCR agonists have been identified as key regulators of both endothelial cell function and atherogenesis. Below we discuss three such agonists that play particularly prominent roles in the pathophysiologic stages of atherogenesis.

#### **3.1 Angiotensin II**

Angiotensin II (Ang II) is a GPCR agonist, long known for its classic role in controlling blood pressure through regulating vascular smooth muscle tension, influencing renal reabsorption of sodium and water, and through stimulating aldosterone release from the adrenal. However, in recent years our understanding of this peptide hormone has broadened, and it is now appreciated that Ang II exerts a much wider spectrum of responses (Luft, 2001). In particular, Ang II is now appreciated for its profound pro-inflammatory effects, exerted on both endothelial and smooth muscle cells of the vasculature (Phillips and Kagiyama, 2002). Through this role, Ang II is thought to promote atherogenesis via mechanisms that are independent from its impact on blood pressure. Consistent with this notion, animal models of Ang II-dependent atherosclerosis, as well as large clinical trials investigating angiotensin converting enzyme (ACE) inhibitors or AGTR1 blockers (eg, HOPE, EUROPA, and LIFE), have demonstrated that the contribution of Ang II to atherogenesis cannot be explained solely by its ability to promote hypertension (Bertrand, 2004; Ferrario and Strawn, 2006; Kintscher et al., 2004). Instead, there is emerging evidence that perhaps the greatest impact of Ang II lies in its ability to directly induce pro-inflammatory signal transduction.

Ultimately, it is the integrity of the surface endothelial lining of the plaque that represents the greatest clinical concern. Any damage to these endothelial cells may then lead to exposure of the extracellular matrix to circulating blood. This includes the exposure of tissue factor (TF), which triggers the coagulation cascade and can lead to life-threatening local thrombosis. Damage to the endothelium may occur via processes occurring in the vessel lumen, for example as a consequence of shear stresses induced by hypertension and plasma turbulence. Alternatively, damage may come from below, as a consequence of inflammation in the plaque. Ongoing inflammation within the lipid core weakens the fibrous cap, in part because inflammatory cells release proteases that degrade the extracellular matrix. For example, macrophages release matrix metalloproteinase 9 (MMP-9), which alone is sufficient to induce rupture of advanced lesions in mice (Gough et al., 2006). T lymphocytes uniquely contribute to plaque instability via the production of IFN- which then downregulates VSMC matrix production (Packard and Libby, 2008). Thus, lesions that are most susceptible to rupture are those that have been weakened over time by the action of matrix-degrading proteases and have a paucity of VSMCs to provide a protective barrier

separating the lipid core from surface endothelial cells (Libby et al., 2011).

**3. Specific GPCR agonists as contributors to vascular inflammation** 

play particularly prominent roles in the pathophysiologic stages of atherogenesis.

its ability to directly induce pro-inflammatory signal transduction.

**3.1 Angiotensin II** 

The GPCR family represents the largest family of cell surface receptors, and includes over 800 known members (Dorsam and Gutkind, 2007; Lappano and Maggiolini, 2011). GPCRs are structurally defined by an extracellular N-terminal tail, seven trans-membrane domains linked together by 6 alternating intracellular and extracellular loops, and a C-terminal tail (Strader et al., 1994). Members of this receptor family respond to a diverse array of ligands including peptides, amines, glycoproteins and enzymes. The receptors relay extracellular signals by activating multiple intracellular signaling pathways which include those for ERK, Akt, JNK, p38MAPK, STAT, and NF-B activation, to name only a few (Dorsam and Gutkind, 2007; Lappano and Maggiolini, 2011). Several GPCR agonists have been identified as key regulators of both endothelial cell function and atherogenesis. Below we discuss three such agonists that

Angiotensin II (Ang II) is a GPCR agonist, long known for its classic role in controlling blood pressure through regulating vascular smooth muscle tension, influencing renal reabsorption of sodium and water, and through stimulating aldosterone release from the adrenal. However, in recent years our understanding of this peptide hormone has broadened, and it is now appreciated that Ang II exerts a much wider spectrum of responses (Luft, 2001). In particular, Ang II is now appreciated for its profound pro-inflammatory effects, exerted on both endothelial and smooth muscle cells of the vasculature (Phillips and Kagiyama, 2002). Through this role, Ang II is thought to promote atherogenesis via mechanisms that are independent from its impact on blood pressure. Consistent with this notion, animal models of Ang II-dependent atherosclerosis, as well as large clinical trials investigating angiotensin converting enzyme (ACE) inhibitors or AGTR1 blockers (eg, HOPE, EUROPA, and LIFE), have demonstrated that the contribution of Ang II to atherogenesis cannot be explained solely by its ability to promote hypertension (Bertrand, 2004; Ferrario and Strawn, 2006; Kintscher et al., 2004). Instead, there is emerging evidence that perhaps the greatest impact of Ang II lies in AGTR1 is expressed on both endothelial cells and VSMCs (Brasier et al., 2002). In addition, all the components of a local renin-angiotensin system (RAS) exist within the vasculature, so that Ang II can be locally produced and act in an autocrine or paracrine fashion, supplementing the effects of systemically circulating Ang II (Sata and Fukuda, 2010). Although Ang II influences numerous aspects of endothelial cell physiology, at least four categories of genes are induced that contribute to atherogenesis (Fig. 2). These include **1)** genes whose products promote recruitment and activation of monocytes and other inflammatory cells (eg, chemokines, cytokines, and adhesion molecules such as MCP-1, IL-6, IL-8, ICAM-1, VCAM-1, and E-selectin), **2)** genes whose products destabilize plaque and promote both proliferation and migration of underlying VSMCs (eg, MMP-9, PAI-1, and IGF-1R), **3)** genes whose products mediate endothelial dysfunction, particularly in the presence of oxidized LDL (eg, LOX-1), and **4)** genes encoding secondary cytokines that can feed back to ECs and VSMCs, further enhancing the pro-inflammatory milieu (eg, TNFα, Il-1β) (Fig. 2) (Brasier et al., 2002; de Winther et al., 2005; Pober and Sessa, 2007). Importantly, activation of LOX-1 sets in motion a destructive feed-forward cycle, whereby it enhances the expression of both AGTR1 and angiotensin converting enzyme (ACE) (Li et al., 2000; Li et al., 2003). This in turn results in enhanced local production of Ang II (Fig. 2). As will be discussed, all the above are NF-B regulated genes, highlighting NF-B activation as a key pro-atherogenic signaling event.

Fig. 2. Pleiotropic effects of Ang II on vascular pathophysiology.

Ang II, either made locally within the vessel wall or present following diffusion from the vessel lumen, can also act on VSMCs. Here, AGTR1 activation results in many of the same pro-inflammatory responses that are seen in endothelial cells. Additionally, MAPK pathways are activated, partly through transactivation of EGF receptors, thereby promoting the hypertrophy and hyperplasia of VSMCs that is characteristic of atherosclerotic lesions (Fig. 2) (Eguchi et al., 1998; Ohtsu et al., 2006; Saito and Berk, 2001).

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 535

plaque NF-κB related pro-atherogenic effects

inflammatory cells Destabilization of plaque

 Expression of VEGF Promotion of plaque neovascularization

 Promotion of plaque neovascularization

VSMCs Proliferation and migration into plaque

Release of pro-inflammatory cytokines

Enhancement of vascular permeability

 Recruitment of inflammatory cells Enhancement of vascular permeability

Stimulation of vasoconstriction

Recruitment and activation of

GPCR Affected cell type in

receptor) Endothelial cells

**4. GPCR connectivity to NF-κB** 

2007; Lappano and Maggiolini, 2011).

Endothelial cells

Endothelial cells

Table 1. Selected effects of three GPCRs and their ligands on atherogenesis.

For ligand activated GPCRs, many, but not all, signaling events are initiated through the activation of heterotrimeric G proteins (Dorsam and Gutkind, 2007; Lappano and Maggiolini, 2011). The cytoplasmic loops within GPCRs serve to recruit these G proteins, consisting of , , and subunits. Upon agonist binding, the receptors promote exchange of GDP for GTP on the G subunit, leading to its dissociation from the G subunits. Both GTP-bound G and G subunits are then able to stimulate a range of downstream effectors. At least part of the specificity in receptor signaling stems from the fact that there are numerous G subtypes, broadly grouped into four classes (Gs, Gi, Gq/11, and G12/13), and GPCRs will preferentially couple to certain subtypes (Dorsam and Gutkind,

Receptors that couple to Gq/11 are known to activate protein kinase C (PKC) isoforms through G protein-dependent stimulation of phospholipase C, Ca2+ mobilization, and DAG generation. These include receptors for agonists described above (Ang II, thrombin, IL-8) as well as others that potentially influence endothelial biology in the context of atherogenesis, including endothelin-1, lysophosphatidic acid (LPA), and SDF-1/CXCL12 . For some time, it has been clear that PKC activation by select GPCRs is a prerequisite for subsequent NF-B activation, but we are only now beginning to unravel the specific mechanistic links between PKC and the NF-B machinery. In order to discuss these links, we will first briefly review the salient features of the NF-B family of transcription factors, and their regulation. For a more nuanced treatment, the reader is referred to one of the more complete reviews of the topic (Hayden and Ghosh, 2008; Oeckinghaus and Ghosh, 2009; Vallabhapurapu and Karin,

Angiotensin II Type 1 receptor

CXCR2 (IL-8

Receptor-1 (thrombin receptor)

2009).

Protease Activated

#### **3.2 Thrombin**

Thrombin is known historically for its role in the clotting cascade. Active thrombin is generated from the inactive precursor, prothrombin, via cleavage by a complex consisting of factor Xa and factor Va, assembled through the actions of tissue factor (TF) (Borissoff et al., 2011). Thrombin then plays a role in generating a stable clot, in part through cleaving fibrinogen to produce fibrin, and through its actions on platelets. However, as is the case for Ang II, there is increasing appreciation for the receptor-mediated, pro-inflammatory effects of thrombin. Unlike most GPCR agonists, thrombin is a serine protease and acts on its cognate receptors through an unusual mechanism. The best studied thrombin receptor on endothelial cells is perhaps the protease activated receptor-1 (PAR-1) (Borissoff et al., 2009). In this case, thrombin binds to an extracellular hirudin-like domain on PAR-1 and cleaves the receptor at a specific site, exposing a cryptic ligand, SFLLRN, present near the Nterminal tail of the receptor. The newly exposed amino acid sequence acts as a tethered ligand by binding to a pocket on extracellular loop 2 and permanently activating the receptor (Borissoff et al., 2009). A synthetic peptide with the same SFLLRN sequence, also known as TRAP-6, can be used to induce the same response from PAR-1 as thrombin (Coughlin, 2005).

Both PAR-1 and its agonist thrombin are major participants in the regulation of endothelial cell biology and atherogenesis, affecting cell signaling, gene expression, endothelial permeability, angiogenesis, and vascular tone (Hirano, 2007). Indeed, the importance of direct pro-atherogenic effects of thrombin on cells of the vessel wall were recently highlighted by a study in mice showing that atherosclerosis can proceed independently of thrombin-induced platelet activation (Hamilton et al., 2009). As with Ang II, many of the effects of thrombin on endothelial cells can be mechanistically linked to NF-B activation.

#### **3.3 IL-8**

A vast array of chemokines and associated GPCRs exist that influence vascular biology (Rosenkilde and Schwartz, 2004). For the purposes of this review, we highlight only one, IL-8 (CXCL8), because of the recent work demonstrating parallels between the molecular signaling pathways activated by IL-8 and those activated by both Ang II and thrombin (Martin et al., 2009). IL-8 is a CXC chemokine with many immunomodulatory functions and a broad range of biological effects. The effects of IL-8 are mediated primarily through CXCR2, a GPCR that is expressed on a broad range of cells, including endothelial cells (Rosenkilde and Schwartz, 2004). IL-8 is upregulated within developing atherosclerotic lesions, in part due to the stimulatory effect of Ox-LDL (Braunersreuther et al., 2007). Among its many effects, IL-8 induces expression of vascular endothelial growth factor (VEGF), which is synthesized and released by endothelial cells and can act in an autocrine/paracrine fashion to induce angiogenesis within the lipid core and to increase vascular permeability (Gavard et al., 2009). As with Ang II and thrombin, many of the pro-inflammatory effects of IL-8 can be attributed to the activation of NF-B. In particular, IL-8 induction of the *VEGF* gene occurs through NF-B binding sites in its promoter (Martin et al., 2009). See Table 1 for a summary of several proatherogenic effects of IL-8, and other GPCR agonists, that have been ascribed to NF-B activation.


Table 1. Selected effects of three GPCRs and their ligands on atherogenesis.

#### **4. GPCR connectivity to NF-κB**

534 Atherogenesis

Thrombin is known historically for its role in the clotting cascade. Active thrombin is generated from the inactive precursor, prothrombin, via cleavage by a complex consisting of factor Xa and factor Va, assembled through the actions of tissue factor (TF) (Borissoff et al., 2011). Thrombin then plays a role in generating a stable clot, in part through cleaving fibrinogen to produce fibrin, and through its actions on platelets. However, as is the case for Ang II, there is increasing appreciation for the receptor-mediated, pro-inflammatory effects of thrombin. Unlike most GPCR agonists, thrombin is a serine protease and acts on its cognate receptors through an unusual mechanism. The best studied thrombin receptor on endothelial cells is perhaps the protease activated receptor-1 (PAR-1) (Borissoff et al., 2009). In this case, thrombin binds to an extracellular hirudin-like domain on PAR-1 and cleaves the receptor at a specific site, exposing a cryptic ligand, SFLLRN, present near the Nterminal tail of the receptor. The newly exposed amino acid sequence acts as a tethered ligand by binding to a pocket on extracellular loop 2 and permanently activating the receptor (Borissoff et al., 2009). A synthetic peptide with the same SFLLRN sequence, also known as TRAP-6, can be used to induce the same response from PAR-1 as thrombin

Both PAR-1 and its agonist thrombin are major participants in the regulation of endothelial cell biology and atherogenesis, affecting cell signaling, gene expression, endothelial permeability, angiogenesis, and vascular tone (Hirano, 2007). Indeed, the importance of direct pro-atherogenic effects of thrombin on cells of the vessel wall were recently highlighted by a study in mice showing that atherosclerosis can proceed independently of thrombin-induced platelet activation (Hamilton et al., 2009). As with Ang II, many of the effects of thrombin on endothelial cells can be mechanistically linked

A vast array of chemokines and associated GPCRs exist that influence vascular biology (Rosenkilde and Schwartz, 2004). For the purposes of this review, we highlight only one, IL-8 (CXCL8), because of the recent work demonstrating parallels between the molecular signaling pathways activated by IL-8 and those activated by both Ang II and thrombin (Martin et al., 2009). IL-8 is a CXC chemokine with many immunomodulatory functions and a broad range of biological effects. The effects of IL-8 are mediated primarily through CXCR2, a GPCR that is expressed on a broad range of cells, including endothelial cells (Rosenkilde and Schwartz, 2004). IL-8 is upregulated within developing atherosclerotic lesions, in part due to the stimulatory effect of Ox-LDL (Braunersreuther et al., 2007). Among its many effects, IL-8 induces expression of vascular endothelial growth factor (VEGF), which is synthesized and released by endothelial cells and can act in an autocrine/paracrine fashion to induce angiogenesis within the lipid core and to increase vascular permeability (Gavard et al., 2009). As with Ang II and thrombin, many of the pro-inflammatory effects of IL-8 can be attributed to the activation of NF-B. In particular, IL-8 induction of the *VEGF* gene occurs through NF-B binding sites in its promoter (Martin et al., 2009). See Table 1 for a summary of several proatherogenic effects of IL-8, and other GPCR agonists, that have been ascribed to NF-B

**3.2 Thrombin** 

(Coughlin, 2005).

to NF-B activation.

**3.3 IL-8** 

activation.

For ligand activated GPCRs, many, but not all, signaling events are initiated through the activation of heterotrimeric G proteins (Dorsam and Gutkind, 2007; Lappano and Maggiolini, 2011). The cytoplasmic loops within GPCRs serve to recruit these G proteins, consisting of , , and subunits. Upon agonist binding, the receptors promote exchange of GDP for GTP on the G subunit, leading to its dissociation from the G subunits. Both GTP-bound G and G subunits are then able to stimulate a range of downstream effectors. At least part of the specificity in receptor signaling stems from the fact that there are numerous G subtypes, broadly grouped into four classes (Gs, Gi, Gq/11, and G12/13), and GPCRs will preferentially couple to certain subtypes (Dorsam and Gutkind, 2007; Lappano and Maggiolini, 2011).

Receptors that couple to Gq/11 are known to activate protein kinase C (PKC) isoforms through G protein-dependent stimulation of phospholipase C, Ca2+ mobilization, and DAG generation. These include receptors for agonists described above (Ang II, thrombin, IL-8) as well as others that potentially influence endothelial biology in the context of atherogenesis, including endothelin-1, lysophosphatidic acid (LPA), and SDF-1/CXCL12 . For some time, it has been clear that PKC activation by select GPCRs is a prerequisite for subsequent NF-B activation, but we are only now beginning to unravel the specific mechanistic links between PKC and the NF-B machinery. In order to discuss these links, we will first briefly review the salient features of the NF-B family of transcription factors, and their regulation. For a more nuanced treatment, the reader is referred to one of the more complete reviews of the topic (Hayden and Ghosh, 2008; Oeckinghaus and Ghosh, 2009; Vallabhapurapu and Karin, 2009).

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 537

an IB, preventing non-canonical NF-B activity. An area of intense interest relates to the question of whether the non-canonical NF-B complex of RelB/p52 regulates a distinct set of genes from those regulated by RelA/p50. Importantly, while GPCRs are known to stimulate the canonical pathway, their potential for activating non-canonical signaling

Fig. 3. Distinct pathways for canonical and non-canonical NF-B activation.

Although the canonical and non-canonical pathways for NF-B activation have historically received the most attention, it is clear that several other mechanisms are in place for activating NF-B subunits. Bearing in mind that several different NF-B heterodimer complexes have been identified, it is likely that our understanding of alternative routes for NF-B activation will only grow as the regulation of these complexes is explored in more detail. For example, recent work in B cells has demonstrated that c-Rel/p50 heterodimers can be regulated by a unique, non-proteosome dependent pathway for IB degradation (O'Connor et al., 2004). Further, kinases other than IKK have been identified that can act to phosphorylate IB proteins, leading to their degradation (McElhinny et al., 1996; Schwarz et al., 1996). Finally, other levels of control exist beyond the simple degradation of IB proteins. Several groups have shown that NF- B subunits are targets of secondary modification, including phosphorylation and acetylation, alterations that can affect their ability to interact with both DNA consensus sites and transcriptional co-regulators. Thus, as the body of experimental data grows, it will no doubt become obvious that the concepts of canonical and non-canonical activation, outlined above and in Fig. 3, represent only a framework for a much more complicated

**4.4 Unique/emerging NF-κB signaling pathways** 

system of regulation.

remains to be elucidated.

#### **4.1 The NF-κB family**

The NF-B family denotes a group of five transcription factors and includes the proteins RelA (p65), RelB, c-Rel, NF-B1 (p105/50), and NF-B2 (p100/52) (Oeckinghaus and Ghosh, 2009). All share a highly conserved Rel homology domain (RHD) which directs their dimerization, nuclear localization, and DNA binding activities (Oeckinghaus and Ghosh, 2009). Upon entering the nucleus, NF-B subunits bind to the NF-B consensus sequence, GGPuNNPyPyCC, present within the regulatory regions of target genes. Along with an array of co-factors, NF-B transcription factors are able to induce or repress transcription of a wide variety of genes. Several pathways exist for activating NF-B, depending upon the specific cellular stimulus, and this affects which NF-B subunits are recruited into action.

