**2. Patients and methods**

#### **2.1. Patients**

physical stimuli that trigger angiogenesis and inflammation (Costa et al., 2007). Further‐ more, there is compelling evidence that HIF-1 contributes to both processes by regulating angiogenesis and functions of inflammatory cells. Many inflammatory stimuli can acti‐ vate the angiogenic programme of endothelial cells. Inflammatory cells, especially mono‐ cytes/macrophages secrete many angiogenic factors such as vascular endothelial growth factor (VEGF), CXCL8 (interleukin-8), granulocyte colony stimulating factor, transforming growth factor-α and β, platelet-derived growth factor, tumor necrosis factor-α, and pros‐ taglandins. The angiogenic factors bind to cognate receptors which are expressed on the surface of vascular endothelial cells and vascular pericytes/smooth muscle cells. Recep‐ tor–ligand interaction activates these cells and promotes the angiogenic response. Com‐ munication between endothelial cells and monocytes/macrophages appears to be bidirectional, because endothelial cell–secreted factors also induce chemotaxis and in‐ creased angiogenic activity in monocytes/macrophages, thus initiating a positive feedback

The angiogenesis are tightly regulated in a complex balance between pro- and anti-angiogenic mechanisms (Carmeliet, 2003; Otrock et al., 2007). The most important proangiogenic growth factors are VEGF and angiopoietins. VEGF and angiopoietins, acting as the modulators of endothelial activation via receptor tyrosine kinase Tie-2, are important for angiogenesis and vascular remodeling. VEGF increases microvascular permeability and induces the prolifera‐ tion, migration, and differentiation of endothelial cells (Hoeben et al., 2009; Stuttfeld &, Ballmer-Hofer, 2009; Olsson et al., 2006). Angiopoietin-2 is a natural endogenous antagonist of the Tie-2, which acts as an autocrine negative regulator of endothelial function (Augustin et al., 2009; Scharpfenecker et al., 2004; Fiedler & Augustin, H2006; Fukuhara et al., 2010). In the presence of VEGF, it mounts an inflammatory response by endothelial activation and induction of permeability, and in the absence of VEGF, it destabilizes the existing vessels and leads to vascular regression. Soluble receptors of angiogenic growth factors which are being released to circulation can act as the inhibitors of angiogenesis and, in some cases, may correlate with the disease severity independently of altered haemodynamics (Findley et al., 2008).

The findings of the large prospective investigations have confirmed the significance of highsensitivity C-reactive protein (hs-CRP) as a marker of progression, functional activity, and adverse cardiovascular outcome in patients with peripheral artery disease (Abdellaoui & Al-

Platelet activating factor acetylhydrolase (PAF-AH; E.C. 3.1.1.47) also named lipoproteinassociated phospholipase A(2) (Lp-PLA(2)) is a novel inflammatory biomarker that has an active role in atherosclerotic development and progression. This enzyme is character‐ ized by its ability to specifically hydrolyze the short acyl group at the sn-2 position of the phospholipids in oxidized LDL, which leads to production of the pro-inflammatory, atherogenic by-products lysophosphatidylcholine and oxidized nonesterified fatty acids. These bioactive lipid mediators act as chemoattractants for monocytes, impair endothelial function, disrupt plasma membranes, and induce apoptosis in smooth muscle cells and macrophages. Epidemiologic studies demonstrate that elevated circulating levels of PAF-AH predict an increased risk of myocardial infarction and stroke, whereas histologic ex‐

cycle (Shireman, 2007).

98 Current Trends in Atherogenesis

Khaffaf, 2007).

