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 <IP_ADDRESS>, 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 <IP_ADDRESS>, 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.<PHONE_NUMBER>-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.<PHONE_NUMBER>-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

00392499

Vol. 81, No.1, pp. 105-11, ISSN 0011-1643

(October 2009), pp. 361-368, ISSN 0971-5916

2006), pp. 59-68, ISSN 846-9558

161, ISSN 009-8981

779, ISSN 0946-2716

ISSN 0028-0836

ISSN 009-7322

Factor Acetylhydrolase Activity as a Genetic Risk Factors in Cerebral Atherosclerosis 525

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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.