#### **4.2 The canonical NF-κB signaling pathway**

In the unstimulated cell, the canonical NF-B subunits, RelA and p50, stand at the ready in the cytoplasm, retained there by a family of regulatory proteins termed inhibitors of B (IBs). These IB proteins conceal the nuclear localization sequences on RelA and p50, preventing their nuclear translocation. Various stimuli, including ligands for the TNF, interleukin, Toll-like, and antigen receptor families, act to induce intracellular signaling pathways that culminate in activation of the chief canonical regulatory complex, termed the IB kinase (IKK) complex (Fig. 3A). The IKK complex is composed of three principal subunits, one regulatory subunit (NEMO/IKK), and two catalytic subunits (IKK and IKK) (Oeckinghaus and Ghosh, 2009). Once activated, the catalytic subunits, particularly IKK, phosphorylate IBs, leading to their ubiquitination and proteosomal degradation. This frees the RelA/p50 complex for nuclear translocation and transcriptional regulation.

While the steps leading from IKK activation to IB phosphorylation and degradation are well-conserved, no matter what the stimulus, specificity is built into the system in that different receptors use vastly different signaling mechanisms for communicating with the IKK complex (dotted lines in Fig. 3A). It is in dissecting these "private pathways" for IKK activation that much of the recent progress in NF-B research has been made. This is a crucial area of discovery, since identifying molecules that specifically mediate IKK activation in response to selected receptor ligands may allow for development of pharmaceuticals that interrupt (or enhance) the response to those ligands and not others. This could be a critically important area of discovery since general inhibition of NF-B can have substantial negative side-effects including the initiation of a generalized state of immunodeficiency, or impairment of growth/development.

#### **4.3 The non-canonical (alternative) NF-κB signaling pathway**

A distinct set of stimuli, including CD40 ligand, BAFF, and lymphotoxin-, work to activate a second set of NF-B subunits (Oeckinghaus and Ghosh, 2009). Activation of their cognate receptors causes phosphorylation of p100, in complex with its partner, RelB (Fig. 3B). This occurs through the kinase activity of IKK, but does not require the other components of the IKK complex. Instead, IKK activation requires the upstream activation of NF-B inducing kinase (NIK), which serves not only to phosphorylate and activate IKK, but also appears to assist in recruiting p100. Once phosphorylated, p100 undergoes partial proteolysis, producing p52, and it is the RelB/p52 complex that is active as a regulator of transcription. Thus, in essence the p100 precursor acts much the same as

The NF-B family denotes a group of five transcription factors and includes the proteins RelA (p65), RelB, c-Rel, NF-B1 (p105/50), and NF-B2 (p100/52) (Oeckinghaus and Ghosh, 2009). All share a highly conserved Rel homology domain (RHD) which directs their dimerization, nuclear localization, and DNA binding activities (Oeckinghaus and Ghosh, 2009). Upon entering the nucleus, NF-B subunits bind to the NF-B consensus sequence, GGPuNNPyPyCC, present within the regulatory regions of target genes. Along with an array of co-factors, NF-B transcription factors are able to induce or repress transcription of a wide variety of genes. Several pathways exist for activating NF-B, depending upon the specific cellular stimulus, and this affects which NF-B subunits are recruited into action.

In the unstimulated cell, the canonical NF-B subunits, RelA and p50, stand at the ready in the cytoplasm, retained there by a family of regulatory proteins termed inhibitors of B (IBs). These IB proteins conceal the nuclear localization sequences on RelA and p50, preventing their nuclear translocation. Various stimuli, including ligands for the TNF, interleukin, Toll-like, and antigen receptor families, act to induce intracellular signaling pathways that culminate in activation of the chief canonical regulatory complex, termed the IB kinase (IKK) complex (Fig. 3A). The IKK complex is composed of three principal subunits, one regulatory subunit (NEMO/IKK), and two catalytic subunits (IKK and IKK) (Oeckinghaus and Ghosh, 2009). Once activated, the catalytic subunits, particularly IKK, phosphorylate IBs, leading to their ubiquitination and proteosomal degradation. This frees the RelA/p50 complex for nuclear translocation and transcriptional regulation. While the steps leading from IKK activation to IB phosphorylation and degradation are well-conserved, no matter what the stimulus, specificity is built into the system in that different receptors use vastly different signaling mechanisms for communicating with the IKK complex (dotted lines in Fig. 3A). It is in dissecting these "private pathways" for IKK activation that much of the recent progress in NF-B research has been made. This is a crucial area of discovery, since identifying molecules that specifically mediate IKK activation in response to selected receptor ligands may allow for development of pharmaceuticals that interrupt (or enhance) the response to those ligands and not others. This could be a critically important area of discovery since general inhibition of NF-B can have substantial negative side-effects including the initiation of a generalized state of

**4.1 The NF-κB family** 

**4.2 The canonical NF-κB signaling pathway** 

immunodeficiency, or impairment of growth/development.

**4.3 The non-canonical (alternative) NF-κB signaling pathway** 

A distinct set of stimuli, including CD40 ligand, BAFF, and lymphotoxin-, work to activate a second set of NF-B subunits (Oeckinghaus and Ghosh, 2009). Activation of their cognate receptors causes phosphorylation of p100, in complex with its partner, RelB (Fig. 3B). This occurs through the kinase activity of IKK, but does not require the other components of the IKK complex. Instead, IKK activation requires the upstream activation of NF-B inducing kinase (NIK), which serves not only to phosphorylate and activate IKK, but also appears to assist in recruiting p100. Once phosphorylated, p100 undergoes partial proteolysis, producing p52, and it is the RelB/p52 complex that is active as a regulator of transcription. Thus, in essence the p100 precursor acts much the same as an IB, preventing non-canonical NF-B activity. An area of intense interest relates to the question of whether the non-canonical NF-B complex of RelB/p52 regulates a distinct set of genes from those regulated by RelA/p50. Importantly, while GPCRs are known to stimulate the canonical pathway, their potential for activating non-canonical signaling remains to be elucidated.

Fig. 3. Distinct pathways for canonical and non-canonical NF-B activation.

#### **4.4 Unique/emerging NF-κB signaling pathways**

Although the canonical and non-canonical pathways for NF-B activation have historically received the most attention, it is clear that several other mechanisms are in place for activating NF-B subunits. Bearing in mind that several different NF-B heterodimer complexes have been identified, it is likely that our understanding of alternative routes for NF-B activation will only grow as the regulation of these complexes is explored in more detail. For example, recent work in B cells has demonstrated that c-Rel/p50 heterodimers can be regulated by a unique, non-proteosome dependent pathway for IB degradation (O'Connor et al., 2004). Further, kinases other than IKK have been identified that can act to phosphorylate IB proteins, leading to their degradation (McElhinny et al., 1996; Schwarz et al., 1996). Finally, other levels of control exist beyond the simple degradation of IB proteins. Several groups have shown that NF- B subunits are targets of secondary modification, including phosphorylation and acetylation, alterations that can affect their ability to interact with both DNA consensus sites and transcriptional co-regulators. Thus, as the body of experimental data grows, it will no doubt become obvious that the concepts of canonical and non-canonical activation, outlined above and in Fig. 3, represent only a framework for a much more complicated system of regulation.

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 539

CARMA1 (also known as Bimp3/CARD11) is a member of the membrane-associated guanylate kinase (MAGUK) superfamily of molecular scaffolds that each utilize multiple discrete protein interaction domains to cluster receptors and cytosolic signaling molecules at the cell membrane (Dimitratos et al., 1999; Fanning and Anderson, 1999). As such, all MAGUKS contain three defining interactions domains: the PSD-95/Dlg/ZO-1 homologous (PDZ) domain, the Src-homology (SH3) domain, and the guanylate kinase (GUK)-like domain (Fig. 4A). CARMA1 is expressed exclusively in lymphocytes, and a few related cells of the immune system, and is one of three known members of the CARMA subfamily. This subfamily is distinguished from members of other MAGUK subfamilies by the presence of additional coiled-coil and caspase recruitment (CARD)

Numerous biochemical and genetic studies have now definitively established that CARMA1 is an essential component of the antigen-induced NF-B signaling pathway in T cells. Data indicate that CARMA1 acts as a molecular bridge, linking PKC activation with stimulation of the downstream signaling proteins, Bcl10 and MALT1 (Fig. 4B) (Lucas et al., 2004). Together, CARMA1, Bcl10, and MALT1 form a complex (referred to as the CBM signalosome) that is recruited to the lymphocyte immunological synapse following receptor engagement. In this complex, the small Bcl10 protein appears to function as an adaptor, capable of oligomerizing MALT1. Finally, MALT1 acts as an effector subunit by stimulating IKK, the IKK regulatory subunit, in part through promoting its K63-linked ubiquitination (Sun et al., 2004; Zhou et al., 2004). This subsequently leads to activation of the catalytic subunits, IKK and IKK, thereby allowing them to phosphorylate IB and free NF-B for nuclear transport. The details as to how MALT1 achieves regulation of the IKK complex are still unfolding, with the current dogma suggesting that the process includes a coordinated series of K63-linked ubiquitination events, not only of IKK but of several other proteins in the complex, as well as IKK phosphorylation, probably via the

A second mechanism of action for MALT1 has recently emerged and gained considerable attention. This relates to the discovery that MALT1 is a substrate-specific protease (McAllister-Lucas and Lucas, 2008). Although such enzymatic activity has long been postulated, based on recognition of a "caspase-like" active site in the C-terminus of MALT1 (Uren et al., 2000), it wasn't until only recently that substrates for MALT1 cleavage were identified. So far, three have been identified, and their cleavage sites mapped (Coornaert et al., 2008; Rebeaud et al., 2008; Staal et al., 2011). Two of the substrates, A20 and CYLD, are deubiquitinases, known for their ability to dampen NF-B signaling through their ubiquitin editing functions, affecting various players in the NF-B machinery. Thus, by targeting these two proteins for cleavage, it is thought that MALT1 proteolytic activity serves to maximize the level of NF-B activation, following antigen stimulation of lymphocytes. Indeed, cleavage of A20 leads to loss of its inhibitory effect and magnified antigen-dependent NF-B activation (Coornaert et al., 2008). In theory, the same could occur with cleavage of CYLD, although initial work has only shown an impact on the related JNK pathway (Staal et al.,

**5. The CARMA3/Bcl10/MALT1 signalosome; missing link for GPCR activity** 

**5.1 Lessons learned from lymphocytes** 

domains.

kinase TAK1.

2011).

#### **4.5 GPCR dependent NF-κB signaling**

Activation of certain GPCRs expressed on vascular cells, including the receptors for Ang II, thrombin, and IL-8, leads to all the hallmarks of canonical NF-B activation. Although the precise mechanisms underlying this response have been unclear, it has long been appreciated that canonical activation requires proximal stimulation of PKC (Fraser, 2008). For example, Ang II induction of NF-B in both endothelial cells, VSMCs, and cardiomyocytes is tightly linked to activation of PKC, although the specific PKC isoform responsible may differ depending on the cell type (Brasier et al., 2000; Hiroki et al., 2004; Kalra et al., 2002; Liao et al., 1997; Parmentier et al., 2006; Rouet-Benzineb et al., 2000). For PAR-1, PKC is known as the primary PKC mediating NF-B activation in endothelial cells (Minami et al., 2004; Rahman et al., 2001). PKC and have both been implicated in LPAdependent NF-B activation, in ovarian cancer cells and in airway epithelial cells, respectively (Cummings et al., 2004; Mahanivong et al., 2008). Finally, several PKC isoforms, including PKC and PKC, have been shown to mediate IL-8/CXCR2-dependent signaling, but these studies have not been performed in endothelial cells or VSMCs (Waugh and Wilson, 2008).

In that upstream PKC activation is a prerequisite for GPCR-responsive NF-B signaling, we and others recognized a parallel theme with the antigen-responsive activation of NF-B in lymphocytes. In B lymphocytes, antigen receptor ligation induces PKC, and this is critical for subsequent NF-B activation, while in T lymphocytes it is PKC that is crucial (Lucas et al., 2004). Over the past decade, a tremendous volume of data has been generated to define the precise molecular steps linking PKC activation with the NF-B machinery in lymphocytes. This work revealed that a multi-protein signaling complex, termed the CARMA1/Bcl10/MALT1 (CBM) signalosome serves as a molecular bridge between the two, and is necessary for lymphocytes to mount a normal, NF-B-dependent immune response to antigenic challenge (Lucas et al., 2004; Thome, 2004; Wegener and Krappmann, 2007). Taking cues from the lymphocyte field, we and others worked to define a novel molecular pathway that explains how GPCR-dependent PKC activation can result in NF-B signaling. This pathway utilizes an analogous CBM signalosome, present in cells outside of the immune system, and is detailed in the next section.

It is important to note, however, that the discovery of a GPCR-responsive CBM signalosome must be viewed in the larger context of GPCR signaling, with the realization that other signaling pathways are active, some of which may influence NF-B through independent mechanisms. For example, Brasier and colleagues have uncovered a distinct mechanism by which ligand-activated AGTR1 induces RelA in VSMCs (Brasier, 2010). In these cells, substantial levels of RelA are found inactive in the nucleus under resting conditions. Ang II stimulation induces a pathway of RhoA and NIK activation, culminating in NIK-dependent phosphorylation of the nuclear RelA species on serine 536 (Choudhary et al., 2007; Cui et al., 2006). This phosphorylated pool of RelA is free from IB regulation and dynamically cycles through the nucleus, interacting with target genes (Bosisio et al., 2006; Sasaki et al., 2005). This mechanism of regulation has been shown to impact the NF-B responsive, *IL-6* gene. Consistent with this observation, we have seen only a partial effect of blocking the CBM signalosome on Ang II-dependent IL-6 induction, underscoring the concept that different NF-B responsive genes may respond to different NF-B transcription factor complexes and/or different modes of NF-B regulation.

#### **5. The CARMA3/Bcl10/MALT1 signalosome; missing link for GPCR activity**

#### **5.1 Lessons learned from lymphocytes**

538 Atherogenesis

Activation of certain GPCRs expressed on vascular cells, including the receptors for Ang II, thrombin, and IL-8, leads to all the hallmarks of canonical NF-B activation. Although the precise mechanisms underlying this response have been unclear, it has long been appreciated that canonical activation requires proximal stimulation of PKC (Fraser, 2008). For example, Ang II induction of NF-B in both endothelial cells, VSMCs, and cardiomyocytes is tightly linked to activation of PKC, although the specific PKC isoform responsible may differ depending on the cell type (Brasier et al., 2000; Hiroki et al., 2004; Kalra et al., 2002; Liao et al., 1997; Parmentier et al., 2006; Rouet-Benzineb et al., 2000). For PAR-1, PKC is known as the primary PKC mediating NF-B activation in endothelial cells (Minami et al., 2004; Rahman et al., 2001). PKC and have both been implicated in LPAdependent NF-B activation, in ovarian cancer cells and in airway epithelial cells, respectively (Cummings et al., 2004; Mahanivong et al., 2008). Finally, several PKC isoforms, including PKC and PKC, have been shown to mediate IL-8/CXCR2-dependent signaling, but these studies have not been performed in endothelial cells or VSMCs (Waugh and

In that upstream PKC activation is a prerequisite for GPCR-responsive NF-B signaling, we and others recognized a parallel theme with the antigen-responsive activation of NF-B in lymphocytes. In B lymphocytes, antigen receptor ligation induces PKC, and this is critical for subsequent NF-B activation, while in T lymphocytes it is PKC that is crucial (Lucas et al., 2004). Over the past decade, a tremendous volume of data has been generated to define the precise molecular steps linking PKC activation with the NF-B machinery in lymphocytes. This work revealed that a multi-protein signaling complex, termed the CARMA1/Bcl10/MALT1 (CBM) signalosome serves as a molecular bridge between the two, and is necessary for lymphocytes to mount a normal, NF-B-dependent immune response to antigenic challenge (Lucas et al., 2004; Thome, 2004; Wegener and Krappmann, 2007). Taking cues from the lymphocyte field, we and others worked to define a novel molecular pathway that explains how GPCR-dependent PKC activation can result in NF-B signaling. This pathway utilizes an analogous CBM signalosome, present in cells outside of the

It is important to note, however, that the discovery of a GPCR-responsive CBM signalosome must be viewed in the larger context of GPCR signaling, with the realization that other signaling pathways are active, some of which may influence NF-B through independent mechanisms. For example, Brasier and colleagues have uncovered a distinct mechanism by which ligand-activated AGTR1 induces RelA in VSMCs (Brasier, 2010). In these cells, substantial levels of RelA are found inactive in the nucleus under resting conditions. Ang II stimulation induces a pathway of RhoA and NIK activation, culminating in NIK-dependent phosphorylation of the nuclear RelA species on serine 536 (Choudhary et al., 2007; Cui et al., 2006). This phosphorylated pool of RelA is free from IB regulation and dynamically cycles through the nucleus, interacting with target genes (Bosisio et al., 2006; Sasaki et al., 2005). This mechanism of regulation has been shown to impact the NF-B responsive, *IL-6* gene. Consistent with this observation, we have seen only a partial effect of blocking the CBM signalosome on Ang II-dependent IL-6 induction, underscoring the concept that different NF-B responsive genes may respond to different NF-B transcription factor complexes

**4.5 GPCR dependent NF-κB signaling** 

immune system, and is detailed in the next section.

and/or different modes of NF-B regulation.

Wilson, 2008).

CARMA1 (also known as Bimp3/CARD11) is a member of the membrane-associated guanylate kinase (MAGUK) superfamily of molecular scaffolds that each utilize multiple discrete protein interaction domains to cluster receptors and cytosolic signaling molecules at the cell membrane (Dimitratos et al., 1999; Fanning and Anderson, 1999). As such, all MAGUKS contain three defining interactions domains: the PSD-95/Dlg/ZO-1 homologous (PDZ) domain, the Src-homology (SH3) domain, and the guanylate kinase (GUK)-like domain (Fig. 4A). CARMA1 is expressed exclusively in lymphocytes, and a few related cells of the immune system, and is one of three known members of the CARMA subfamily. This subfamily is distinguished from members of other MAGUK subfamilies by the presence of additional coiled-coil and caspase recruitment (CARD) domains.

Numerous biochemical and genetic studies have now definitively established that CARMA1 is an essential component of the antigen-induced NF-B signaling pathway in T cells. Data indicate that CARMA1 acts as a molecular bridge, linking PKC activation with stimulation of the downstream signaling proteins, Bcl10 and MALT1 (Fig. 4B) (Lucas et al., 2004). Together, CARMA1, Bcl10, and MALT1 form a complex (referred to as the CBM signalosome) that is recruited to the lymphocyte immunological synapse following receptor engagement. In this complex, the small Bcl10 protein appears to function as an adaptor, capable of oligomerizing MALT1. Finally, MALT1 acts as an effector subunit by stimulating IKK, the IKK regulatory subunit, in part through promoting its K63-linked ubiquitination (Sun et al., 2004; Zhou et al., 2004). This subsequently leads to activation of the catalytic subunits, IKK and IKK, thereby allowing them to phosphorylate IB and free NF-B for nuclear transport. The details as to how MALT1 achieves regulation of the IKK complex are still unfolding, with the current dogma suggesting that the process includes a coordinated series of K63-linked ubiquitination events, not only of IKK but of several other proteins in the complex, as well as IKK phosphorylation, probably via the kinase TAK1.

A second mechanism of action for MALT1 has recently emerged and gained considerable attention. This relates to the discovery that MALT1 is a substrate-specific protease (McAllister-Lucas and Lucas, 2008). Although such enzymatic activity has long been postulated, based on recognition of a "caspase-like" active site in the C-terminus of MALT1 (Uren et al., 2000), it wasn't until only recently that substrates for MALT1 cleavage were identified. So far, three have been identified, and their cleavage sites mapped (Coornaert et al., 2008; Rebeaud et al., 2008; Staal et al., 2011). Two of the substrates, A20 and CYLD, are deubiquitinases, known for their ability to dampen NF-B signaling through their ubiquitin editing functions, affecting various players in the NF-B machinery. Thus, by targeting these two proteins for cleavage, it is thought that MALT1 proteolytic activity serves to maximize the level of NF-B activation, following antigen stimulation of lymphocytes. Indeed, cleavage of A20 leads to loss of its inhibitory effect and magnified antigen-dependent NF-B activation (Coornaert et al., 2008). In theory, the same could occur with cleavage of CYLD, although initial work has only shown an impact on the related JNK pathway (Staal et al., 2011).