The study included 110 patients, 19 women and 91 men, with clinically and angiographically confirmed diagnosis of peripheral arterial disease. The study population was referred to the Digital subtraction angiography (DSA) in order to determine the precise extent and localization of peripheral limb atherosclerosis and assess the technical possibility to perform percutaneous transluminal angioplasty (PTA). Based on the angiographic findings, for the purpose of the present investigation the angiographic score was assessed for each patient. The angiographic score takes into consideration the extent (percentage of vessel lumen reduction) and diffusion of peripheral arterial disease (involved segments of vascular tree). The distal aorta plus 10 segments (common iliac artery, external iliac artery, common femoral artery, profunda femoral artery, superficial femoral artery, popliteal artery, truncus tibiofibularis, anterior tibial artery, posterior tibial artery and fibular artery) on each side were scored on the basis of vessel lumen reduction: 1 if stenoses involved a reduction in the vessel lumen of <50%, 2 if stenoses involved 50 to 99% reduction, and 3 if total occlusion was present. The sum of the points assigned to each of these arteries was called the angiographic score.

The control group consisted of 118 patients, 61 female and 57 male with suspected symptoms of peripheral arterial disease referred to Doppler examination. At the Doppler examination, all of them had normal triphasic waveforms of the peripheral arteries.

All Doppler and DSA procedures were performed at the Institute for Diagnostic and Inter‐ ventional Radiology of the Merkur University Hospital. Doppler examinations were per‐ formed at a center of excellence with more than 3,000 examinations performed per year. DSA was performed by an experienced vascular interventional radiologist. All participants gave their informed written consent. This study was approved by the Ethics Committee of the Merkur University Hospital, Zagreb, Croatia.

catalytic concentrations of PAF-AH are expressed in international units per liter of serum and

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101

Commercially available ELISA kits for VEGF (DVE00), Ang2 (DANG 20) and Tie2 (DTE 200) were purchased from R&D Systems (Minneapolis, MN, USA) and used according to the manufacturer's instruction. Serum samples were kept frozen at -80°C until the day of analysis. Briefly, the microtitre plates were coated with monoclonal antibodies specific for either VEGF-A, Ang-2 or Tie-2 and the first step was to add standards and samples to the wells. During the following incubation period, the VEGF-A, Ang-2 or Tie-2 present in standards and samples were bound to the immobilized antibody. After a thorough wash, an a horseradish peroxidaselinked polyclonal antibody specific for VEGF, Ang-2 or Tie-2 was pipetted into the wells and following a second incubation and wash step a substrate solution was added and colour developed in proportion to the amount of VEGF-A, Ang-2 or Tie-2. After further washings to remove any unbound antibody–enzyme reagent, tetramethylbenzidine was added. The colour development was subsequently stopped and the intensity of colour was measured by using Stat Fax®2100, Microplate reader, Awareness Technology Inc., Palm City, FL, USA. The values were calculated using a standard curve generated with specific standards provided by the manufacturer. The detection limit for VEGF, Ang-2 and Tie-2 was 9 ng/L, 8,3 ng/L, and 14 ng/ L, respectively. The intra-assay and interassay coefficients of variation were in the range given by the manufacturer <6,7% and <8,8% for VEGF, < 6,9% and <10,4% for Ang-2 and< 5,3% and

Statistical analyses were performed using the SPSS software package for Windows, version 13 (SPSS Inc, Chicago, IL, USA). Descriptive analyses were performed and data were presented as mean, median, S.D. and percentile. Normal distribution of the study variables was tested using Kolmogorov–Smirnov test. Student *t* test and Mann-Whitney U test or the Kruskal– Wallis test applied according to the normal or non-normal distribution. Spearman coefficient

of correlation was calculated to evaluate relationships between different variables.

Demographic and clinical characteristics of the participants are shown in Table 1.

The patients had significantly higher concentrations of CRP, triglyceride, index of atheroscle‐ rosis, the ratio of total and HDL cholesterol, and lower concentrations of total, LDL and HDL-

*2.3.3. The concentrations of angiogenesis biomarkers: VEGF, Ang-2 and Tie-2 receptor*

standardized against concentration of LDL-cholesterol.

<8,5% for Tie-2 receptor.

**2.4. Statistical analysis**

**3. Results**

**3.1. Patients**

**3.2. The lipid status and CRP**

cholesterol (Table2).

#### **2.2. Samples**

Blood samples were taken under controlled pre-analytical conditions in the morning after 12 h fast. Serum was separated by centrifuging the samples at 4°C at 3000 rpm for 15 minutes.