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 541

Many of the details concerning how the CBM complex is recruited and activated at the T cell immunological synapse have become clear only recently. First, Ghosh and co-workers demonstrated that, following T cell receptor stimulation, the enzyme 3-phosphoinositidedependent kinase 1 (PDK1) serves to anchor both activated PKC and CARMA1 within close proximity to one another, at the immunological synapse (Fig. 4B) (Lee et al., 2005). PDK1 is a kinase known to phosphorylate PKC at a specific site within its "activation loop" (Belham et al., 1999; Mora et al., 2004; Newton, 2001, 2003). This phosphorylation appears to serve as a priming reaction, allowing PKC to then respond to activating signals, such as Ca2+ or DAG, depending on the specific isoform. However, the ability of PDK1 to interact with CARMA1 represents a newly defined role for this kinase. Although PDK1 plays a key role in recruiting the CBM signalosome to the T cell receptor, it may not represent the only molecular link; for example, recent work has demonstrated that the protein ADAP is also

A second major finding was that PKC acts to phosphorylate specific sites within the linker region of CARMA1, which resides between the CARD/coiled-coil domains and the domains present in all MAGUK proteins (PDZ/SH3/GUK) (Matsumoto et al., 2005; Sommer et al., 2005). This appears to result in a conformational change in CARMA1, allowing for exposure of the CARD domain (Sommer et al., 2005). Consequently, Bcl10 and MALT1 can then be effectively recruited to the immunological synapse because their recruitment depends primarily on a CARD-CARD interaction between CARMA1 and Bcl10. Finally, the IKK complex is recruited and can thereby be activated by the fully assembled CBM complex (Shinohara et al., 2005; Stilo et al., 2004; Weil et al., 2003). It should be noted that the majority of the work defining the mechanism of action of the CBM signalosome has been carried out using T cell models. Interestingly, not all of the concepts are likely to hold true for B cells. In particular, based on phenotypic differences between *MALT1-/-* mice and *BCL10-/-* or *CARMA1-/-* mice, there is some debate as to whether MALT1 is an obligate player in B cell

**5.2 CARMA3, a CARMA homologue expressed in cells outside the immune system**  Except for CARMA1, the key molecules mediating antigen-dependent NF-B activation in lymphocytes are ubiquitously expressed in a diverse array of cells. However, we and others had noted that a highly related protein, CARMA3 (Bimp1/CARD10), is expressed more broadly than the immune cell-specific CARMA1 (McAllister-Lucas et al., 2001; Wang et al., 2001b). The *CARMA1* and *CARMA3* genes encode proteins that are highly similar; the CARDS and coiled-coil domains share approximately 60% and 50% sequence identity with one another, respectively, while the PDZ, SH3 and GUK domains share approximately 20- 30% identity. The functional similarities between CARMA3 and CARMA1 are illustrated by the fact that CARMA3 can rescue antigen-induced NF-B activation in CARMA1-deficient T cells (Matsumoto et al., 2005). Of potential importance to cardiovascular pathophysiology, all three proteins of a putative CARMA3-containing CBM complex are abundant in heart and aorta, and western blotting confirms their presence in those tissues at the protein level (McAllister-Lucas et al., 2010; McAllister-Lucas et al., 2007). As a result, we wondered if CARMA3 might scaffold an analogous CBM signalosome in cells outside the immune system. The known dependence on PKC activation for GPCR-responsive NF-B stimulation led to the hypothesis that specific GPCRs might represent candidates for receptors that

could communicate with CARMA3 and it associated signaling molecules.

crucial in this regard (Medeiros et al., 2007).

receptor-dependent NF-B activation.

Fig. 4. A, Schematic diagram of proteins that make up the CBM signalosome. B and C, Similarities and differences exist between the mechanisms through which the CARMA1 and CARMA3-containing signalosomes act to stimulate the IKK complex; see text for description.

Fig. 4. A, Schematic diagram of proteins that make up the CBM signalosome. B and C, Similarities and differences exist between the mechanisms through which the CARMA1 and CARMA3-containing signalosomes act to stimulate the IKK complex; see text for

description.

Many of the details concerning how the CBM complex is recruited and activated at the T cell immunological synapse have become clear only recently. First, Ghosh and co-workers demonstrated that, following T cell receptor stimulation, the enzyme 3-phosphoinositidedependent kinase 1 (PDK1) serves to anchor both activated PKC and CARMA1 within close proximity to one another, at the immunological synapse (Fig. 4B) (Lee et al., 2005). PDK1 is a kinase known to phosphorylate PKC at a specific site within its "activation loop" (Belham et al., 1999; Mora et al., 2004; Newton, 2001, 2003). This phosphorylation appears to serve as a priming reaction, allowing PKC to then respond to activating signals, such as Ca2+ or DAG, depending on the specific isoform. However, the ability of PDK1 to interact with CARMA1 represents a newly defined role for this kinase. Although PDK1 plays a key role in recruiting the CBM signalosome to the T cell receptor, it may not represent the only molecular link; for example, recent work has demonstrated that the protein ADAP is also crucial in this regard (Medeiros et al., 2007).

A second major finding was that PKC acts to phosphorylate specific sites within the linker region of CARMA1, which resides between the CARD/coiled-coil domains and the domains present in all MAGUK proteins (PDZ/SH3/GUK) (Matsumoto et al., 2005; Sommer et al., 2005). This appears to result in a conformational change in CARMA1, allowing for exposure of the CARD domain (Sommer et al., 2005). Consequently, Bcl10 and MALT1 can then be effectively recruited to the immunological synapse because their recruitment depends primarily on a CARD-CARD interaction between CARMA1 and Bcl10. Finally, the IKK complex is recruited and can thereby be activated by the fully assembled CBM complex (Shinohara et al., 2005; Stilo et al., 2004; Weil et al., 2003). It should be noted that the majority of the work defining the mechanism of action of the CBM signalosome has been carried out using T cell models. Interestingly, not all of the concepts are likely to hold true for B cells. In particular, based on phenotypic differences between *MALT1-/-* mice and *BCL10-/-* or *CARMA1-/-* mice, there is some debate as to whether MALT1 is an obligate player in B cell receptor-dependent NF-B activation.

#### **5.2 CARMA3, a CARMA homologue expressed in cells outside the immune system**

Except for CARMA1, the key molecules mediating antigen-dependent NF-B activation in lymphocytes are ubiquitously expressed in a diverse array of cells. However, we and others had noted that a highly related protein, CARMA3 (Bimp1/CARD10), is expressed more broadly than the immune cell-specific CARMA1 (McAllister-Lucas et al., 2001; Wang et al., 2001b). The *CARMA1* and *CARMA3* genes encode proteins that are highly similar; the CARDS and coiled-coil domains share approximately 60% and 50% sequence identity with one another, respectively, while the PDZ, SH3 and GUK domains share approximately 20- 30% identity. The functional similarities between CARMA3 and CARMA1 are illustrated by the fact that CARMA3 can rescue antigen-induced NF-B activation in CARMA1-deficient T cells (Matsumoto et al., 2005). Of potential importance to cardiovascular pathophysiology, all three proteins of a putative CARMA3-containing CBM complex are abundant in heart and aorta, and western blotting confirms their presence in those tissues at the protein level (McAllister-Lucas et al., 2010; McAllister-Lucas et al., 2007). As a result, we wondered if CARMA3 might scaffold an analogous CBM signalosome in cells outside the immune system. The known dependence on PKC activation for GPCR-responsive NF-B stimulation led to the hypothesis that specific GPCRs might represent candidates for receptors that could communicate with CARMA3 and it associated signaling molecules.

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 543

which then allows recruitment of arrestins to the receptors. This helps to uncouple G proteins from the receptors and assists in receptor internalization through clathrin-coated pits. But more recently, arrestins have become known as scaffold proteins that facilitate the recruitment and activation of a number of distinct secondary signaling molecules (DeFea, 2011). In this way, arrestins are now appreciated not just for their ability to terminate GPCR signaling, but also for their ability to promote a second layer of GPCR-

Lin and colleagues originally showed that -arrestin 2 deficient MEFs were unable to respond to LPA treatment with an NF-B signal (Sun and Lin, 2008). They then determined that -arrestin 2 bound to CARMA3. Further, co-immunoprecipitation experiments demonstrated that CARMA3 could interact with the LPA receptor only when -arrestin 2 was present to act as a bridge. Subsequent work showed that -arrestin 2 deficient MEFs are also defective in thrombin-dependent NF-B activation (Delekta et al., 2010). Thus, it is likely that for GPCR signaling, -arrestin 2 serves a scaffolding role, analogous to what has

Despite the progress that has been made in understanding the regulation of CARMA3 containing CBM signalosomes, much remains to be learned. The CARMA1-containing complex has been studied for a much longer period of time, and many of the finer details have been explored in more detail. For example, the sites of PKC-dependent CARMA1 phosphorylation, which allow for unfolding of CARMA1 and exposure of the CARD domain, have been mapped (Matsumoto et al., 2005; Sommer et al., 2005). In addition, other kinases have been identified that can positively or negatively regulate CARMA1 (Bidere et al., 2009; Brenner et al., 2009; Ishiguro et al., 2006; Shinohara et al., 2007). In contrast,

mechanisms for regulation of CARMA3 remain mostly speculative at this time.

**5.4 Distinct mechanisms for CARMA1- and CARMA3-dependent IKK activation** 

Another area of potential distinction, differentiating CARMA1- and CARMA3-containing signalosomes, relates to their ability to facilitate IKK complex phosphorylation, a necessary step for full IKK activation. As described previously, MALT1 is thought to play a major role as an "effector" protein in the CBM complex, coordinating the activation of the IKK complex. This occurs at least in part through the ability of MALT1 to direct multiple K63 linked ubiquitin modifications, targeting IKK, Bcl10, and even MALT1 itself. In the lymphocyte, these ubiquitin chains may then serve as a scaffold to recruit the kinase, TAK1, which completes the activation of the IKK complex through phosphorylation of IKK at specific residues within its activation loop (Shinohara et al., 2005; Wang et al., 2001a). As for the activation of TAK1, this appears to occur through a parallel pathway, initiated by the antigen receptor, that does not depend upon the CBM complex (Shambharkar et al., 2007)

Interestingly, recent work has shown that TAK1 is dispensable for LPA-dependent NF-B activation. Instead, another mitogen-activated protein kinase, MEKK3, takes its place (Sun et al., 2009). Thus, the ligand-activated LPA receptor induces a parallel pathway, independent of the CARMA3-containing CBM complex, that causes MEKK3 activation, subsequently leading to IKK phosphorylation (Fig. 4C). It remains an open question as to whether MEKK3 will be involved in IKK complex activation downstream of all GPCRs, or whether distinct kinases will act in concert with the CARMA3-containing CBM complex, depending

been shown for PDK1 in the lymphocyte system (Fig. 4C).

dependent responses.

(Fig. 4B).

on the specific GPCR being induced.

Using distinct systems and approaches, we and two other groups simultaneously demonstrated that two GPCRs could harness a CARMA3-containing CBM signalosome for the purposes of NF-B activation (Klemm et al., 2007; McAllister-Lucas et al., 2007; Wang et al., 2007) (Fig. 4C). Our group demonstrated the essential role of CARMA3, Bcl10, and MALT1 in the Ang II-dependent activation of canonical NF-B (McAllister-Lucas et al., 2007). This initial work focused on hepatocytes as a model, but subsequent work demonstrated that the same CBM machinery is active in endothelial cells following Ang II stimulation (McAllister-Lucas et al., 2010). Individually knocking down each component of the putative CARMA3/Bcl10/MALT1 complex completely blocked Ang II-dependent IB phosphorylation, a marker of canonical NF-B activation, or induction of an NF-B responsive reporter gene. In addition, expression of a dominant negative mutant of CARMA3 was sufficient to impair Ang II-dependent K63-linked IKK polyubiquitination.

The other two studies focused on mouse embryonic fibroblasts (MEFs) from *BCL10-/-* and *MALT1-/-* mouse strains. In contrast to wild-type MEFs, these knockout cells showed a complete lack of NF-B activation when stimulated with lysophosphatidic acid (LPA) (Klemm et al., 2007; Wang et al., 2007). Follow-up work revealed the same phenomenon with *CARMA3-/-* MEFs (Grabiner et al., 2007). Like the receptor for Ang II (AGTR1), receptors for LPA (LPA1-4) are prototypical GPCRs, coupled primarily with Gq/11 subunits. With regard to pathophysiology, LPA receptor-induced NF-B activation has been linked to a variety of consequences, depending upon the cell type affected, which include the promotion of carcinoma cell survival and spread, as well as endothelial dysfunction. However, despite the potential importance of the CBM complex in mediating LPAdependent effects in endothelial cells or VSMCs, studies have yet to be published that specifically link LPA receptors to the CBM components in the vasculature.

Following these initial studies, several groups have added to the list of GPCRs capable of harnessing the CARMA3-containing CBM signalosome. To date, however, the only receptors that have been specifically shown to utilize the signalosome in endothelial cells are those for Ang II (AGTR1), thrombin (PAR-1) and IL-8 (CXCR2) (Delekta et al., 2010; Martin et al., 2009; McAllister-Lucas et al., 2010).

#### **5.3 Distinct mechanisms for recruiting CARMA1- and CARMA3-containing signalosomes**

Although there are strong parallels between the mechanisms underlying recruitment and activation of the CARMA1-containing CBM signalosome of lymphocytes and the CARMA3 containing CBM signalosome of endothelial cells, there are also notable differences. Most striking are the differences in how the signalosomes communicate with their cognate receptors. We have already described work demonstrating a crucial role for PDK1 in scaffolding an interaction with antigen receptors in the T cell. In contrast, PDK1 may have no role in coordinating GPCR-dependent CBM assembly; at least for PAR-1, knockdown of PDK1 in endothelial cells has no effect on thrombin-dependent NF-B activation (Delekta et al., 2010). Instead, we and others have implicated -arrestin 2 as a protein that could serve the function of scaffolding CARMA3 to select GPCRs (Delekta et al., 2010; Sun and Lin, 2008).

Traditionally, the -arrestin proteins have been known for their role in down-regulating activated GPCRs through receptor endocytosis, leading to their recycling or degradation. Activated GPCRs are phosphorylated by various G protein receptor kinases (GRKs),

Using distinct systems and approaches, we and two other groups simultaneously demonstrated that two GPCRs could harness a CARMA3-containing CBM signalosome for the purposes of NF-B activation (Klemm et al., 2007; McAllister-Lucas et al., 2007; Wang et al., 2007) (Fig. 4C). Our group demonstrated the essential role of CARMA3, Bcl10, and MALT1 in the Ang II-dependent activation of canonical NF-B (McAllister-Lucas et al., 2007). This initial work focused on hepatocytes as a model, but subsequent work demonstrated that the same CBM machinery is active in endothelial cells following Ang II stimulation (McAllister-Lucas et al., 2010). Individually knocking down each component of the putative CARMA3/Bcl10/MALT1 complex completely blocked Ang II-dependent IB phosphorylation, a marker of canonical NF-B activation, or induction of an NF-B responsive reporter gene. In addition, expression of a dominant negative mutant of CARMA3 was sufficient to impair Ang II-dependent K63-linked IKK polyubiquitination. The other two studies focused on mouse embryonic fibroblasts (MEFs) from *BCL10-/-* and *MALT1-/-* mouse strains. In contrast to wild-type MEFs, these knockout cells showed a complete lack of NF-B activation when stimulated with lysophosphatidic acid (LPA) (Klemm et al., 2007; Wang et al., 2007). Follow-up work revealed the same phenomenon with *CARMA3-/-* MEFs (Grabiner et al., 2007). Like the receptor for Ang II (AGTR1), receptors for LPA (LPA1-4) are prototypical GPCRs, coupled primarily with Gq/11 subunits. With regard to pathophysiology, LPA receptor-induced NF-B activation has been linked to a variety of consequences, depending upon the cell type affected, which include the promotion of carcinoma cell survival and spread, as well as endothelial dysfunction. However, despite the potential importance of the CBM complex in mediating LPAdependent effects in endothelial cells or VSMCs, studies have yet to be published that

specifically link LPA receptors to the CBM components in the vasculature.

**5.3 Distinct mechanisms for recruiting CARMA1- and CARMA3-containing** 

et al., 2009; McAllister-Lucas et al., 2010).

**signalosomes** 

2008).

Following these initial studies, several groups have added to the list of GPCRs capable of harnessing the CARMA3-containing CBM signalosome. To date, however, the only receptors that have been specifically shown to utilize the signalosome in endothelial cells are those for Ang II (AGTR1), thrombin (PAR-1) and IL-8 (CXCR2) (Delekta et al., 2010; Martin

Although there are strong parallels between the mechanisms underlying recruitment and activation of the CARMA1-containing CBM signalosome of lymphocytes and the CARMA3 containing CBM signalosome of endothelial cells, there are also notable differences. Most striking are the differences in how the signalosomes communicate with their cognate receptors. We have already described work demonstrating a crucial role for PDK1 in scaffolding an interaction with antigen receptors in the T cell. In contrast, PDK1 may have no role in coordinating GPCR-dependent CBM assembly; at least for PAR-1, knockdown of PDK1 in endothelial cells has no effect on thrombin-dependent NF-B activation (Delekta et al., 2010). Instead, we and others have implicated -arrestin 2 as a protein that could serve the function of scaffolding CARMA3 to select GPCRs (Delekta et al., 2010; Sun and Lin,

Traditionally, the -arrestin proteins have been known for their role in down-regulating activated GPCRs through receptor endocytosis, leading to their recycling or degradation. Activated GPCRs are phosphorylated by various G protein receptor kinases (GRKs), which then allows recruitment of arrestins to the receptors. This helps to uncouple G proteins from the receptors and assists in receptor internalization through clathrin-coated pits. But more recently, arrestins have become known as scaffold proteins that facilitate the recruitment and activation of a number of distinct secondary signaling molecules (DeFea, 2011). In this way, arrestins are now appreciated not just for their ability to terminate GPCR signaling, but also for their ability to promote a second layer of GPCRdependent responses.

Lin and colleagues originally showed that -arrestin 2 deficient MEFs were unable to respond to LPA treatment with an NF-B signal (Sun and Lin, 2008). They then determined that -arrestin 2 bound to CARMA3. Further, co-immunoprecipitation experiments demonstrated that CARMA3 could interact with the LPA receptor only when -arrestin 2 was present to act as a bridge. Subsequent work showed that -arrestin 2 deficient MEFs are also defective in thrombin-dependent NF-B activation (Delekta et al., 2010). Thus, it is likely that for GPCR signaling, -arrestin 2 serves a scaffolding role, analogous to what has been shown for PDK1 in the lymphocyte system (Fig. 4C).

Despite the progress that has been made in understanding the regulation of CARMA3 containing CBM signalosomes, much remains to be learned. The CARMA1-containing complex has been studied for a much longer period of time, and many of the finer details have been explored in more detail. For example, the sites of PKC-dependent CARMA1 phosphorylation, which allow for unfolding of CARMA1 and exposure of the CARD domain, have been mapped (Matsumoto et al., 2005; Sommer et al., 2005). In addition, other kinases have been identified that can positively or negatively regulate CARMA1 (Bidere et al., 2009; Brenner et al., 2009; Ishiguro et al., 2006; Shinohara et al., 2007). In contrast, mechanisms for regulation of CARMA3 remain mostly speculative at this time.

#### **5.4 Distinct mechanisms for CARMA1- and CARMA3-dependent IKK activation**

Another area of potential distinction, differentiating CARMA1- and CARMA3-containing signalosomes, relates to their ability to facilitate IKK complex phosphorylation, a necessary step for full IKK activation. As described previously, MALT1 is thought to play a major role as an "effector" protein in the CBM complex, coordinating the activation of the IKK complex. This occurs at least in part through the ability of MALT1 to direct multiple K63 linked ubiquitin modifications, targeting IKK, Bcl10, and even MALT1 itself. In the lymphocyte, these ubiquitin chains may then serve as a scaffold to recruit the kinase, TAK1, which completes the activation of the IKK complex through phosphorylation of IKK at specific residues within its activation loop (Shinohara et al., 2005; Wang et al., 2001a). As for the activation of TAK1, this appears to occur through a parallel pathway, initiated by the antigen receptor, that does not depend upon the CBM complex (Shambharkar et al., 2007) (Fig. 4B).

Interestingly, recent work has shown that TAK1 is dispensable for LPA-dependent NF-B activation. Instead, another mitogen-activated protein kinase, MEKK3, takes its place (Sun et al., 2009). Thus, the ligand-activated LPA receptor induces a parallel pathway, independent of the CARMA3-containing CBM complex, that causes MEKK3 activation, subsequently leading to IKK phosphorylation (Fig. 4C). It remains an open question as to whether MEKK3 will be involved in IKK complex activation downstream of all GPCRs, or whether distinct kinases will act in concert with the CARMA3-containing CBM complex, depending on the specific GPCR being induced.

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 545

Under certain conditions such as tissue hypoxia, VEGF synthesis and secretion is regulated via activation of the transcription factor, hypoxia inducible factor-1 (HIF-1) (Semenza, 2007). Although hypoxia is a potent stimulus for VEGF expression, inflammatory cytokines have also been reported to stimulate VEGF expression through mechanisms that have not been fully delineated. One recent study, however, revealed that regulation of VEGF expression in endothelial cells can occur via CBM signalosome-mediated activation of NF-B (Martin et al., 2009). These investigators showed that the pro-inflammatory cytokine IL-8 (CXCL8) stimulated VEGF production and secretion through activation of its cognate GPCR, CXCR2. This receptor, in turn, was linked to the CBM signaling complex and NF-B activation. Importantly, the effect of IL-8 on VEGF induction was independent of HIF-1 but entirely dependent on NF-B. Inhibition of any of the CBM components by siRNA was effective at reducing NF-B activation and resulted in a marked inhibition of VEGF mRNA expression and protein secretion. Further, this was accompanied by decreased autocrine VEGFR2 activation. These results suggest that the CBM signalosome is necessary for regulating VEGF production in endothelial cells in response to certain inflammatory cytokines, and might indirectly contribute to the VEGF-dependent transition of the endothelium into a pro-

As discussed earlier, thrombin and its receptor, PAR-1, are thought to induce a variety of pro-inflammatory responses that may also contribute to endothelial phenotype changes (Hirano, 2007). Thrombin levels are increased at sites of vascular injury and thrombosis, where the persistent stimulation of its receptor leads to endothelial dysfunction, thereby increasing inflammatory responses leading to further vessel wall damage and atherosclerotic lesion progression. One of the key responses to local thrombin production is the increase in endothelial cell expression of adhesion molecules, allowing for firm adhesion of circulating monocytes and other leukocytes (Minami et al., 2004). Our lab has specifically shown that thrombin-dependent activation of the CBM-NF-B signaling axis in endothelial cells results in upregulation of two such adhesion molecules, VCAM-1 and ICAM-1, at both the mRNA and protein levels (Delekta et al., 2010). Further, we showed that thrombininduced adhesion of monocytes to endothelial cells requires the intact CBM signalosome; siRNA-mediated knockdown of Bcl10 in endothelial cells altered their phenotype to completely abolish thrombin-dependent monocyte adherence. Further work will be required to test the role of the CBM proteins in modulating other aspects of thrombin-

Similar to thrombin, Ang II produced locally in the vasculature has been reported to induce a number of inflammatory responses, including the expression of NF-B-sensitive adhesion molecules and cytokines in endothelial cells, and the recruitment of inflammatory cells to the vessel wall (Daugherty and Cassis, 2004). Our group recently showed that Ang II activation of its receptor, present on endothelial cells and on VSMCs, stimulates NF-B

Further, we tested the effects of manipulating the signalosome *in vivo*. In these studies, we utilized the *ApoE-/-* mouse strain described earlier, which is hyperlipidemic and prone to developing atherosclerosis. The development of lesions, however, can be dramatically

angiogenic phenotype.

dependent endothelial dysfunction.

**6.2.3 Ang II and** *in vivo* **atherogenesis** 

through the CBM signalosome (McAllister-Lucas et al., 2010).

**6.2.2 Thrombin and endothelial cell/monocyte adhesion** 

#### **6. The CARMA3/Bcl10/MALT1 signalosome and endothelial phenotype**

#### **6.1 Role for endothelial NF-κB activation in atherogenesis**

As discussed earlier, many pieces of evidence implicate endothelial cell NF-B activation as an important GPCR-mediated signaling event favoring atherogenesis. Recently, this concept was reinforced by elegant studies using two related mouse models (Gareus et al., 2008). These researchers first generated an endothelial-specific *IKK-/-* mouse, to disrupt any NF-B signaling in this cell type. These *IKK-/-* mice were crossed with a mouse model of atherosclerosis (*ApoE-/-*) and fed a cholesterol-rich diet. After ten weeks, these mice showed a 30% reduction in plaque size, 40% reduction of T cells in plaques, and an overall retardation in the progression to advanced plaques as compared to *IKK+/+/ApoE-/-* mice under the same diet. To further demonstrate the specific role of endothelial NF-B in atherogenesis, an additional transgenic mouse model was created, expressing dominant negative IB (IB-SR) under control of the Tie2 promoter. This effectively targeted expression of the dominant negative mutant to endothelial cells. Since the dominant negative mutant lacks phosphorylation acceptor sites for IKK, its expression effectively keeps NF-B subunits sequestered in an inactive state, regardless of whether or not the cell is being stimulated by any of the classic NF-B inducers. These mice were once again backcrossed with *ApoE-/-* mice and placed on a high cholesterol diet for ten weeks, after which they showed a 60% reduction in plaque size and a significant reduction in plaque progression as compared to *ApoE-/-* mice with normally functioning NF-B (Gareus et al., 2008). The endothelium of IB-SR/*ApoE-/-* mice was almost completely free from expression of most cytokines, chemokines and adhesion molecules. Taken together, this study provides exceptionally strong evidence that NF-B activation within the endothelium alone is necessary to drive a significant atherogenesis response.

#### **6.2 The GPCR-CBM-NF-κB axis in endothelial dysfunction and atherogenesis**

To date, three GPCRs have been linked to the CBM signalosome and NF-B activation, specifically in endothelial cells. These are the receptors for Ang II, thrombin, and IL-8. Others are sure to follow; for example, clear evidence exists for an important role for LPA receptor-dependent NF-B activation in endothelial biology, but to date the connections between this receptor and the CBM signalosome have been explored only in cell models outside vascular biology. In the following sections, we describe the specific work that has been done to investigate the GPCR-CBM-NF-B signaling axis in endothelial cells, focusing on the cellular and pathophysiologic consequences of activating this signaling axis.

#### **6.2.1 IL-8 and VEGF induction**

Vascular endothelial growth factor (VEGF) is a key endothelial-specific growth factor that is induced in response to tissue damage. VEGF modulates endothelial cell phenotypes by inducing cell proliferation, promoting cell migration, and inhibiting apoptosis, and is regarded as a key regulator of angiogenesis (Ferrara et al., 2003). In atherogenesis, VEGF may play a role in promoting the pathologic ingrowth of the neovascular network from the vasa vasorum, into plaque developing in the subintimal space. These effects are mediated through VEGF binding to its own receptors, VEGFR-1/2 (Ferrara et al., 2003).

As discussed earlier, many pieces of evidence implicate endothelial cell NF-B activation as an important GPCR-mediated signaling event favoring atherogenesis. Recently, this concept was reinforced by elegant studies using two related mouse models (Gareus et al., 2008).

atherosclerosis (*ApoE-/-*) and fed a cholesterol-rich diet. After ten weeks, these mice showed a 30% reduction in plaque size, 40% reduction of T cells in plaques, and an overall retardation

diet. To further demonstrate the specific role of endothelial NF-B in atherogenesis, an additional transgenic mouse model was created, expressing dominant negative IB (IB-SR) under control of the Tie2 promoter. This effectively targeted expression of the dominant negative mutant to endothelial cells. Since the dominant negative mutant lacks phosphorylation acceptor sites for IKK, its expression effectively keeps NF-B subunits sequestered in an inactive state, regardless of whether or not the cell is being stimulated by any of the classic NF-B inducers. These mice were once again backcrossed with *ApoE-/-* mice and placed on a high cholesterol diet for ten weeks, after which they showed a 60% reduction in plaque size and a significant reduction in plaque progression as compared to *ApoE-/-* mice with normally functioning NF-B (Gareus et al., 2008). The endothelium of IB-SR/*ApoE-/-* mice was almost completely free from expression of most cytokines, chemokines and adhesion molecules. Taken together, this study provides exceptionally strong evidence that NF-B activation within the endothelium alone is necessary to drive a significant

*-/-* mice were crossed with a mouse model of

*-/-* mouse, to disrupt any NF-B

*+/+/ApoE-/-* mice under the same

**6. The CARMA3/Bcl10/MALT1 signalosome and endothelial phenotype** 

**6.2 The GPCR-CBM-NF-κB axis in endothelial dysfunction and atherogenesis** 

To date, three GPCRs have been linked to the CBM signalosome and NF-B activation, specifically in endothelial cells. These are the receptors for Ang II, thrombin, and IL-8. Others are sure to follow; for example, clear evidence exists for an important role for LPA receptor-dependent NF-B activation in endothelial biology, but to date the connections between this receptor and the CBM signalosome have been explored only in cell models outside vascular biology. In the following sections, we describe the specific work that has been done to investigate the GPCR-CBM-NF-B signaling axis in endothelial cells, focusing on the cellular and pathophysiologic consequences of activating this signaling

Vascular endothelial growth factor (VEGF) is a key endothelial-specific growth factor that is induced in response to tissue damage. VEGF modulates endothelial cell phenotypes by inducing cell proliferation, promoting cell migration, and inhibiting apoptosis, and is regarded as a key regulator of angiogenesis (Ferrara et al., 2003). In atherogenesis, VEGF may play a role in promoting the pathologic ingrowth of the neovascular network from the vasa vasorum, into plaque developing in the subintimal space. These effects are mediated through VEGF binding to its own receptors, VEGFR-

**6.1 Role for endothelial NF-κB activation in atherogenesis** 

These researchers first generated an endothelial-specific *IKK*

in the progression to advanced plaques as compared to *IKK*

signaling in this cell type. These *IKK*

atherogenesis response.

**6.2.1 IL-8 and VEGF induction** 

1/2 (Ferrara et al., 2003).

axis.

Under certain conditions such as tissue hypoxia, VEGF synthesis and secretion is regulated via activation of the transcription factor, hypoxia inducible factor-1 (HIF-1) (Semenza, 2007). Although hypoxia is a potent stimulus for VEGF expression, inflammatory cytokines have also been reported to stimulate VEGF expression through mechanisms that have not been fully delineated. One recent study, however, revealed that regulation of VEGF expression in endothelial cells can occur via CBM signalosome-mediated activation of NF-B (Martin et al., 2009). These investigators showed that the pro-inflammatory cytokine IL-8 (CXCL8) stimulated VEGF production and secretion through activation of its cognate GPCR, CXCR2. This receptor, in turn, was linked to the CBM signaling complex and NF-B activation. Importantly, the effect of IL-8 on VEGF induction was independent of HIF-1 but entirely dependent on NF-B. Inhibition of any of the CBM components by siRNA was effective at reducing NF-B activation and resulted in a marked inhibition of VEGF mRNA expression and protein secretion. Further, this was accompanied by decreased autocrine VEGFR2 activation. These results suggest that the CBM signalosome is necessary for regulating VEGF production in endothelial cells in response to certain inflammatory cytokines, and might indirectly contribute to the VEGF-dependent transition of the endothelium into a proangiogenic phenotype.

#### **6.2.2 Thrombin and endothelial cell/monocyte adhesion**

As discussed earlier, thrombin and its receptor, PAR-1, are thought to induce a variety of pro-inflammatory responses that may also contribute to endothelial phenotype changes (Hirano, 2007). Thrombin levels are increased at sites of vascular injury and thrombosis, where the persistent stimulation of its receptor leads to endothelial dysfunction, thereby increasing inflammatory responses leading to further vessel wall damage and atherosclerotic lesion progression. One of the key responses to local thrombin production is the increase in endothelial cell expression of adhesion molecules, allowing for firm adhesion of circulating monocytes and other leukocytes (Minami et al., 2004). Our lab has specifically shown that thrombin-dependent activation of the CBM-NF-B signaling axis in endothelial cells results in upregulation of two such adhesion molecules, VCAM-1 and ICAM-1, at both the mRNA and protein levels (Delekta et al., 2010). Further, we showed that thrombininduced adhesion of monocytes to endothelial cells requires the intact CBM signalosome; siRNA-mediated knockdown of Bcl10 in endothelial cells altered their phenotype to completely abolish thrombin-dependent monocyte adherence. Further work will be required to test the role of the CBM proteins in modulating other aspects of thrombindependent endothelial dysfunction.

#### **6.2.3 Ang II and** *in vivo* **atherogenesis**

Similar to thrombin, Ang II produced locally in the vasculature has been reported to induce a number of inflammatory responses, including the expression of NF-B-sensitive adhesion molecules and cytokines in endothelial cells, and the recruitment of inflammatory cells to the vessel wall (Daugherty and Cassis, 2004). Our group recently showed that Ang II activation of its receptor, present on endothelial cells and on VSMCs, stimulates NF-B through the CBM signalosome (McAllister-Lucas et al., 2010).

Further, we tested the effects of manipulating the signalosome *in vivo*. In these studies, we utilized the *ApoE-/-* mouse strain described earlier, which is hyperlipidemic and prone to developing atherosclerosis. The development of lesions, however, can be dramatically

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 547

stimulated NF-B activation has been shown to require the CBM complex in MEFs and in ovarian cancer cells (Grabiner et al., 2007; Klemm et al., 2007; Mahanivong et al., 2008; Wang

Identification of the CBM signalosome as a critical mediator of GPCR-dependent proinflammatory effects suggests that pharmaceutical targeting of the CBM proteins could represent a new strategy for preventing or treating atherosclerosis. Since disruption of the CBM signalosome blocks NF-B activation and inflammatory signaling downstream of AGTR1, PAR-1, CXCR2 and probably other GPCRs within the vessel wall that contribute to endothelial dysfunction and atherogenesis, inhibiting vascular CBM activity may prove beneficial. Potential pharmaceutical approaches include: **1)** preventing specific upstream events that link GPCR stimulation to CBM activation, **2)** blocking key post-translational modifications of CBM components, and **3)** directly targeting the activity of CBM

The specific upstream molecular mechanisms by which GPCR stimulation promotes assembly and activation of the CBM have not yet been extensively investigated, and these mechanisms likely vary significantly depending on ligand, GPCR and cell type. However, there are some clues to potential therapeutic targets that could be critical for GPCR-induced CBM activation in the vasculature. For example, arrestin 2 associates with CARMA3, and studies thus far demonstrate that arrestin 2 is required for both LPA and thrombin to induce GPCR-dependent NF-B activation (Delekta et al., 2010; Sun and Lin, 2008). Intriguingly, recent studies demonstrate that in VSMCs, arrestin 2 mediates AGTR1 dependent prevention of apoptosis and is required for both LPA and thrombin-induced vascular smooth muscle cell proliferation (Ahn et al., 2009; Kim et al., 2008). Furthermore, deficiency of arrestin 2 protects LDL receptor knockout (*ldlr-/-*) mice from aortic atherosclerosis (Kim et al., 2008). Together, these studies suggest that somehow targeting arrestin 2 could represent a rational therapeutic strategy for preventing GPCR-dependent CBM activation and combating atherosclerosis. Precisely how to inhibit arrestin 2 dependent CBM activation remains to be investigated, but one potential approach would be to block GRK-mediated phosphorylation of GPCRs, thus preventing phosphorylationdependent recruitment of arrestin 2 to the GPCR (DeFea, 2011). In addition, "G-proteinbiased ligands" which selectively activate G-protein-mediated signaling downstream of specific GPCRs, while inhibiting arrestin-mediated signaling, are currently under development, and such agents may prove to be useful in modulating arrestin 2/CBM-

In addition to arrestin 2-mediated recruitment of CARMA3, another critical step in GPCRinduced CBM activation that represents a potential therapeutic target is the PKC-mediated phosphorylation of CARMA3. As discussed above, T-cell receptor (TCR) or B-cell receptor (BCR) stimulation induces PKC-dependent phosphorylation of CARMA1, thus causing a conformational change that allows CARMA1 to recruit Bcl10/MALT1 to the receptor and

et al., 2007), it is likely that the same will hold true for endothelial cells.

dependent vascular inflammatory disease (Whalen et al., 2011).

**7.2 Targets involved in CBM modification** 

**7. Therapeutic opportunities** 

components themselves.

**7.1 Upstream targets** 

accelerated through infusion with Ang II, even at subpressor doses. When infusions are carried out for as little as 4 weeks, the mice develop prominent and premature atherosclerotic lesions, even in the absence of a high-fat diet; these can be visualized grossly by staining the intimal surfaces of the aorta with Oil-red-O, a stain that reacts to lipid-laden lesions (Fig. 5). This effect of Ang II infusion is generally accepted to be the result of its direct pro-inflammatory effects on the vessel wall, which conspires with hyperlipidemia to cause accelerated atherogenesis. We tested the role of the CBM signalosome by crossing *ApoE-/-* and *Bcl10-/-* mice to generate a double knock-out line (McAllister-Lucas et al., 2010). The absence of Bcl10 in the ApoE-deficient strain revealed a dramatic phenotype in which the mice were protected from developing Ang II-induced atherosclerosis and aortic aneurysms (Fig. 5). Additionally, the reduction in atherosclerotic lesions in aortas from *ApoE-/-/Bcl10-/-* mice was associated with reduced aortic gene expression of several proinflammatory molecules, as compared to *ApoE-/-* mice infused with Ang II in the same way.

Fig. 5. Representative aortic arches from mice infused with Ang II for 4 weeks. Genotypes are as indicated. Aortas are stained with Oil-red-O to highlight lipid-laden intimal lesions (fatty streaks-advanced lesions). See text and McAllister-Lucas et al., 2010 for details.

#### **6.2.4 LPA and the CBM signalosome in atherogenesis?**

As mentioned, it is likely that other GPCRs will be linked to the CBM-NF-B signaling axis in endothelial cells, since several have already been linked in this way through work on other cell types. In particular, the receptors for LPA are likely to harness the CBM proteins in endothelial cells, considering their prominent role in affecting endothelial cell biology. A new study by Schober and colleagues demonstrates that LPA, produced via oxidation of LDL particles, enhances atherosclerotic lesion formation in *ApoE-/-* mice (Zhou et al., 2011). This effect is mediated largely via LPA receptor-dependent elaboration of CXCL1 (GRO-) on the surface of endothelial cells. CXCL1 is a chemokine that acts to promote monocyte recruitment to the endothelial wall, and thus plays a role in promoting atherogenesis. The authors showed that CXCL1 expression was in part NF-B dependent. Thus, since LPA-

stimulated NF-B activation has been shown to require the CBM complex in MEFs and in ovarian cancer cells (Grabiner et al., 2007; Klemm et al., 2007; Mahanivong et al., 2008; Wang et al., 2007), it is likely that the same will hold true for endothelial cells.