### **2.3. Methods**

#### *2.3.1. The lipid status and CRP*

Analytical methods for measurement of the lipid status, including serum triglyceride, total cholesterol, LDL and HDL-cholesterol concentrations as well as CRP used in this study have been accredited according to ISO 15189, Medical laboratories - Particular requirements for quality and competence (ISO 15189, 2008) (Flegar-Meštrić et al., 2010). 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 (Olym‐ pus Mishima Co., Ltd., Shizuoka, Japan). Serum triglyceride and total cholesterol were measured by enzymatic PAP- method. HDL cholesterol was measured with direct method based on selective inhibition of the non-HDL fractions by means of polyanions. A homogene‐ ous assay for the selective measurement of LDL cholesterol in serum was used. The index of atherosclerosis and the established risk factor were calculated as the ratio of LDL cholesterol to HDL cholesterol and total cholesterol to HDL cholesterol. CRP concentrations were determined by high-sensitivity latex-enhanced immunoturbidimetric assay.

#### *2.3.2. The catalytic concentrations of PAF-AH*

The catalytic concentrations of PAF-AH were determined in serum by spectrophotometric method described by Kosaka T. et al. (2000) using the AZWELL Auto PAF-AH Assay Kits (AZWELL Inc., Osaka, Japan) on a biochemical analyzer Olympus AU600 (Olympus Mishima Co., Ltd., Shizuoka, Japan). Serum samples were kept frozen at -80°C until the day of analysis. PAF-AH hydrolyzes the sn-2 position of the substrate (1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine), producing 4-nitrophenyl succinate. This compound immediately degrades in aqueous solution and liberates 4-nitrophenol. In the first phase, 2 µL 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 *4* nitrophenol was measured by reading differences in absorbance at 405 nm (main wavelength) and 505 nm (subwavelength) between 1 and 3 minutes after addition of the substrate. The catalytic concentrations of PAF-AH are expressed in international units per liter of serum and standardized against concentration of LDL-cholesterol.

#### *2.3.3. The concentrations of angiogenesis biomarkers: VEGF, Ang-2 and Tie-2 receptor*

Commercially available ELISA kits for VEGF (DVE00), Ang2 (DANG 20) and Tie2 (DTE 200) were purchased from R&D Systems (Minneapolis, MN, USA) and used according to the manufacturer's instruction. Serum samples were kept frozen at -80°C until the day of analysis. Briefly, the microtitre plates were coated with monoclonal antibodies specific for either VEGF-A, Ang-2 or Tie-2 and the first step was to add standards and samples to the wells. During the following incubation period, the VEGF-A, Ang-2 or Tie-2 present in standards and samples were bound to the immobilized antibody. After a thorough wash, an a horseradish peroxidaselinked polyclonal antibody specific for VEGF, Ang-2 or Tie-2 was pipetted into the wells and following a second incubation and wash step a substrate solution was added and colour developed in proportion to the amount of VEGF-A, Ang-2 or Tie-2. After further washings to remove any unbound antibody–enzyme reagent, tetramethylbenzidine was added. The colour development was subsequently stopped and the intensity of colour was measured by using Stat Fax®2100, Microplate reader, Awareness Technology Inc., Palm City, FL, USA. The values were calculated using a standard curve generated with specific standards provided by the manufacturer. The detection limit for VEGF, Ang-2 and Tie-2 was 9 ng/L, 8,3 ng/L, and 14 ng/ L, respectively. The intra-assay and interassay coefficients of variation were in the range given by the manufacturer <6,7% and <8,8% for VEGF, < 6,9% and <10,4% for Ang-2 and< 5,3% and <8,5% for Tie-2 receptor.

#### **2.4. Statistical analysis**

All Doppler and DSA procedures were performed at the Institute for Diagnostic and Inter‐ ventional Radiology of the Merkur University Hospital. Doppler examinations were per‐ formed at a center of excellence with more than 3,000 examinations performed per year. DSA was performed by an experienced vascular interventional radiologist. All participants gave their informed written consent. This study was approved by the Ethics Committee of the

Blood samples were taken under controlled pre-analytical conditions in the morning after 12 h fast. Serum was separated by centrifuging the samples at 4°C at 3000 rpm for 15 minutes.

Analytical methods for measurement of the lipid status, including serum triglyceride, total cholesterol, LDL and HDL-cholesterol concentrations as well as CRP used in this study have been accredited according to ISO 15189, Medical laboratories - Particular requirements for quality and competence (ISO 15189, 2008) (Flegar-Meštrić et al., 2010). 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 (Olym‐ pus Mishima Co., Ltd., Shizuoka, Japan). Serum triglyceride and total cholesterol were measured by enzymatic PAP- method. HDL cholesterol was measured with direct method based on selective inhibition of the non-HDL fractions by means of polyanions. A homogene‐ ous assay for the selective measurement of LDL cholesterol in serum was used. The index of atherosclerosis and the established risk factor were calculated as the ratio of LDL cholesterol to HDL cholesterol and total cholesterol to HDL cholesterol. CRP concentrations were

The catalytic concentrations of PAF-AH were determined in serum by spectrophotometric method described by Kosaka T. et al. (2000) using the AZWELL Auto PAF-AH Assay Kits (AZWELL Inc., Osaka, Japan) on a biochemical analyzer Olympus AU600 (Olympus Mishima Co., Ltd., Shizuoka, Japan). Serum samples were kept frozen at -80°C until the day of analysis. PAF-AH hydrolyzes the sn-2 position of the substrate (1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine), producing 4-nitrophenyl succinate. This compound immediately degrades in aqueous solution and liberates 4-nitrophenol. In the first phase, 2 µL 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 *4* nitrophenol was measured by reading differences in absorbance at 405 nm (main wavelength) and 505 nm (subwavelength) between 1 and 3 minutes after addition of the substrate. The

determined by high-sensitivity latex-enhanced immunoturbidimetric assay.

Merkur University Hospital, Zagreb, Croatia.

*2.3.2. The catalytic concentrations of PAF-AH*

**2.2. Samples**

100 Current Trends in Atherogenesis

**2.3. Methods**

*2.3.1. The lipid status and CRP*

Statistical analyses were performed using the SPSS software package for Windows, version 13 (SPSS Inc, Chicago, IL, USA). Descriptive analyses were performed and data were presented as mean, median, S.D. and percentile. Normal distribution of the study variables was tested using Kolmogorov–Smirnov test. Student *t* test and Mann-Whitney U test or the Kruskal– Wallis test applied according to the normal or non-normal distribution. Spearman coefficient of correlation was calculated to evaluate relationships between different variables.

## **3. Results**

#### **3.1. Patients**

Demographic and clinical characteristics of the participants are shown in Table 1.

#### **3.2. The lipid status and CRP**

The patients had significantly higher concentrations of CRP, triglyceride, index of atheroscle‐ rosis, the ratio of total and HDL cholesterol, and lower concentrations of total, LDL and HDLcholesterol (Table2).


**3.3. The catalytic concentrations of PAF-AH**

arterial disease (Table 5.).

**Biochemical parameters (units)**

**Biochemical parameters (units)**

subjects. Data are given as median (interquartile range).

off therapy.

(Table 6.).

Mann–Whitney's tests

Mann–Whitney's tests

(interquartile range).

The catalytic concentrations of PAF-AH did not differ between the two groups, while LDL standardized catalytic concentrations of PAF-AH (U/mmol) showed significant difference (Table 3). The catalytic concentrations of PAF-AH were higher in men than in women in control subjects (Table 4.), whereas no gender difference was observed in patients with peripheral

The Evaluation of New Biomarkers of Inflammation and Angiogenesis in Peripheral Arterial Disease

A significant difference in the catalytic concentrations of PAF-AH was found between subjects on lipolythic therapy and subjects off therapy (P=0,032), with the median concentration of PAF-AH in subjects off therapy being higher than that observed in subjects on lipolythic therapy: 425, interquartile range 351-494 U/L vs 364, interquartile range, 316-427 U/L. There was no difference in catalytic concentrations of PAF-AH between smokers and non-smokers, diabetic and nondiabetic subjects nor between the subjects on antihypertensive therapy and subjects