#### **7. Therapeutic opportunities**

546 Atherogenesis

accelerated through infusion with Ang II, even at subpressor doses. When infusions are carried out for as little as 4 weeks, the mice develop prominent and premature atherosclerotic lesions, even in the absence of a high-fat diet; these can be visualized grossly by staining the intimal surfaces of the aorta with Oil-red-O, a stain that reacts to lipid-laden lesions (Fig. 5). This effect of Ang II infusion is generally accepted to be the result of its direct pro-inflammatory effects on the vessel wall, which conspires with hyperlipidemia to cause accelerated atherogenesis. We tested the role of the CBM signalosome by crossing *ApoE-/-* and *Bcl10-/-* mice to generate a double knock-out line (McAllister-Lucas et al., 2010). The absence of Bcl10 in the ApoE-deficient strain revealed a dramatic phenotype in which the mice were protected from developing Ang II-induced atherosclerosis and aortic aneurysms (Fig. 5). Additionally, the reduction in atherosclerotic lesions in aortas from *ApoE-/-/Bcl10-/-* mice was associated with reduced aortic gene expression of several proinflammatory molecules, as compared to *ApoE-/-* mice infused with Ang II in the same way.

Fig. 5. Representative aortic arches from mice infused with Ang II for 4 weeks. Genotypes are as indicated. Aortas are stained with Oil-red-O to highlight lipid-laden intimal lesions (fatty streaks-advanced lesions). See text and McAllister-Lucas et al., 2010 for details.

As mentioned, it is likely that other GPCRs will be linked to the CBM-NF-B signaling axis in endothelial cells, since several have already been linked in this way through work on other cell types. In particular, the receptors for LPA are likely to harness the CBM proteins in endothelial cells, considering their prominent role in affecting endothelial cell biology. A new study by Schober and colleagues demonstrates that LPA, produced via oxidation of LDL particles, enhances atherosclerotic lesion formation in *ApoE-/-* mice (Zhou et al., 2011). This effect is mediated largely via LPA receptor-dependent elaboration of CXCL1 (GRO-) on the surface of endothelial cells. CXCL1 is a chemokine that acts to promote monocyte recruitment to the endothelial wall, and thus plays a role in promoting atherogenesis. The authors showed that CXCL1 expression was in part NF-B dependent. Thus, since LPA-

**6.2.4 LPA and the CBM signalosome in atherogenesis?** 

Identification of the CBM signalosome as a critical mediator of GPCR-dependent proinflammatory effects suggests that pharmaceutical targeting of the CBM proteins could represent a new strategy for preventing or treating atherosclerosis. Since disruption of the CBM signalosome blocks NF-B activation and inflammatory signaling downstream of AGTR1, PAR-1, CXCR2 and probably other GPCRs within the vessel wall that contribute to endothelial dysfunction and atherogenesis, inhibiting vascular CBM activity may prove beneficial. Potential pharmaceutical approaches include: **1)** preventing specific upstream events that link GPCR stimulation to CBM activation, **2)** blocking key post-translational modifications of CBM components, and **3)** directly targeting the activity of CBM components themselves.

#### **7.1 Upstream targets**

The specific upstream molecular mechanisms by which GPCR stimulation promotes assembly and activation of the CBM have not yet been extensively investigated, and these mechanisms likely vary significantly depending on ligand, GPCR and cell type. However, there are some clues to potential therapeutic targets that could be critical for GPCR-induced CBM activation in the vasculature. For example, arrestin 2 associates with CARMA3, and studies thus far demonstrate that arrestin 2 is required for both LPA and thrombin to induce GPCR-dependent NF-B activation (Delekta et al., 2010; Sun and Lin, 2008). Intriguingly, recent studies demonstrate that in VSMCs, arrestin 2 mediates AGTR1 dependent prevention of apoptosis and is required for both LPA and thrombin-induced vascular smooth muscle cell proliferation (Ahn et al., 2009; Kim et al., 2008). Furthermore, deficiency of arrestin 2 protects LDL receptor knockout (*ldlr-/-*) mice from aortic atherosclerosis (Kim et al., 2008). Together, these studies suggest that somehow targeting arrestin 2 could represent a rational therapeutic strategy for preventing GPCR-dependent CBM activation and combating atherosclerosis. Precisely how to inhibit arrestin 2 dependent CBM activation remains to be investigated, but one potential approach would be to block GRK-mediated phosphorylation of GPCRs, thus preventing phosphorylationdependent recruitment of arrestin 2 to the GPCR (DeFea, 2011). In addition, "G-proteinbiased ligands" which selectively activate G-protein-mediated signaling downstream of specific GPCRs, while inhibiting arrestin-mediated signaling, are currently under development, and such agents may prove to be useful in modulating arrestin 2/CBMdependent vascular inflammatory disease (Whalen et al., 2011).

#### **7.2 Targets involved in CBM modification**

In addition to arrestin 2-mediated recruitment of CARMA3, another critical step in GPCRinduced CBM activation that represents a potential therapeutic target is the PKC-mediated phosphorylation of CARMA3. As discussed above, T-cell receptor (TCR) or B-cell receptor (BCR) stimulation induces PKC-dependent phosphorylation of CARMA1, thus causing a conformational change that allows CARMA1 to recruit Bcl10/MALT1 to the receptor and

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 549

GPCR stimulation can induce MALT1-dependent cleavage of Bcl10 has not yet been investigated, and how Bcl10 cleavage might impact endothelial or VSMC function is totally unknown. TCR stimulation also induces the cleavage of A20 by MALT1, and this results in loss of A20's ability to inhibit TCR-dependent NF-B activation. It is speculated that A20 cleavage, which separates the N-terminal deubiquitination domain from the Cterminal substrate interaction domain, prevents the removal of activating K63-linked polyubiquitin from A20's substrates, which include TRAFs 2 and 6, Bcl10, IKK and MALT1 itself, and that preserving these activating ubiquitination events promotes NF-B activity. In this way, MALT1-dependent A20 cleavage can amplify the degree of NF-B dependent gene expression (Coornaert et al., 2008). Whether GPCRs within the vasculature such as AGTR1, PAR-1, CXCR2 or LPA receptors induce MALT1-dependent A20 cleavage is not known. Intriguingly, A20 appears to have a protective effect against atherosclerosis in both mice and humans. In *ApoE-/-* mice, A20 haploinsufficiency results in a significant increase in atherosclerosis compared to normal A20 controls, whereas transgenic overexpression of A20 results in decreased atherosclerosis (Wolfrum et al., 2007). Moreover, in human diabetic patients, polymorphisms at the *A20* locus leading to reduced levels of A20 expression are associated with increased coronary artery disease (Boonyasrisawat et al., 2007). Whether inhibition of MALT1-mediated cleavage of A20 might impact GPCR-driven atherogenesis remains to be investigated, but based on these studies, one might predict that preventing A20 cleavage could be protective. Initial studies suggest that MALT1-induced cleavage of CYLD is required for TCR-induced JNK activation (Staal et al., 2011), but MALT1-dependent CYLD cleavage has not been studied in the vasculature. Interestingly, CYLD overexpression attenuates neointimal formation in a rat model of carotid artery injury (Takami et al., 2008). How these studies of CYLD relate to GPCR/CBM-mediated atherogenesis remain to be investigated. Clearly much remains to be learned about the role of MALT1 proteolytic activity and its biologic affects. Future studies will hopefully elucidate whether MALT1 proteolytic activity contributes to vascular pathobiology and whether inhibiting this activity represents a rational

There is a pressing need to understand the molecular mechanisms underlying cardiovascular disease, as it is the leading cause of disease-related deaths worldwide. Atherosclerosis is a chronic inflammatory disease of the vasculature in which the proinflammatory transcription factor, NF-B, is a chief driving force. NF-B-dependent plaque formation is mediated via the expression of cytokines, chemokines, and adhesion molecules, and via changes in endothelial biology including a reduction in NO production. While many inducers of NF-B have been identified that act on endothelial cells, selective GPCR ligands clearly represent major players in the process of atherogenesis. Up until recently, little was known about the precise molecular mechanisms through which GPCRs communicate NF-B activation in endothelial cells. However, dramatic progress has now been made in understanding this process, and we have outlined much of this work here in this chapter. It is hoped that delineating the molecules mediating GPCR-dependent NF-B activation will provide new avenues for pharmaceutical development, adding a new layer of therapeutic

therapeutic approach to atherogenesis.

opportunity in our efforts to combat atherogenesis.

**8. Conclusion** 

form the CARMA1-Bcl10-MALT1 (CBM) complex (Matsumoto et al., 2005; Sommer et al., 2005). It is not yet known if a similar mechanism of PKC-induced CARMA3 phosphorylation occurs downstream of GPCRs, although it is well established that GPCR stimulation leads to phosphorylation and activation of various PKC isoforms and treatment with broad-spectrum PKC inhibitors can block GPCR-dependent NF-B activation. However, a pharmaceutical approach targeting PKC in atherogenesis is likely to be complex (Ding et al., 2011), since as we described earlier, each GPCR may utilize distinct PKC isoforms to communicate with the CBM complex. Nevertheless, progress is being made on this front; a recent report demonstrated that treatment with the DAG/calcium-dependent PKC inhibitor, Go6976, and siRNA-mediated silencing of PKC�both blocked AGTR1 dependent NF-B signaling in VSMCs (Doyon and Servant, 2010). Likewise, PKC inhibitors RO318220 and GF109203X have been shown to abrogate thrombin-dependent proinflammatory signaling in human aortic VSMCs (Chung et al., 2010). Perhaps the PKC isoform has been most thoroughly studied in the context of atherosclerosis. For example, genetic knockdown of PKC�or treatment with the PKC inhibitor, ruboxistaurin, results in decreased atherosclerosis in ApoE-deficient mice (Harja et al., 2009), and this same PKC inhibitor has also been shown to reduce endothelial dysfunction in human patients (Mehta et al., 2009). Because there is much evidence supporting a critical role for PKCs in atherogenesis and there are multiple isoform-specific PKC inhibitors already available, it will be of great interest to determine whether inhibition of particular PKCs blocks CBM activation by specific GPCRs within the vasculature and whether these effects are associated with a pharmaceutical benefit in the setting of atherosclerosis.

Like PKC-mediated phosphorylation of CARMA3, other post-translational modifications of CBM components may be critical to GPCR-induced CBM activity and could therefore represent potential targets for pharmaceutical intervention in GPCR-driven atherosclerosis. In lymphocytes, several kinases and phosphatases have been implicated in regulating the phosphorylation status of CARMA1 and Bcl10, and similarly, several ubiquitin ligases and deubiquitinases have been implicated in regulating the ubiquitination status of all three components of the CBM complex. In contrast to antigen receptor-dependent CBM activation in lymphocytes, GPCR-dependent regulation of the phosphorylation and ubiquitination of CARMA3, Bcl10 and MALT1 has not yet been investigated, although it seems likely that at least some of the same processes that regulate CBM activity in response to antigen receptor stimulation will also play a role in GPCR-dependent CBM regulation. Future studies may identify specific kinases, phosphatases, ubiquitin ligases and/or deubiquitinases that could be targeted in an effort to treat atherosclerosis by inhibiting GPCR/CBM pro-inflammatory activity in the vasculature.

#### **7.3 Targeting the enzymatic activity of the CBM signalosome itself**

MALT1, recently discovered to be a protease, is the only component of the CBM complex that is known to possess intrinsic enzymatic activity, and inhibition of MALT1 proteolytic activity may indeed represent a promising new therapeutic target for the treatment of atherosclerosis. As described in section 5.1, three proteolytic substrates for MALT1 have been identified so far: the MALT1 binding partner Bcl10, and the NF-B-inhibiting deubiquitinases A20 and CYLD (Coornaert et al., 2008; Rebeaud et al., 2008; Staal et al., 2011). In T-cells, MALT1-dependent cleavage of Bcl10 is induced by TCR stimulation and may play a role in integrin-mediated T-cell adhesion (Rebeaud et al., 2008). Whether

GPCR stimulation can induce MALT1-dependent cleavage of Bcl10 has not yet been investigated, and how Bcl10 cleavage might impact endothelial or VSMC function is totally unknown. TCR stimulation also induces the cleavage of A20 by MALT1, and this results in loss of A20's ability to inhibit TCR-dependent NF-B activation. It is speculated that A20 cleavage, which separates the N-terminal deubiquitination domain from the Cterminal substrate interaction domain, prevents the removal of activating K63-linked polyubiquitin from A20's substrates, which include TRAFs 2 and 6, Bcl10, IKK and MALT1 itself, and that preserving these activating ubiquitination events promotes NF-B activity. In this way, MALT1-dependent A20 cleavage can amplify the degree of NF-B dependent gene expression (Coornaert et al., 2008). Whether GPCRs within the vasculature such as AGTR1, PAR-1, CXCR2 or LPA receptors induce MALT1-dependent A20 cleavage is not known. Intriguingly, A20 appears to have a protective effect against atherosclerosis in both mice and humans. In *ApoE-/-* mice, A20 haploinsufficiency results in a significant increase in atherosclerosis compared to normal A20 controls, whereas transgenic overexpression of A20 results in decreased atherosclerosis (Wolfrum et al., 2007). Moreover, in human diabetic patients, polymorphisms at the *A20* locus leading to reduced levels of A20 expression are associated with increased coronary artery disease (Boonyasrisawat et al., 2007). Whether inhibition of MALT1-mediated cleavage of A20 might impact GPCR-driven atherogenesis remains to be investigated, but based on these studies, one might predict that preventing A20 cleavage could be protective. Initial studies suggest that MALT1-induced cleavage of CYLD is required for TCR-induced JNK activation (Staal et al., 2011), but MALT1-dependent CYLD cleavage has not been studied in the vasculature. Interestingly, CYLD overexpression attenuates neointimal formation in a rat model of carotid artery injury (Takami et al., 2008). How these studies of CYLD relate to GPCR/CBM-mediated atherogenesis remain to be investigated. Clearly much remains to be learned about the role of MALT1 proteolytic activity and its biologic affects. Future studies will hopefully elucidate whether MALT1 proteolytic activity contributes to vascular pathobiology and whether inhibiting this activity represents a rational therapeutic approach to atherogenesis.

#### **8. Conclusion**

548 Atherogenesis

form the CARMA1-Bcl10-MALT1 (CBM) complex (Matsumoto et al., 2005; Sommer et al., 2005). It is not yet known if a similar mechanism of PKC-induced CARMA3 phosphorylation occurs downstream of GPCRs, although it is well established that GPCR stimulation leads to phosphorylation and activation of various PKC isoforms and treatment with broad-spectrum PKC inhibitors can block GPCR-dependent NF-B activation. However, a pharmaceutical approach targeting PKC in atherogenesis is likely to be complex (Ding et al., 2011), since as we described earlier, each GPCR may utilize distinct PKC isoforms to communicate with the CBM complex. Nevertheless, progress is being made on this front; a recent report demonstrated that treatment with the DAG/calcium-dependent PKC inhibitor, Go6976, and siRNA-mediated silencing of PKC�both blocked AGTR1 dependent NF-B signaling in VSMCs (Doyon and Servant, 2010). Likewise, PKC inhibitors RO318220 and GF109203X have been shown to abrogate thrombin-dependent proinflammatory signaling in human aortic VSMCs (Chung et al., 2010). Perhaps the PKC isoform has been most thoroughly studied in the context of atherosclerosis. For example, genetic knockdown of PKC�or treatment with the PKC inhibitor, ruboxistaurin, results in decreased atherosclerosis in ApoE-deficient mice (Harja et al., 2009), and this same PKC inhibitor has also been shown to reduce endothelial dysfunction in human patients (Mehta et al., 2009). Because there is much evidence supporting a critical role for PKCs in atherogenesis and there are multiple isoform-specific PKC inhibitors already available, it will be of great interest to determine whether inhibition of particular PKCs blocks CBM activation by specific GPCRs within the vasculature and whether these effects are associated

Like PKC-mediated phosphorylation of CARMA3, other post-translational modifications of CBM components may be critical to GPCR-induced CBM activity and could therefore represent potential targets for pharmaceutical intervention in GPCR-driven atherosclerosis. In lymphocytes, several kinases and phosphatases have been implicated in regulating the phosphorylation status of CARMA1 and Bcl10, and similarly, several ubiquitin ligases and deubiquitinases have been implicated in regulating the ubiquitination status of all three components of the CBM complex. In contrast to antigen receptor-dependent CBM activation in lymphocytes, GPCR-dependent regulation of the phosphorylation and ubiquitination of CARMA3, Bcl10 and MALT1 has not yet been investigated, although it seems likely that at least some of the same processes that regulate CBM activity in response to antigen receptor stimulation will also play a role in GPCR-dependent CBM regulation. Future studies may identify specific kinases, phosphatases, ubiquitin ligases and/or deubiquitinases that could be targeted in an effort to treat atherosclerosis by inhibiting GPCR/CBM pro-inflammatory

MALT1, recently discovered to be a protease, is the only component of the CBM complex that is known to possess intrinsic enzymatic activity, and inhibition of MALT1 proteolytic activity may indeed represent a promising new therapeutic target for the treatment of atherosclerosis. As described in section 5.1, three proteolytic substrates for MALT1 have been identified so far: the MALT1 binding partner Bcl10, and the NF-B-inhibiting deubiquitinases A20 and CYLD (Coornaert et al., 2008; Rebeaud et al., 2008; Staal et al., 2011). In T-cells, MALT1-dependent cleavage of Bcl10 is induced by TCR stimulation and may play a role in integrin-mediated T-cell adhesion (Rebeaud et al., 2008). Whether

with a pharmaceutical benefit in the setting of atherosclerosis.

**7.3 Targeting the enzymatic activity of the CBM signalosome itself** 

activity in the vasculature.

There is a pressing need to understand the molecular mechanisms underlying cardiovascular disease, as it is the leading cause of disease-related deaths worldwide. Atherosclerosis is a chronic inflammatory disease of the vasculature in which the proinflammatory transcription factor, NF-B, is a chief driving force. NF-B-dependent plaque formation is mediated via the expression of cytokines, chemokines, and adhesion molecules, and via changes in endothelial biology including a reduction in NO production. While many inducers of NF-B have been identified that act on endothelial cells, selective GPCR ligands clearly represent major players in the process of atherogenesis. Up until recently, little was known about the precise molecular mechanisms through which GPCRs communicate NF-B activation in endothelial cells. However, dramatic progress has now been made in understanding this process, and we have outlined much of this work here in this chapter. It is hoped that delineating the molecules mediating GPCR-dependent NF-B activation will provide new avenues for pharmaceutical development, adding a new layer of therapeutic opportunity in our efforts to combat atherogenesis.

G Protein-Coupled Receptor Dependent NF-κB Signaling in Atherogenesis 551

Chung, S.W., Park, J.W., Lee, S.A., Eo, S.K., & Kim, K. (2010). Thrombin promotes

Coornaert, B., Baens, M., Heyninck, K., Bekaert, T., Haegman, M., Staal, J., Sun, L., Chen,

Coughlin, S.R. (2005). Protease-activated receptors in hemostasis, thrombosis and vascular

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This work was supported by grants from the National Institutes of Health (NIH), U.S.A., R01HL082914 and R01DK079973.