A statistically significant correlation was found between the catalytic concentration of PAF-AH and the concentration of triglycerides, total and LDL-cholesterol in both groups studied

PAF-AH (U/L) 405 (330-471) 406 (359-479) 0,591

PAF-AH/LDL (U/mmol ) 121 (107-139) 98 (86-120) <0,001

**Table 3.** The catalytic concentrations of PAF-AH in the patients with peripheral arterial disease (PAD) and control

PAF-AH (U/L) 459 (383- 519) 385 (319-437) 0,005

PAF-AH/LDL (U/mmol ) 121 (95-137) 92 (79-103) <0,001

**Table 4.** The catalytic concentrations of PAF-AH in the male and female control subjects. Data are given as median

**Male (N=28)** **Control subjects (N=64)**

> **Female (N=36)**

**P**

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103

**P**

**Patients with PAD (N=93)**

**Table 1.** Demographic and clinical characteristics of the study groups: patients with peripheral arterial disease (PAD) and controls. Data are given as mean ± standard deviation, unless otherwise stated.


**Table 2.** The lipid status and CRP concentrations in the patients with peripheral arterial disease (PAD) and controls. Data are given as median (interquartile range).

#### **3.3. The catalytic concentrations of PAF-AH**

**Parameter Patients with PAD**

Diastolic and systolic blood pressure >90 mm

102 Current Trends in Atherogenesis

**Biochemical parameters (units)**

Mann–Whitney's tests

Data are given as median (interquartile range).

**(N=110)**

Body mass index (kg/m2), x±sd 26,63 ± 4,04 26,51± 3,08 P = 0,795

Systolic blood pressure >140 mm Hg, n(%) 74 (67%) 0 P<0,001

Age (years), x±sd 64,33 ± 9,79 59,11± 7,31 P<0,001

Male sex, n (%) 91 (83%) 57 (48%) P<0,001

Hg, n (%) 30 (27%) <sup>0</sup> P<0,001

Diabetes, n (%) 39 (35%) 0 P<0,001

Active smokers, n (%) 49 (45%) 13 (11%) P<0,001

Hypolipemic therapy, n (%) 64 (58%) 0 P<0,001

Antihypertensive therapy, n (%) 69 (63%) 0 P<0,001

Cerebrovascular simptoms, (%) 11 (10%) 0 P<0,001

Coronary artery disease symptoms, n (%) 26 (24%) 0 P<0,001

**Table 1.** Demographic and clinical characteristics of the study groups: patients with peripheral arterial disease (PAD)

Triglyceride (mmol/L) 1,89 (1,30-2,36) 1,42 (1,03-1,76) <0,001

Total cholesterol (mmol/L) 5,45 (4,68-6,10) 6,35 (5,66-6,89) <0,001

HDL-cholesterol (mmol/L) 1,10 (0,98-1,30) 1,60 (1,39-1,81) <0,001

LDL- cholesterol (mmol/L) 3,30 (2,70-3,90) 3,96 (3,40-4,55) <0,001

**Table 2.** The lipid status and CRP concentrations in the patients with peripheral arterial disease (PAD) and controls.

CRP (mg/L) 3,70 (1,78-7,40) 1,40 (0,60-2,43) <0,001

and controls. Data are given as mean ± standard deviation, unless otherwise stated.

**Patients with PAD (N=110)**

**Control subjects (N=118)**

**Control subjects (N=118)**

**P**

**P**

The catalytic concentrations of PAF-AH did not differ between the two groups, while LDL standardized catalytic concentrations of PAF-AH (U/mmol) showed significant difference (Table 3). The catalytic concentrations of PAF-AH were higher in men than in women in control subjects (Table 4.), whereas no gender difference was observed in patients with peripheral arterial disease (Table 5.).

A significant difference in the catalytic concentrations of PAF-AH was found between subjects on lipolythic therapy and subjects off therapy (P=0,032), with the median concentration of PAF-AH in subjects off therapy being higher than that observed in subjects on lipolythic therapy: 425, interquartile range 351-494 U/L vs 364, interquartile range, 316-427 U/L. There was no difference in catalytic concentrations of PAF-AH between smokers and non-smokers, diabetic and nondiabetic subjects nor between the subjects on antihypertensive therapy and subjects off therapy.

A statistically significant correlation was found between the catalytic concentration of PAF-AH and the concentration of triglycerides, total and LDL-cholesterol in both groups studied (Table 6.).


**Table 3.** The catalytic concentrations of PAF-AH in the patients with peripheral arterial disease (PAD) and control subjects. Data are given as median (interquartile range).


**Table 4.** The catalytic concentrations of PAF-AH in the male and female control subjects. Data are given as median (interquartile range).


and Tie-2 receptor (P=0,005) between low and high risk subjects, as well as in the concentrations of VEGF (P=0,012), Ang-2 (P<0,001), and Tie-2 receptor (P=0,02) between the moderate and high cardiovascular risk subjects, whereas there were no statistically significant differences in the concentrations of VEGF (P=0,377), Ang-2 (P=0,438), and Tie-2 receptor (P=0,673) between

The Evaluation of New Biomarkers of Inflammation and Angiogenesis in Peripheral Arterial Disease

VEGF (ng/L) 263 (142-403) 287 (115-483) 0,983 Ang-2 (ng/L) 2018 (1613-2689) 1603 (1452-2138) 0,001 Tie-2 (μg/L) 21,4 (18,6-23,9) 19,6 (18,1-22,2)\* 0,049

**Table 7.** Biochemical parameters in the patients with peripheral arterial disease (PAD) and controls. Data are given as

**PAD CONTROL**

**Figure 1.** Comparison of VEGF concentrations (median, interquartile range) in the patients with peripheral arterial dis‐

**Control subjects (N=54)**

**P**

105

http://dx.doi.org/10.5772/53341

the groups of low and moderate cardiovascular risk subjects.

**Patients with PAD (N=110)**

**Biochemical parameters (units)**

Mann–Whitney's tests, \*N=43

median (interquartile range)..

0

ease (PAD) and control subjects.

200

400

**VEGF (ng/L)**

600

800

**Table 5.** The catalytic concentrations of PAF-AH in male and female patients with peripheral arterial disease. Data are given as median (interquartile range)..


**Table 6.** Relationships between the catalytic concentrations of PAF-AH and serum lipids parameters and CRP concentrations in the study groups: patients with peripheral arterial disease (PAD) and controls

#### **3.4. Serum VEGF, Ang-2 and Tie-2 concentrations**

The concentration of VEGF did not differ significantly between groups (Figure 1., Table 7.). The patients had higher concentrations of Ang-2 and Tie-2 receptor. (Figure 2.,3., Table 7.).

A significant difference in the concentrations of VEGF was found between diabetic and nondiabetic subjects (P= 0,006), with the median (interquartile range) concentration of VEGF in diabetics being higher than that observed in nondiabetic subjects: 358 (210-463) vs. 197 (130-335) ng/L. There was no difference in concentrations of VEGF, Ang-2, and Tie-2 receptor between smokers and non-smokers, nor between the subjects on lipolythic and antihyperten‐ sive therapy and subjects off therapy. All three serum biomarkers of angiogenesis correlated with the CRP concentrations (Table 8). The concentrations of HDL- cholesterol, VEGF, Ang-2, and Tie-2 were statistically significantly different among the subjects with various cardiovas‐ cular risk according to CRP concentrations (Table 9.). Post hoc tests (Mann–Whitney's test) suggested a significant difference in HDL -cholesterol values between the low risk subjects (CRP<1,0 mg/L) compared with the moderate (CRP between 1,0-3,0 mg/L) (P=0,004) and high risk (P=0,011) subjects (CRP >3,0 mg/L). The subject groups of moderate and high cardiovas‐ cular risk did not differ significantly in the HDL cholesterol concentration (P=0,666). Statisti‐ cally significant difference was found in the concentrations of VEGF (P=0,011), Ang-2 (P<0,001), and Tie-2 receptor (P=0,005) between low and high risk subjects, as well as in the concentrations of VEGF (P=0,012), Ang-2 (P<0,001), and Tie-2 receptor (P=0,02) between the moderate and high cardiovascular risk subjects, whereas there were no statistically significant differences in the concentrations of VEGF (P=0,377), Ang-2 (P=0,438), and Tie-2 receptor (P=0,673) between the groups of low and moderate cardiovascular risk subjects.