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**26** 

*Japan* 

**Vasoprotective Effect of Foods as Treatments:** 

Collagen is a major protein in living organisms and accounts for about one-third of all protein in mammalian bodies, including the human body. Recently, collagen peptides have been used as foods that take advantage of their tertiary functions. We have been focusing on

Chicken collagen hydrolysate (CCH) is obtained by treating chicken feet with enzymes to produce an angiotensin-converting enzyme (ACE) inhibitory peptide. Administration of this CCH for 12 weeks reduces blood pressure in humans. We therefore investigated the mechanism of the vasoprotective effect of CCH. We tested whether prolonged CCH treatment of rats or mice would restore endothelial cell function and improve proinflammatory cytokine levels. We found that CCH treatment improved the vasorelaxation of rat aorta damaged with L-NG-nitroarginine methyl ester , an NO synthesis inhibitor. CCH treatment also reduced the serum levels of IL-6, sICAM-1, and TNF-α in an

These findings indicate the usefulness of collagen peptides as foods promoting anti-

Years have passed since functional foods and their tertiary function first attracted attention. The primary function of foods is to supply the nutrients required to sustain life, and the secondary function is to satisfy taste preferences. The tertiary function of foods is to exert biological regulatory effects, such as biophylaxis, homeostatic maintenance, and disease prevention, which are activated upon food intake. Purified food ingredients that have tertiary functions are widely consumed as supplements. Multitudes of supplements are available on today's market: besides common vitamins, minerals, and amino acids, there are catechins, which are antioxidant constituents of tea (Katiyar, 2003), soy isoflavones, which have female hormone–like actions (Weijer, 2002), and docosahexaenoic acids and eicosapentaenoic acids, which decrease triglyceride levels (Tamai, 2004). Collagen is being used widely, not only in supplements but

also as an ingredient of common food products such as beverages, yogurts, and breads.

Collagen is a major protein in living organisms and accounts for about one-third of all protein in mammalian bodies, including the human body. It forms an extracellular matrix that plays a role in the formation of connective tissues and acts as a scaffold for cells, but its accumulation declines with age. The majority of the collagen in the body exhibits a triple

**1. Introduction** 

the vasoprotective effect of collagen peptides.

atherosclerotic mouse model, C57BL/6.KOR-ApoEsh1.

atherogenesis via a vasoprotective effect.

**Chicken Collagen Hydrolysate** 

Mikako Sato, Yoshihisa Takahata and Fumiki Morimatsu

Tomomi Kouguchi, Youzuo Zhang,

*Nippon Meat Packers, Inc., R & D Center* 


### **Vasoprotective Effect of Foods as Treatments: Chicken Collagen Hydrolysate**

Tomomi Kouguchi, Youzuo Zhang, Mikako Sato, Yoshihisa Takahata and Fumiki Morimatsu *Nippon Meat Packers, Inc., R & D Center Japan* 

#### **1. Introduction**

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V.M. (2000). Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. *Mol* 

plays a critical role in NF-kappaB activation induced by G protein-coupled

associated guanylate kinase family member that interacts with BCL10 and activates

Induction of the NF-kappaB cascade by recruitment of the scaffold molecule

A20 on atherosclerosis in apolipoprotein E-deficient mice is associated with reduced expression of NF-kappaB target genes. *Proc Natl Acad Sci U S A.* 104,

(2004). Bcl10 activates the NF-kappaB pathway through ubiquitination of NEMO.

TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. *Mol* 

kappaB activation. *Proc Natl Acad Sci U S A.* 105, 17085-17090

*Cell.* 14, 289-301

*Cell.* 6, 961-967

6735-6741

18601-18606

*Nature.* 427, 167-171

activation. *Cell Signal.* 21, 1488-1494

activation. *Nat Rev Immunol.* 4, 348-359

enzyme in vascular cells. *Am J Pathol.* 172, 818-829

receptors. *Proc Natl Acad Sci U S A.* 104, 145-150

NEMO to the T cell receptor. *Immunity.* 18, 13-26

and G protein-biased agonists. *Trends Mol Med.* 17, 126-139

NF-kappa B. *J Biol Chem.* 276, 21405-21409

kappaB. *Sci STKE.* 2007, pe21

Collagen is a major protein in living organisms and accounts for about one-third of all protein in mammalian bodies, including the human body. Recently, collagen peptides have been used as foods that take advantage of their tertiary functions. We have been focusing on the vasoprotective effect of collagen peptides.

Chicken collagen hydrolysate (CCH) is obtained by treating chicken feet with enzymes to produce an angiotensin-converting enzyme (ACE) inhibitory peptide. Administration of this CCH for 12 weeks reduces blood pressure in humans. We therefore investigated the mechanism of the vasoprotective effect of CCH. We tested whether prolonged CCH treatment of rats or mice would restore endothelial cell function and improve proinflammatory cytokine levels. We found that CCH treatment improved the vasorelaxation of rat aorta damaged with L-NG-nitroarginine methyl ester , an NO synthesis inhibitor. CCH treatment also reduced the serum levels of IL-6, sICAM-1, and TNF-α in an atherosclerotic mouse model, C57BL/6.KOR-ApoEsh1.

These findings indicate the usefulness of collagen peptides as foods promoting antiatherogenesis via a vasoprotective effect.

Years have passed since functional foods and their tertiary function first attracted attention. The primary function of foods is to supply the nutrients required to sustain life, and the secondary function is to satisfy taste preferences. The tertiary function of foods is to exert biological regulatory effects, such as biophylaxis, homeostatic maintenance, and disease prevention, which are activated upon food intake. Purified food ingredients that have tertiary functions are widely consumed as supplements. Multitudes of supplements are available on today's market: besides common vitamins, minerals, and amino acids, there are catechins, which are antioxidant constituents of tea (Katiyar, 2003), soy isoflavones, which have female hormone–like actions (Weijer, 2002), and docosahexaenoic acids and eicosapentaenoic acids, which decrease triglyceride levels (Tamai, 2004). Collagen is being used widely, not only in supplements but also as an ingredient of common food products such as beverages, yogurts, and breads.

Collagen is a major protein in living organisms and accounts for about one-third of all protein in mammalian bodies, including the human body. It forms an extracellular matrix that plays a role in the formation of connective tissues and acts as a scaffold for cells, but its accumulation declines with age. The majority of the collagen in the body exhibits a triple

Vasoprotective Effect of Foods as Treatments: Chicken Collagen Hydrolysate 559

weigh 1.5 kg; they are therefore as heavy as the liver. Although blood vessels were once seen as simply the "pipes" that circulate blood, it has become increasingly clear that the vascular endothelial cells receive signals from organs and control blood supply and the secretion of

There are many diseases caused by vascular abnormalities, especially in Japan. According to the cause-specific death rates reported by the Ministry of Health, Labor, and Welfare of Japan in 2006, death rates due to circulatory system diseases are extremely high: after malignant neoplasms (30.4%), cardiovascular diseases account for 15.9% of all deaths and cerebrovascular diseases account for 11.8%. From this perspective, protecting the blood vessels from disease should increase the quality of life of many people. We therefore took advantage of the absorbability of collagen peptides and aimed to develop ones targeting the

Collagen peptides are generally extracted from pig skin or fish scales. However, here we used chicken legs as sources of the new collagen peptides. This was because, although gelatin is known to be allergenic, our previous study showed that the allergenicity of

Chicken legs were solubilized by acid treatment and the extracted collagen was processed by proteases. The resulting low-molecular-weight collagen peptides were then dried and powdered for subsequent use as low-molecular-weight chicken collagen hydrolysate (CCH) (Saiga, 2008) (Fig. 2). Our preliminary *in vitro* experiments showed that CCH strongly inhibits angiotensin-converting enzyme (ACE). Production of angiotensin II, a vasopressor, is suppressed by the inhibition of ACE in the blood and organs, thereby resulting in a hypotensive effect (Gupta, 2010). Because hypertension is closely related to arteriosclerosis, the inhibition of blood pressure elevation is expected to have a protective effect on the blood vessels. In addition, ACE serves as a kininase II (Sharma, 2009). Because kininase II degrades bradykinin, a vasodilator, inhibition of ACE (or kininase II) by CCH causes bradykinin accumulation in the body. Bradykinin activates endothelial nitric oxide synthase (eNOS) and increases the production of nitric oxide (NO), a vasodilator. In this manner, CCH was expected to have a vasoprotective function—a novel tertiary function of foods—

**1.3 Development of a low-molecular-weight chicken collagen hydrolysate** 

chicken-derived gelatin is the lowest among a number of types (Taguchi, 2002).

**2. Hypotensive effects of chicken collagen hydrolysate in subjects with** 

Arteriosclerosis and hypertension are closely associated with each other. If strong pressure is applied continuously to an artery because of hypertension, the arterial walls are damaged and blood cholesterols infiltrate the walls through the damaged areas and cause arteriosclerosis. In addition, advanced arteriosclerosis narrows the blood vessels and causes blood flow to deteriorate. The heartbeat is then enhanced to improve blood flow, and this causes the blood pressure to increase. In this manner, hypertension accelerates arteriosclerosis and produces a vicious cycle. If we could alleviate hypertension, we would thus also be able to ameliorate arteriosclerosis. We therefore initially conducted a clinical trial to verify the hypotensive effect of CCH in humans

various cytokines on demand (Kato, 2004).

protection of blood vessels.

through its ACE inhibitory activity.

**hypertension** 

(Kouguchi, 2008).

helix structure; with heating, this structure is lost and the collagen becomes gelatin like. Moreover, as a result of enzymatic degradation that eliminates its gelation ability, the gelatin increases in solubility and becomes collagen peptides (Fig. 1), which are frequently consumed by women, in particular. Collagen peptides are consumed as a food product to supply the collagen lost from the body with age, and a substantial number of reports have shown that treatment with collagen peptides increases well-being.

Fig. 1. Collagen in the body exhibits a triple helix structure but is denatured and becomes gelatin-like if heated. Enzyme treatment of denatured collagen produces collagen peptides, which are composed of atypical repetitions of -Gly-X-Y-Gly-X-Y- and are consumed as functional foods.

#### **1.1 The tertiary function of collagen peptides**

In recent years, vigorous research has been conducted to elucidate both the mechanism by which collagen peptides are absorbed from food into the body and the tertiary functions of this protein. Orally administered collagen peptides are transferred to the blood in the form of dipeptides or tripeptides, without being completely degraded to amino acids (Iwai, 2009 and Shigemura,2009). A double-blind placebo-controlled trial has confirmed that collagen peptide treatment increases the skin's moisture content (Ohara, 2009). The primary structure of collagen consists of atypical repetitions of -Gly-X-Y- and characteristically includes hydroxyproline, which is produced by posttranslational modification. Many studies have suggested that this particular sequence enables collagen to exert multiple bioactivities, not only in skin and bones, but also in blood vessels, which contain large amounts of collagen (Arborelius, 1999). Accordingly, collagen peptides are expected to have tertiary functions additional to those already known.

#### **1.2 Targeting blood vessels**

The blood vessels are referred to as the largest organ in the body, because the vascular endothelial cells, which line the vessel lumens, cover an area as large as six tennis courts and

helix structure; with heating, this structure is lost and the collagen becomes gelatin like. Moreover, as a result of enzymatic degradation that eliminates its gelation ability, the gelatin increases in solubility and becomes collagen peptides (Fig. 1), which are frequently consumed by women, in particular. Collagen peptides are consumed as a food product to supply the collagen lost from the body with age, and a substantial number of reports have

Collagen

Gelatin

**Collagen peptides**

shown that treatment with collagen peptides increases well-being.

Heat treatment

Enzymatic digestion

Fig. 1. Collagen in the body exhibits a triple helix structure but is denatured and becomes gelatin-like if heated. Enzyme treatment of denatured collagen produces collagen peptides, which are composed of atypical repetitions of -Gly-X-Y-Gly-X-Y- and are consumed as

In recent years, vigorous research has been conducted to elucidate both the mechanism by which collagen peptides are absorbed from food into the body and the tertiary functions of this protein. Orally administered collagen peptides are transferred to the blood in the form of dipeptides or tripeptides, without being completely degraded to amino acids (Iwai, 2009 and Shigemura,2009). A double-blind placebo-controlled trial has confirmed that collagen peptide treatment increases the skin's moisture content (Ohara, 2009). The primary structure of collagen consists of atypical repetitions of -Gly-X-Y- and characteristically includes hydroxyproline, which is produced by posttranslational modification. Many studies have suggested that this particular sequence enables collagen to exert multiple bioactivities, not only in skin and bones, but also in blood vessels, which contain large amounts of collagen (Arborelius, 1999). Accordingly, collagen peptides are expected to have tertiary functions

The blood vessels are referred to as the largest organ in the body, because the vascular endothelial cells, which line the vessel lumens, cover an area as large as six tennis courts and

Triple helix

Denaturation

Digestion

functional foods.

**Gly Gly X Y X Y**

additional to those already known.

**1.2 Targeting blood vessels** 

**1.1 The tertiary function of collagen peptides** 

weigh 1.5 kg; they are therefore as heavy as the liver. Although blood vessels were once seen as simply the "pipes" that circulate blood, it has become increasingly clear that the vascular endothelial cells receive signals from organs and control blood supply and the secretion of various cytokines on demand (Kato, 2004).

There are many diseases caused by vascular abnormalities, especially in Japan. According to the cause-specific death rates reported by the Ministry of Health, Labor, and Welfare of Japan in 2006, death rates due to circulatory system diseases are extremely high: after malignant neoplasms (30.4%), cardiovascular diseases account for 15.9% of all deaths and cerebrovascular diseases account for 11.8%. From this perspective, protecting the blood vessels from disease should increase the quality of life of many people. We therefore took advantage of the absorbability of collagen peptides and aimed to develop ones targeting the protection of blood vessels.

#### **1.3 Development of a low-molecular-weight chicken collagen hydrolysate**

Collagen peptides are generally extracted from pig skin or fish scales. However, here we used chicken legs as sources of the new collagen peptides. This was because, although gelatin is known to be allergenic, our previous study showed that the allergenicity of chicken-derived gelatin is the lowest among a number of types (Taguchi, 2002).

Chicken legs were solubilized by acid treatment and the extracted collagen was processed by proteases. The resulting low-molecular-weight collagen peptides were then dried and powdered for subsequent use as low-molecular-weight chicken collagen hydrolysate (CCH) (Saiga, 2008) (Fig. 2). Our preliminary *in vitro* experiments showed that CCH strongly inhibits angiotensin-converting enzyme (ACE). Production of angiotensin II, a vasopressor, is suppressed by the inhibition of ACE in the blood and organs, thereby resulting in a hypotensive effect (Gupta, 2010). Because hypertension is closely related to arteriosclerosis, the inhibition of blood pressure elevation is expected to have a protective effect on the blood vessels. In addition, ACE serves as a kininase II (Sharma, 2009). Because kininase II degrades bradykinin, a vasodilator, inhibition of ACE (or kininase II) by CCH causes bradykinin accumulation in the body. Bradykinin activates endothelial nitric oxide synthase (eNOS) and increases the production of nitric oxide (NO), a vasodilator. In this manner, CCH was expected to have a vasoprotective function—a novel tertiary function of foods through its ACE inhibitory activity.

#### **2. Hypotensive effects of chicken collagen hydrolysate in subjects with hypertension**

Arteriosclerosis and hypertension are closely associated with each other. If strong pressure is applied continuously to an artery because of hypertension, the arterial walls are damaged and blood cholesterols infiltrate the walls through the damaged areas and cause arteriosclerosis. In addition, advanced arteriosclerosis narrows the blood vessels and causes blood flow to deteriorate. The heartbeat is then enhanced to improve blood flow, and this causes the blood pressure to increase. In this manner, hypertension accelerates arteriosclerosis and produces a vicious cycle. If we could alleviate hypertension, we would thus also be able to ameliorate arteriosclerosis. We therefore initially conducted a clinical trial to verify the hypotensive effect of CCH in humans (Kouguchi, 2008).

Vasoprotective Effect of Foods as Treatments: Chicken Collagen Hydrolysate 561

CCH at 2.9 g/day


Fig. 3. Clinical trial schedule for CCH administration. The 120 subjects were assigned to two

Blood pressure was measured a total of 11 times in the course of the experiment: twice in the pre-treatment observation period, 7 times in the treatment period, and twice in the posttreatment observation period. The subjects were kept at rest for at least 10 min before the measurement. Blood pressure in the left cubital fossa was measured while the subjects were seated. Blood pressure was measured more than once with a mercury manometer. The average value of 2 stable measurements (i.e. when the difference of the values was less than 5 mmHg) was recorded as the value recorded. Pulse rate was measured once at each visit. The subjects' condition was also interviewed by a doctor at the time of measurement of

Systolic blood pressures in the test food group were non-significantly lower (*P* < 0.1) than those of the placebo group after 2 weeks of treatment and were significantly lower (*P* < 0.05) than in the placebo group after 12 weeks of treatment (Fig. 4). In the test food group, in comparison with the mean pre-treatment blood pressure (139.7 mm Hg), the blood pressure was significantly lower after 2 weeks (133.9 mm Hg; *P* < 0.001), 4 weeks (135.7 mm Hg; *P* < 0.01), 6 weeks (134.6 mm Hg; *P* < 0.001), 8 weeks (134.4 mm Hg; *P* < 0.01), 10 weeks (134.6 mm Hg; *P* < 0.001), and 12 weeks (133.5 mm Hg; *P* < 0.001). After 2 weeks of treatment, the blood pressure in the test food group was 135.5 mm Hg; this was non-significantly lower than the pre-treatment blood pressure (*P* < 0.1). In the placebo group, blood pressure after 6 weeks of treatment (135.9 mm Hg) was significantly lower than the pre-treatment blood

Blood pressures in the test food group decreased continuously during the treatment period. Because the compositional difference between the test food and placebo in this experiment was only the presence or the absence of CCH, the observed antihypertensive effect was considered to be due to CCH treatment. We had previously confirmed that CCH exhibits ACE inhibitory activity and antihypertensive effects in rats (Saiga, 2008). The results of this study indicated that CCH had a similar antihypertensive effect in humans. Moreover, on

12weeks

groups and given the experimental or placebo diet for 12 weeks. Blood pressure was measured a total of 11 times in the course of the experiment: twice in the pre-treatment observation period, 7 times in the treatment period, and twice in the post-treatment

or Placebo

60 subjects 60 subjects

Post-treatment observation period

+4 weeks

and : Measurement of blood pressure

Pre-treatment observation period


observation period.

blood pressure.

**2.6 Discussion** 

**2.5 Results** 

Treatment period

0

**2.4 Measurement of blood pressure** 

pressure (139.8 mm Hg) (*P* < 0.05).

Extract in hot water Drying and powderization Protease treatment Chicken legs Chicken collagen hydrolysate ; CCH Acid treatment

Fig. 2. Process of production of chicken collagen hydrolysate (CCH). Chicken legs are used as the basic ingredient and are treated with acid and then hot water to extract collagen, which was then processed with proteases. The resulting low-molecular-weight collagen peptides are then dried and powered for subsequent use as CCH.

#### **2.1 Subjects**

Subjects for the test were 120 healthy, antihypertensive drug–free, adult males and females with mild hypertension or high-normal blood pressure. The subjects (males, 59; females, 61) were randomly assigned to two groups. No significant differences in subject characteristics, including sex, age, height, body weight, body mass index, systolic blood pressure, diastolic blood pressure, and pulse rate, were observed between the two groups (P > 0.2).

The study was approved by the institutional review board and was performed under the close supervision of the study investigators. The subjects were well informed about the test contents and methods by the study investigators, and they provided written informed consent to protect their rights in accordance with the spirit of the Declaration of Helsinki.

#### **2.2 Experimental diets**

A drink containing CCH (hereafter, referred to as the test food) or its counterpart without CCH (hereafter, referred to as the placebo) was used in the experiment. The test food contained 2.9 g of CCH; for the placebo, the raw material composition was the same as that of the test food, but without the CCH.

#### **2.3 Trial design**

The trial was designed as a placebo-controlled, double-blind, parallel-group comparison study. The study ran for a total of 18 weeks: 2 observational weeks before the treatment (pre-treatment observation period), a 12-week treatment period, and 4 weeks for posttreatment observation (post-treatment observation period). All subjects were given a bottle of drink daily during the treatment period. All subjects were directed not to change their daily diets and exercise regimens (Fig.3). They were advised strongly not to overeat, overdrink, or over-exercise.

and : Measurement of blood pressure

Fig. 3. Clinical trial schedule for CCH administration. The 120 subjects were assigned to two groups and given the experimental or placebo diet for 12 weeks. Blood pressure was measured a total of 11 times in the course of the experiment: twice in the pre-treatment observation period, 7 times in the treatment period, and twice in the post-treatment observation period.

#### **2.4 Measurement of blood pressure**

Blood pressure was measured a total of 11 times in the course of the experiment: twice in the pre-treatment observation period, 7 times in the treatment period, and twice in the posttreatment observation period. The subjects were kept at rest for at least 10 min before the measurement. Blood pressure in the left cubital fossa was measured while the subjects were seated. Blood pressure was measured more than once with a mercury manometer. The average value of 2 stable measurements (i.e. when the difference of the values was less than 5 mmHg) was recorded as the value recorded. Pulse rate was measured once at each visit. The subjects' condition was also interviewed by a doctor at the time of measurement of blood pressure.

#### **2.5 Results**

560 Atherogenesis

Chicken legs

Fig. 2. Process of production of chicken collagen hydrolysate (CCH). Chicken legs are used as the basic ingredient and are treated with acid and then hot water to extract collagen, which was then processed with proteases. The resulting low-molecular-weight collagen

Subjects for the test were 120 healthy, antihypertensive drug–free, adult males and females with mild hypertension or high-normal blood pressure. The subjects (males, 59; females, 61) were randomly assigned to two groups. No significant differences in subject characteristics, including sex, age, height, body weight, body mass index, systolic blood pressure, diastolic

The study was approved by the institutional review board and was performed under the close supervision of the study investigators. The subjects were well informed about the test contents and methods by the study investigators, and they provided written informed consent to protect their rights in accordance with the spirit of the Declaration

A drink containing CCH (hereafter, referred to as the test food) or its counterpart without CCH (hereafter, referred to as the placebo) was used in the experiment. The test food contained 2.9 g of CCH; for the placebo, the raw material composition was the same as that

The trial was designed as a placebo-controlled, double-blind, parallel-group comparison study. The study ran for a total of 18 weeks: 2 observational weeks before the treatment (pre-treatment observation period), a 12-week treatment period, and 4 weeks for posttreatment observation (post-treatment observation period). All subjects were given a bottle of drink daily during the treatment period. All subjects were directed not to change their daily diets and exercise regimens (Fig.3). They were advised strongly not to overeat, over-

blood pressure, and pulse rate, were observed between the two groups (P > 0.2).

peptides are then dried and powered for subsequent use as CCH.

**2.1 Subjects** 

of Helsinki.

**2.3 Trial design** 

drink, or over-exercise.

**2.2 Experimental diets** 

of the test food, but without the CCH.

Extract in hot water

Acid treatment

Protease treatment

Drying and powderization

Chicken collagen hydrolysate ; CCH

Systolic blood pressures in the test food group were non-significantly lower (*P* < 0.1) than those of the placebo group after 2 weeks of treatment and were significantly lower (*P* < 0.05) than in the placebo group after 12 weeks of treatment (Fig. 4). In the test food group, in comparison with the mean pre-treatment blood pressure (139.7 mm Hg), the blood pressure was significantly lower after 2 weeks (133.9 mm Hg; *P* < 0.001), 4 weeks (135.7 mm Hg; *P* < 0.01), 6 weeks (134.6 mm Hg; *P* < 0.001), 8 weeks (134.4 mm Hg; *P* < 0.01), 10 weeks (134.6 mm Hg; *P* < 0.001), and 12 weeks (133.5 mm Hg; *P* < 0.001). After 2 weeks of treatment, the blood pressure in the test food group was 135.5 mm Hg; this was non-significantly lower than the pre-treatment blood pressure (*P* < 0.1). In the placebo group, blood pressure after 6 weeks of treatment (135.9 mm Hg) was significantly lower than the pre-treatment blood pressure (139.8 mm Hg) (*P* < 0.05).

#### **2.6 Discussion**

Blood pressures in the test food group decreased continuously during the treatment period. Because the compositional difference between the test food and placebo in this experiment was only the presence or the absence of CCH, the observed antihypertensive effect was considered to be due to CCH treatment. We had previously confirmed that CCH exhibits ACE inhibitory activity and antihypertensive effects in rats (Saiga, 2008). The results of this study indicated that CCH had a similar antihypertensive effect in humans. Moreover, on

Vasoprotective Effect of Foods as Treatments: Chicken Collagen Hydrolysate 563

The clinical trial described in the preceding section suggested that CCH protects the blood vessels by inducing NO production. Therefore, we next directly investigated the vasodilatory effect of CCH *ex vivo* by using rat blood vessels. We administered L-NGnitroarginine methyl ester (L-NAME), an NO synthesis inhibitor, to rats to trigger vascular endothelial dysfunction. We then tested whether prolonged CCH treatment of the rats

Thirty-six male WKY rats (10 weeks old) were randomly allocated to three groups. The first group (control group) received untreated chow and drinking water. The second group (L-NAME group) received L-NAME in their drinking water (0.5 g/L) for 8 weeks. The third group (L-NAME+CCH group) received L-NAME in their drinking water and CCH (2.0 g/kg daily) via a metal oral Zonde needle. All animal procedures were performed in accordance with the Animal Experimentation Guidelines of the Japanese Association for Laboratory Animal Science and were approved by the Animal Use and Care Committee of

A vasorelaxation assay was performed on the tissue of eight or nine rats from each group after 8 weeks of treatment. The rats were anesthetized with diethyl ether and the thoracic aorta was removed. The surrounding connective tissue and fat were carefully removed from the thoracic aorta, which was then cut into 2- to 3-mm-wide rings. Segments of thoracic aorta were mounted between two steel hooks in isolated tissue chambers containing Krebs-Henseleit solution at 37 °C. The isometric tension was recorded with an isometric forcedisplacement transducer. After an equilibration period, L-norepinephrine bitartrate was added to cause contraction. This was followed by the addition of cumulative doses of acetylcholine chloride to the bath solution to produce relaxation. Vascular relaxation was

After 8 weeks of treatment, the survival rate of the L-NAME group rats, which had received L-NAME in their drinking water, was 66.7% of that of the control rats. However, rats that had ingested CCH (L-NAME+CCH group) had a significantly better survival rate (91.7% of that of the control group) than the L-NAME rats (*P* < 0.05) (Fig.6). During all of the experiments, monitoring revealed that the rats drank 17 to 30 mL of water and ate 16 to 30 g of chow every day, confirming that their drinking and eating patterns were unaffected by the treatment protocols. Body weight gains did not differ among groups (data not shown). We measured the vasorelaxant effects of CCH treatment after 8 weeks of treatment (Fig. 7). Treatment with acetylcholine chloride caused concentration-dependent relaxation of the thoracic aorta preparations from all groups after the preparations had been caused to contract by the addition of L-norepinephrine bitartrate. The acetylcholine chloride induced a relaxation response in the thoracic aortas from the L-NAME group (12.7% vasorelaxation); this was significantly less than that in preparations from the control group (69.5%).

**3. CCH treatment improves vascular endothelial function in rats and thus** 

**exerts protective effects on organs** 

**3.1 Experimental animals** 

Nippon Meat Packers, Inc.

**3.2 Vasorelaxation assay** 

**3.3 Results** 

would restore their endothelial function (Zhang, 2010).

expressed as a percentage of tension development (Fig.5).

medical examination some subjects reported a dry cough. Dry cough is typically observed with ACE inhibitor administration and is attributed to bradykinin accumulation in the body. This raises the possibility that the CCH inhibited kininase II and thus caused accumulation of bradykinin, a vasoprotector, which then induced NO production via the stimulation of eNOS. The results suggest that CCH exerts vasoprotective effects by ameliorating blood pressure in humans.

Fig. 4. Time-course of changes in systolic blood pressure in the subjects. Systolic blood pressures in the test food group were non-significantly lower than those in the placebo group after 2 weeks of treatment and were significantly lower than in the placebo group after 12 weeks of treatment. In comparison with the pre-treatment blood pressure (mean of the values at −2, −1, and 0 weeks), the blood pressure in the test food group was consistently and significantly lower throughout the treatment period. Data are mean ± SE values . + *P*< 0.1, *# P*< 0.05 versus placebo group. † *P*< 0.1, \**P*< 0.05, \*\* *P*< 0.01, \*\*\* *P*< 0.001 versus pre-treatment blood pressure.

In our previous *in vitro* studies, we found that CCH treatment of human umbilical vein endothelial cells directly increased eNOS activation (data not shown). When eNOS expressed in vascular endothelial cells is activated, the cells produce NO. The NO functions as a signal to relax adjacent vascular smooth muscle cells; consequently, this dilates arteries and increases blood flow. Other than NO, vascular endothelial cells excrete vasoactive substances such as endothelin, a vasopressor, and maintain the balance of constriction and dilation of blood vessels. NO production via eNOS is particularly important in maintaining the homeostasis of blood vessels. Taken together, these findings indicate that oral administration of CCH improves blood pressure by inhibiting ACE and protects blood vessels by inducing NO production, thereby inhibiting the development of arteriosclerosis.

#### **3. CCH treatment improves vascular endothelial function in rats and thus exerts protective effects on organs**

The clinical trial described in the preceding section suggested that CCH protects the blood vessels by inducing NO production. Therefore, we next directly investigated the vasodilatory effect of CCH *ex vivo* by using rat blood vessels. We administered L-NGnitroarginine methyl ester (L-NAME), an NO synthesis inhibitor, to rats to trigger vascular endothelial dysfunction. We then tested whether prolonged CCH treatment of the rats would restore their endothelial function (Zhang, 2010).

#### **3.1 Experimental animals**

562 Atherogenesis

medical examination some subjects reported a dry cough. Dry cough is typically observed with ACE inhibitor administration and is attributed to bradykinin accumulation in the body. This raises the possibility that the CCH inhibited kininase II and thus caused accumulation of bradykinin, a vasoprotector, which then induced NO production via the stimulation of eNOS. The results suggest that CCH exerts vasoprotective effects by ameliorating blood


Treatment period

\* \*\*

\*\*

Fig. 4. Time-course of changes in systolic blood pressure in the subjects. Systolic blood pressures in the test food group were non-significantly lower than those in the placebo group after 2 weeks of treatment and were significantly lower than in the placebo group after 12 weeks of treatment. In comparison with the pre-treatment blood pressure (mean of

consistently and significantly lower throughout the treatment period. Data are mean ± SE values . + *P*< 0.1, *# P*< 0.05 versus placebo group. † *P*< 0.1, \**P*< 0.05, \*\* *P*< 0.01, \*\*\*

In our previous *in vitro* studies, we found that CCH treatment of human umbilical vein endothelial cells directly increased eNOS activation (data not shown). When eNOS expressed in vascular endothelial cells is activated, the cells produce NO. The NO functions as a signal to relax adjacent vascular smooth muscle cells; consequently, this dilates arteries and increases blood flow. Other than NO, vascular endothelial cells excrete vasoactive substances such as endothelin, a vasopressor, and maintain the balance of constriction and dilation of blood vessels. NO production via eNOS is particularly important in maintaining the homeostasis of blood vessels. Taken together, these findings indicate that oral administration of CCH improves blood pressure by inhibiting ACE and protects blood vessels by inducing NO production, thereby inhibiting the development of

the values at −2, −1, and 0 weeks), the blood pressure in the test food group was

+ ♯

\*

\* \*\* \*\*\*

\*\*

\* \*\*

†

(weeks)

pressure in humans.

(mmHg)

125

arteriosclerosis.

130

135

140

145

150

Systolic blood pressure

*P*< 0.001 versus pre-treatment blood pressure.

Thirty-six male WKY rats (10 weeks old) were randomly allocated to three groups. The first group (control group) received untreated chow and drinking water. The second group (L-NAME group) received L-NAME in their drinking water (0.5 g/L) for 8 weeks. The third group (L-NAME+CCH group) received L-NAME in their drinking water and CCH (2.0 g/kg daily) via a metal oral Zonde needle. All animal procedures were performed in accordance with the Animal Experimentation Guidelines of the Japanese Association for Laboratory Animal Science and were approved by the Animal Use and Care Committee of Nippon Meat Packers, Inc.

#### **3.2 Vasorelaxation assay**

A vasorelaxation assay was performed on the tissue of eight or nine rats from each group after 8 weeks of treatment. The rats were anesthetized with diethyl ether and the thoracic aorta was removed. The surrounding connective tissue and fat were carefully removed from the thoracic aorta, which was then cut into 2- to 3-mm-wide rings. Segments of thoracic aorta were mounted between two steel hooks in isolated tissue chambers containing Krebs-Henseleit solution at 37 °C. The isometric tension was recorded with an isometric forcedisplacement transducer. After an equilibration period, L-norepinephrine bitartrate was added to cause contraction. This was followed by the addition of cumulative doses of acetylcholine chloride to the bath solution to produce relaxation. Vascular relaxation was expressed as a percentage of tension development (Fig.5).

#### **3.3 Results**

After 8 weeks of treatment, the survival rate of the L-NAME group rats, which had received L-NAME in their drinking water, was 66.7% of that of the control rats. However, rats that had ingested CCH (L-NAME+CCH group) had a significantly better survival rate (91.7% of that of the control group) than the L-NAME rats (*P* < 0.05) (Fig.6). During all of the experiments, monitoring revealed that the rats drank 17 to 30 mL of water and ate 16 to 30 g of chow every day, confirming that their drinking and eating patterns were unaffected by the treatment protocols. Body weight gains did not differ among groups (data not shown). We measured the vasorelaxant effects of CCH treatment after 8 weeks of treatment (Fig. 7). Treatment with acetylcholine chloride caused concentration-dependent relaxation of the thoracic aorta preparations from all groups after the preparations had been caused to

contract by the addition of L-norepinephrine bitartrate. The acetylcholine chloride induced a relaxation response in the thoracic aortas from the L-NAME group (12.7% vasorelaxation); this was significantly less than that in preparations from the control group (69.5%).

Vasoprotective Effect of Foods as Treatments: Chicken Collagen Hydrolysate 565


Fig. 7. Vasorelaxation of rat thoracic aortas over the 8 weeks of the test period . Treatment with acetylcholine chloride caused concentration-dependent relaxation of the thoracic aorta

vasorelaxation was significantly higher in the L-NAME + CCH group than in the L-NAME

We found that CCH treatment improved vascular endothelial function. Acetylcholine activates eNOS expressed in vascular endothelial cells and induces NO production, thereby dilating blood vessels. In L-NAME-treated rats, the vasodilation response associated with NO production induced by an acetylcholine stimulus was inhibited; however, CCH treatment improved this response. As stated earlier, our previous studies have confirmed that CCH activates eNOS in vascular endothelial cells *in vitro*; this result was again supported by our study. In essence, therefore, CCH treatment strongly activated eNOS,

Moreover, the survival curves showed that the survival rate of L-NAME-treated rats was significantly enhanced by CCH administration. This may have been because CCH treatment alleviated the various organ failures caused by L-NAME-induced vascular disorders. We previously conducted the same experiment by using a higher concentration of L-NAME (1 g/L) and prepared tissue sections for observation. We identified substantial tissue damage associated with L-NAME treatment in the blood vessels, kidney, and heart ; this damage was alleviated by CCH treatment (Fig. 8). The substantial fibrosis observed, especially in the heart and liver, was relieved by CCH treatment. Although further investigations of this attenuation effect of CCH treatment on tissue damage are required, we consider that it

preparations from all groups. Especially at high acetylcholine concentrations,

promoted NO production, and thus triggered a vasodilatory response.

results from tissue protection via the vasoprotective effects of CCH.

group. Data are mean ± SE values (n=8-9 rats). \* *P*< 0.05 versus L-NAME group.

ACh

(Log Molar)

\*

20

0

**3.4 Discussion** 

40

60

80

100

(%)

Vasorelaxation

Control group

L-NAME group

L-NAME+CCH group

Compared with that of the L-NAME group, vasorelaxation of the thoracic aortas from the L-NAME+CCH group (36.0%) was significantly improved by long-term administration of CCH (*P* < 0.05).

Fig. 5. Schematic of the Magnus apparatus. The excised rat thoracic aorta was cut into 2- to 3-mm-wide rings and the segments were mounted between two steel hooks in isolated tissue chambers containing Krebs-Henseleit solution at 37C. Drops of L-norepinephrine bitartrate were then added to the tissue chamber to cause the aorta to contract. This was followed by the addition of various doses of acetylcholine chloride to trigger aortic relaxation. The electrical signals for this contraction–relaxation reaction were amplified via a transducer and recorded.

Fig. 6. Survival rates of rats during the test period. Eight weeks into the test period, the survival rate of the L-NAME + CCH group was significantly higher than that of the L-NAME group. Data are mean ± SE values (n=12 rats). \* *P*< 0.05 versus L-NAME group.

Fig. 7. Vasorelaxation of rat thoracic aortas over the 8 weeks of the test period . Treatment with acetylcholine chloride caused concentration-dependent relaxation of the thoracic aorta preparations from all groups. Especially at high acetylcholine concentrations, vasorelaxation was significantly higher in the L-NAME + CCH group than in the L-NAME group. Data are mean ± SE values (n=8-9 rats). \* *P*< 0.05 versus L-NAME group.

#### **3.4 Discussion**

564 Atherogenesis

Compared with that of the L-NAME group, vasorelaxation of the thoracic aortas from the L-NAME+CCH group (36.0%) was significantly improved by long-term administration of

Fig. 5. Schematic of the Magnus apparatus. The excised rat thoracic aorta was cut into 2- to 3-mm-wide rings and the segments were mounted between two steel hooks in isolated tissue chambers containing Krebs-Henseleit solution at 37C. Drops of L-norepinephrine bitartrate were then added to the tissue chamber to cause the aorta to contract. This was followed by the addition of various doses of acetylcholine chloride to trigger aortic

relaxation. The electrical signals for this contraction–relaxation reaction were amplified via a

Constrictor : L-norepinephrine bitartrate

Contraction / Relaxation

Thoracic

aorta

Krebs-Henseleit solution at 37℃

Control group

L-NAME group

L-NAME+CCH group

\*

Relaxant : acetylcholine chloride

(fixed)

CCH (*P* < 0.05).

Tension

transducer and recorded.

Survival rate

20

0

40

60

80

100

(%)

**Recorder Amplifier Transducer**

Constrictor Relaxant Wash out

Time

012345678

Fig. 6. Survival rates of rats during the test period. Eight weeks into the test period, the survival rate of the L-NAME + CCH group was significantly higher than that of the L-NAME group. Data are mean ± SE values (n=12 rats). \* *P*< 0.05 versus L-NAME group.

Weeks of treatment

(weeks)

We found that CCH treatment improved vascular endothelial function. Acetylcholine activates eNOS expressed in vascular endothelial cells and induces NO production, thereby dilating blood vessels. In L-NAME-treated rats, the vasodilation response associated with NO production induced by an acetylcholine stimulus was inhibited; however, CCH treatment improved this response. As stated earlier, our previous studies have confirmed that CCH activates eNOS in vascular endothelial cells *in vitro*; this result was again supported by our study. In essence, therefore, CCH treatment strongly activated eNOS, promoted NO production, and thus triggered a vasodilatory response.

Moreover, the survival curves showed that the survival rate of L-NAME-treated rats was significantly enhanced by CCH administration. This may have been because CCH treatment alleviated the various organ failures caused by L-NAME-induced vascular disorders. We previously conducted the same experiment by using a higher concentration of L-NAME (1 g/L) and prepared tissue sections for observation. We identified substantial tissue damage associated with L-NAME treatment in the blood vessels, kidney, and heart ; this damage was alleviated by CCH treatment (Fig. 8). The substantial fibrosis observed, especially in the heart and liver, was relieved by CCH treatment. Although further investigations of this attenuation effect of CCH treatment on tissue damage are required, we consider that it results from tissue protection via the vasoprotective effects of CCH.

Vasoprotective Effect of Foods as Treatments: Chicken Collagen Hydrolysate 567

Levels of TC, triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and highdensity lipoprotein cholesterol (HDL-C) in the plasma and liver were determined. Total

In addition, plasma levels of interleukin-6 (IL-6), soluble intercellular adhesion molecule-1

At the end of the 12-week test period, the thoracic aorta and liver were excised from the dissected rats and were fixed in formalin, paraffin-embedded, and sliced with a microtome to prepare thin sections, which were then stained with Oil Red O or hematoxylin-eosin for

The mice were treated with CCH for 12 weeks and then sacrificed for analysis. Compared with those in the controls, the amounts of plasma TC and hepatic lipid and TG in the CCH group were reduced by 14.4%, 24.7%, and 42.8%, respectively (Table 1). However, CCH administration had no obvious influence on the concentrations of TG, LDL-C, and HDL-C in

Control 1208±93 308±72 678±68 10.2±1.5 75.3±6.6 0.31±0.11 4.9±2.4 10% CCH 880±73 306 \* ±64 550±80 11.3±0.9 56.7±4.8 0.27 \* ±0.07 2.8±1.5 \*

Table 1. Effect of CCH treatment on plasma concentrations of TC, TG, LDL-C, and HDL-C and on hepatic total lipid, TC, and TG in C57BL/6.KOR-ApoEsh1 mice at the end of the 12 week test period. Plasma TC, hepatic total lipid, and hepatic TG concentrations were significantly lower in the 10% CCH group than in the control group. Data are mean ± SE

We also investigated the effects of CCH treatment on plasma proinflammatory cytokine levels in C57BL/6.KOR-ApoEshl mice. Administration of CCH resulted in decreases in plasma levels of IL-6 (by 43.4%, *P* < 0.01), sICAM-1 (by 17.9%, *P* < 0.05), and TNF-α (by

To investigate whether CCH had a preventive and therapeutic effect on arteriosclerosis, atherosclerotic lesions in the aorta were observed by microscopy with Oil Red O staining (Fig.10). There were no obvious differences in the aortas of the CCH and control groups. We then tested whether CCH treatment had alleviated liver damage in the C57BL/6.KOR-ApoEshl mouse model. Sections of paraffin-embedded liver were stained with hematoxylin-eosin or Oil Red O. Treatment with 10% CCH for 12 weeks decreased the abundance of diffuse lipid droplets and fat vacuoles compared with that in the control

Plasma (mg/100ml) Liver (mg/g) TC TG LDL-C HDL-C Lipid TC TG

(sICAM-1), and tumor necrosis factor-α (TNF-α) were measured by ELISA.

**4.2 Measurement of plasma and hepatic lipids** 

lipids extracted from the liver were also analyzed.

values (n=9 mice). \* *P*< 0.05 versus L-NAME group.

**4.3 Observation of tissue sections** 

histological observation.

the plasma or of TC in the liver.

24.1%, *P* < 0.01) (Fig.9).

group (Fig.10).

**4.4 Results** 

Fig. 8. Tissue sections of blood vessels (A, ×160), kidney (B, ×80) and heart (C, ×80) after treatment with L-NAME at a high concentration (1 g/L). Tissues were stained with Masson trichrome. These sections are from a similarly designed previous experiment of ours. Significant tissue damage caused by L-NAME was observed in the blood vessels, kidneys, and heart tissues, whereas CCH treatment alleviated these damages. Arrows indicate signs of fibrosis.

#### **4. CCH treatment inhibits proinflammatory cytokine expression in a mouse model of arteriosclerosis**

Previous studies have indicated that CCH exerts vasoprotective effects and thus organ protective effects. We therefore investigated the effects of CCH in an atherosclerosis mouse model, C57BL/6.KOR-ApoEsh1. This mouse is spontaneously hyperlipidemic and characteristically has high total cholesterol (TC) levels and arteriosclerotic lesions. Using this mouse model, we examined the changes in blood cholesterol levels and proinflammatory cytokine expression in response to prolonged CCH treatment (Zhang, 2010).

#### **4.1 Experimental animals**

Eighteen male C57BL/6.KOR-ApoEshl mice (7 weeks old) were randomly allocated to two groups (n = 9) and fed on a normal diet or a diet supplemented with 10% CCH for 12 weeks. At the end of the 12-week experiment, the mice were sacrificed, blood was obtained from their veins, and tissues were collected for further analysis. All animal procedures were performed in accordance with the Animal Experimentation Guidelines of the Japanese Association for Laboratory Animal Science and were approved by the Animal Use and Care Committee of Nippon Meat Packers, Inc.

#### **4.2 Measurement of plasma and hepatic lipids**

Levels of TC, triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and highdensity lipoprotein cholesterol (HDL-C) in the plasma and liver were determined. Total lipids extracted from the liver were also analyzed.

In addition, plasma levels of interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1), and tumor necrosis factor-α (TNF-α) were measured by ELISA.

#### **4.3 Observation of tissue sections**

At the end of the 12-week test period, the thoracic aorta and liver were excised from the dissected rats and were fixed in formalin, paraffin-embedded, and sliced with a microtome to prepare thin sections, which were then stained with Oil Red O or hematoxylin-eosin for histological observation.

#### **4.4 Results**

566 Atherogenesis

blood vessels

heart

kidney

Control group L-NAME group L-NAME+CCH group

Fig. 8. Tissue sections of blood vessels (A, ×160), kidney (B, ×80) and heart (C, ×80) after treatment with L-NAME at a high concentration (1 g/L). Tissues were stained with Masson trichrome. These sections are from a similarly designed previous experiment of ours. Significant tissue damage caused by L-NAME was observed in the blood vessels, kidneys, and heart tissues, whereas CCH treatment alleviated these damages. Arrows indicate signs

**4. CCH treatment inhibits proinflammatory cytokine expression in a mouse** 

cytokine expression in response to prolonged CCH treatment (Zhang, 2010).

Previous studies have indicated that CCH exerts vasoprotective effects and thus organ protective effects. We therefore investigated the effects of CCH in an atherosclerosis mouse model, C57BL/6.KOR-ApoEsh1. This mouse is spontaneously hyperlipidemic and characteristically has high total cholesterol (TC) levels and arteriosclerotic lesions. Using this mouse model, we examined the changes in blood cholesterol levels and proinflammatory

Eighteen male C57BL/6.KOR-ApoEshl mice (7 weeks old) were randomly allocated to two groups (n = 9) and fed on a normal diet or a diet supplemented with 10% CCH for 12 weeks. At the end of the 12-week experiment, the mice were sacrificed, blood was obtained from their veins, and tissues were collected for further analysis. All animal procedures were performed in accordance with the Animal Experimentation Guidelines of the Japanese Association for Laboratory Animal Science and were approved by the Animal Use and Care

A

B

C

of fibrosis.

**model of arteriosclerosis** 

**4.1 Experimental animals** 

Committee of Nippon Meat Packers, Inc.

The mice were treated with CCH for 12 weeks and then sacrificed for analysis. Compared with those in the controls, the amounts of plasma TC and hepatic lipid and TG in the CCH group were reduced by 14.4%, 24.7%, and 42.8%, respectively (Table 1). However, CCH administration had no obvious influence on the concentrations of TG, LDL-C, and HDL-C in the plasma or of TC in the liver.


Table 1. Effect of CCH treatment on plasma concentrations of TC, TG, LDL-C, and HDL-C and on hepatic total lipid, TC, and TG in C57BL/6.KOR-ApoEsh1 mice at the end of the 12 week test period. Plasma TC, hepatic total lipid, and hepatic TG concentrations were significantly lower in the 10% CCH group than in the control group. Data are mean ± SE values (n=9 mice). \* *P*< 0.05 versus L-NAME group.

We also investigated the effects of CCH treatment on plasma proinflammatory cytokine levels in C57BL/6.KOR-ApoEshl mice. Administration of CCH resulted in decreases in plasma levels of IL-6 (by 43.4%, *P* < 0.01), sICAM-1 (by 17.9%, *P* < 0.05), and TNF-α (by 24.1%, *P* < 0.01) (Fig.9).

To investigate whether CCH had a preventive and therapeutic effect on arteriosclerosis, atherosclerotic lesions in the aorta were observed by microscopy with Oil Red O staining (Fig.10). There were no obvious differences in the aortas of the CCH and control groups. We then tested whether CCH treatment had alleviated liver damage in the C57BL/6.KOR-ApoEshl mouse model. Sections of paraffin-embedded liver were stained with hematoxylin-eosin or Oil Red O. Treatment with 10% CCH for 12 weeks decreased the abundance of diffuse lipid droplets and fat vacuoles compared with that in the control group (Fig.10).

Vasoprotective Effect of Foods as Treatments: Chicken Collagen Hydrolysate 569

Our results suggested that, as well as lowering plasma TC, CCH had a lipid-lowering effect through regulation of hepatic lipid biosynthesis to suppress TG levels. In humans, collagenspecific peptides are absorbed into the blood as a result of CCH treatment (Iwai, 2009). Once absorbed into the body, the CCH peptides function as regulatory factors to influence cholesterol homeostasis. This effect may have contributed to the decrease in the abundance

Because inflammation plays an important role in the development of arteriosclerosis, inflammatory markers were also examined to investigate the anti-inflammatory function of dietary intervention. IL-6, sICAM-1, and TNF-α are the major proinflammatory cytokines secreted by adipocytes. At the same time, NO inhibits the expression of these proinflammatory cytokines in the vascular endothelium. Our previous studies indicate that orally ingested CCH induces NO production in the body. Hence, the results imply that CCH treatment downregulates several proinflammatory cytokines via NO production, thereby having beneficial effects on the fat tissues. Further detailed investigations are, however,

Unfortunately, no direct therapeutic effect of CCH on arteriosclerotic plaques was observed in this study. Nevertheless, the data demonstrated that CCH treatment substantially reduced both the total lipid content in the liver and the production of proinflammatory cytokines such as IL-6, TNF-α, and sICAM-1 in a mouse model highly susceptible to arteriosclerosis. High levels of expression of these factors lead to the progression of arteriosclerosis. From this perspective, long-term CCH treatment may be effective as a simple dietary, rather than drug, treatment for preventing

**5. Conclusion: The availability of collagen peptides as a food providing anti-**

It has been frequently reported that externally applied collagen peptides help to increase water retention owing to their high water retentivity. On the other hand, the functionality of orally ingested collagen is not fully understood. However, much of the evidence reported in recent years, including the results of this study, supports the specific physiological activities

In this study, we examined the impacts of collagen peptides on blood vessels from various perspectives. We demonstrated that collagen peptides exhibit vasoprotective functions via

Functional foods will not replace pharmaceuticals. However, what humans continue to do regularly for survival is to eat. Whereas a balanced diet obviously supports healthy life, elucidation of the tertiary function of food ingredients by precisely following their mechanisms is a long-term mission for food researchers. We focused on collagen and analyzed the whole process from development of, to research into, novel chicken-derived collagen peptides. We clarified the efficacy of vasoprotection, which is a novel tertiary function of collagen peptides. We intend to continue our efforts to demonstrate the beneficial functionalities of collagen in the hope of improving the global quality of life

of lipid droplets and fat vacuoles observed in the liver tissues.

necessary to elucidate more of the direct effects of CCH on fat cells.

**atherogenesis via a vasoprotective effect** 

through the consumption of this food product.

NO production and effectively protect against atherogenesis.

of collagen absorbed by the body.

**4.5 Discussion** 

arteriosclerosis.

Fig. 9. Effect of CCH treatment on plasma proinflammatory cytokine levels in C57BL/6.KOR-ApoEsh1 mice at the end of the 12-week test period. Interleukin-6 (IL-6) (A), soluble intercellular adhesion molecule-1 (sICAM-1 ) (B), tumor necrosis factor alpha (TNFα) (C). The levels of all plasma proinflammatory cytokines were significantly lower in the 10% CCH group than in the control group. Data are mean ± SE values (n=9 mice). \* *P*< 0.05, \*\* *P*< 0.01 versus control group.

Fig. 10. Tissue sections of aortic root (A) and liver (B and C) at the end of the 12-week test period. Tissues were stained with Oil Red O (A, ×80; C, ×140) or hematoxylin-eosin (B , ×140). No obvious change was observed in the aortic root of the 10% CCH group; however, diffuse lipid droplets and fat vacuoles in the livers of the treatment group were less abundant than in those of the control group.

#### **4.5 Discussion**

568 Atherogenesis

AB C

Control 10% CCH

C57BL/6.KOR-ApoEsh1 mice at the end of the 12-week test period. Interleukin-6 (IL-6) (A), soluble intercellular adhesion molecule-1 (sICAM-1 ) (B), tumor necrosis factor alpha (TNFα) (C). The levels of all plasma proinflammatory cytokines were significantly lower in the 10% CCH group than in the control group. Data are mean ± SE values (n=9 mice). \* *P*<

\*

0

Control 10% CCH

Aoratic root

Liver

Liver

\*

200 300

100

500

400

700

TNF-a (pg/ml)

600

0

Fig. 9. Effect of CCH treatment on plasma proinflammatory cytokine levels in

Control group 10%CCH group

Fig. 10. Tissue sections of aortic root (A) and liver (B and C) at the end of the 12-week test period. Tissues were stained with Oil Red O (A, ×80; C, ×140) or hematoxylin-eosin (B , ×140). No obvious change was observed in the aortic root of the 10% CCH group; however,

diffuse lipid droplets and fat vacuoles in the livers of the treatment group were less

100

200

300

400

sICAM-1 (ng/ml)

IL-6 (pg/ml)

Control 10% CCH

A

B

C

abundant than in those of the control group.

\*\*

0.05, \*\* *P*< 0.01 versus control group.

Our results suggested that, as well as lowering plasma TC, CCH had a lipid-lowering effect through regulation of hepatic lipid biosynthesis to suppress TG levels. In humans, collagenspecific peptides are absorbed into the blood as a result of CCH treatment (Iwai, 2009). Once absorbed into the body, the CCH peptides function as regulatory factors to influence cholesterol homeostasis. This effect may have contributed to the decrease in the abundance of lipid droplets and fat vacuoles observed in the liver tissues.

Because inflammation plays an important role in the development of arteriosclerosis, inflammatory markers were also examined to investigate the anti-inflammatory function of dietary intervention. IL-6, sICAM-1, and TNF-α are the major proinflammatory cytokines secreted by adipocytes. At the same time, NO inhibits the expression of these proinflammatory cytokines in the vascular endothelium. Our previous studies indicate that orally ingested CCH induces NO production in the body. Hence, the results imply that CCH treatment downregulates several proinflammatory cytokines via NO production, thereby having beneficial effects on the fat tissues. Further detailed investigations are, however, necessary to elucidate more of the direct effects of CCH on fat cells.

Unfortunately, no direct therapeutic effect of CCH on arteriosclerotic plaques was observed in this study. Nevertheless, the data demonstrated that CCH treatment substantially reduced both the total lipid content in the liver and the production of proinflammatory cytokines such as IL-6, TNF-α, and sICAM-1 in a mouse model highly susceptible to arteriosclerosis. High levels of expression of these factors lead to the progression of arteriosclerosis. From this perspective, long-term CCH treatment may be effective as a simple dietary, rather than drug, treatment for preventing arteriosclerosis.

#### **5. Conclusion: The availability of collagen peptides as a food providing antiatherogenesis via a vasoprotective effect**

It has been frequently reported that externally applied collagen peptides help to increase water retention owing to their high water retentivity. On the other hand, the functionality of orally ingested collagen is not fully understood. However, much of the evidence reported in recent years, including the results of this study, supports the specific physiological activities of collagen absorbed by the body.

In this study, we examined the impacts of collagen peptides on blood vessels from various perspectives. We demonstrated that collagen peptides exhibit vasoprotective functions via NO production and effectively protect against atherogenesis.

Functional foods will not replace pharmaceuticals. However, what humans continue to do regularly for survival is to eat. Whereas a balanced diet obviously supports healthy life, elucidation of the tertiary function of food ingredients by precisely following their mechanisms is a long-term mission for food researchers. We focused on collagen and analyzed the whole process from development of, to research into, novel chicken-derived collagen peptides. We clarified the efficacy of vasoprotection, which is a novel tertiary function of collagen peptides. We intend to continue our efforts to demonstrate the beneficial functionalities of collagen in the hope of improving the global quality of life through the consumption of this food product.

#### **6. References**


Katiyar SK. 2003. Skin photoprotection by green tea: antioxidant and immunomodulatory effects. Curr Drug Targets Immune Endocr Metabol Disord 3(3):234-42. Weijer P, Barentsen R. 2002. Isoflavones from red clover (Promensil) significantly reduce

Tamai T, Ikematsu H, Shionoya K, Murota I, Baba T, Hiura N, Sato R. 2004. Effect of fish

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Shigemura Y, Iwai K, Morimatsu F, Iwamoto T, Mori T, Oda C, Taira T, Park E, Nakamura

Ohara H, Ito K, Iida H, Matsumoto H. 2009. Improvement in the moisture content of the

Arborelius M, Konttinen Y, Nordström D, Solovieva S. 1999. Gly-X-Y repeat sequences in the treatment of active rheumatoid arthritis. Rheumatol Int 18(4):129-35. Kato T, Node K. 2004. Frontier of vascular failure. Node K. (Eds.). Concept of vasucular

Taguchi Y. 2002. Development of a low allergic chicken gelatin. Packaging of foodstuff.

Saiga A, Iwai K, Hayakawa T, Takahata Y, Kitamura S, Nishimura T, Morimatsu F. 2008.

Gupta R, Guptha S. 2010. Strategies for initial management of hypertension. Indian J Med

Sharma JN. 2009. Hypertension and the bradykinin system. Curr Hypertens Rep 11(3):178-

Kouguchi T, Ohmori T, Hayakawa T, Takahata Y, Maeyama Y, Kajimoto Y, Kitakaze M,

Zhang Y, Kouguchi T, Shimizu M, Ohmori T, Takahata Y, Morimatsu F. 2010. Chiken

Zhang Y, Kouguchi T, Shimizu K, Sato M, Takahata Y, Morimatsu F. 2010. Chicken collagen

high-normal blood pressure. Jpn Pharmacol Ther 36(6):561–75.

Angiotensin I-converting enzyme inhibitory peptides obtained from chicken

Morimatsu F. 2008. Hypotensive effects and safety of intake of lactic acid beverage containing chicken collagen hydrolysate in subjects with mild hypertension or

collagen hydrolysate protects rats from hypertension and cardiovascular damage. J

hydrolysate reduces proinflammatory cytokine production in C57BL/6.KOR-

menopausal hot flush symptoms compared with placebo. Maturitas 25;42(3):187-93.

sausage enriched with DHA (docosahexaenoic acid) on serum lpids (II): effect of three month-long intake on serum lipids, and safety evaluation. J Jpn Soc Clin Nutr

F. 2009. Blood concentration of food-derived peptides following oral intake of chicken collagen hydrolysate and its angiotensin-converting enzyme inhibitory activity in healthy volunteers. Nippon Shokuhin Kagaku Kogaku Kaishi 56(6):326–

Y, Sato K. 2009. Effect of Prolyl-hydroxyproline (Pro-Hyp), a food-derived collagen peptide in human blood, on growth of fibroblasts from mouse skin. J Agric Food

stratum corneum following 4 weeks of collagen hydrolysate ingestion. Nippon

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Med Food 13(2):399-405.

Shokuhin Kagaku Kogaku Kaishi 56:137–45.

collagen hydrolysate. J Agric Food Chem 56:9586–91.

ApoEshl mice. J Nutr Sci Vitaminol 56(3):208-10.

failure. 21-2. Tokyo. Medical review.

30.

### *Edited by Sampath Parthasarathy*

This monograph will bring out the state-of-the-art advances in the dynamics of cholesterol transport and will address several important issues that pertain to oxidative stress and inflammation. The book is divided into three major sections. The book will offer insights into the roles of specific cytokines, inflammation, and oxidative stress in atherosclerosis and is intended for new researchers who are curious about atherosclerosis as well as for established senior researchers and clinicians who would be interested in novel findings that may link various aspects of the disease.

Atherogenesis

*Edited by Sampath Parthasarathy*

ISBN 978-953-307-992-9

ISBN 978-953-51-6784-6

Atherogenesis

Photo by Ugreen / iStock