**Biochemical parameters (units)**

given as median (interquartile range)..

Mann–Whitney's tests

104 Current Trends in Atherogenesis

**Male (N=75)**

PAF-AH (U/L) 405 (331- 477) 409 (329-442) 0,722 PAF-AH/LDL (U/mmol ) 123 (108-141) 110 (100- 119) 0,031

**Table 5.** The catalytic concentrations of PAF-AH in male and female patients with peripheral arterial disease. Data are

**Correlation coefficient Patients with PAD (N=93)**

Triglyceride (mmol/L) 0,33 0,001 0,41 0,001 Total cholesterol (mmol/L) 0,70 <0,001 0,32 0,010 HDL-cholesterol (mmol/L) -0,22 0,035 -0,33 0,009 LDL- cholesterol (mmol/L) 0,70 <0,001 0,33 0,009

CRP (mg/L) -0,09 0,371 -0,06 0,617

The concentration of VEGF did not differ significantly between groups (Figure 1., Table 7.). The patients had higher concentrations of Ang-2 and Tie-2 receptor. (Figure 2.,3., Table 7.).

A significant difference in the concentrations of VEGF was found between diabetic and nondiabetic subjects (P= 0,006), with the median (interquartile range) concentration of VEGF in diabetics being higher than that observed in nondiabetic subjects: 358 (210-463) vs. 197 (130-335) ng/L. There was no difference in concentrations of VEGF, Ang-2, and Tie-2 receptor between smokers and non-smokers, nor between the subjects on lipolythic and antihyperten‐ sive therapy and subjects off therapy. All three serum biomarkers of angiogenesis correlated with the CRP concentrations (Table 8). The concentrations of HDL- cholesterol, VEGF, Ang-2, and Tie-2 were statistically significantly different among the subjects with various cardiovas‐ cular risk according to CRP concentrations (Table 9.). Post hoc tests (Mann–Whitney's test) suggested a significant difference in HDL -cholesterol values between the low risk subjects (CRP<1,0 mg/L) compared with the moderate (CRP between 1,0-3,0 mg/L) (P=0,004) and high risk (P=0,011) subjects (CRP >3,0 mg/L). The subject groups of moderate and high cardiovas‐ cular risk did not differ significantly in the HDL cholesterol concentration (P=0,666). Statisti‐ cally significant difference was found in the concentrations of VEGF (P=0,011), Ang-2 (P<0,001),

**Table 6.** Relationships between the catalytic concentrations of PAF-AH and serum lipids parameters and CRP

concentrations in the study groups: patients with peripheral arterial disease (PAD) and controls

**3.4. Serum VEGF, Ang-2 and Tie-2 concentrations**

**Female (N=18)**

**r P r P**

**P**

**Control subjects (N=64)**


**Table 7.** Biochemical parameters in the patients with peripheral arterial disease (PAD) and controls. Data are given as median (interquartile range)..

**Figure 1.** Comparison of VEGF concentrations (median, interquartile range) in the patients with peripheral arterial dis‐ ease (PAD) and control subjects.

**Figure 2.** Comparison of Ang-2 concentrations (median, interquartile range) in the patients with peripheral arterial disease (PAD) and control subjects.


**Table 8.** Spearman coefficient of correlation between the lipid profile, CRP and biomarkers of angiogenesis in patients with peripheral arterial disease (n=110).

**Figure 4.** Correlation between serum concentrations of VEGF and CRP in patients with peripheral arterial disease.

**PAD CONTROL**

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**Figure 3.** Comparison of Tie-2 concentrations (median, interquartile range) in the patients with peripheral arterial dis‐

Sperman coefficient of correlation r= 0,45; P<0,001.

15

ease (PAD) and control subjects.

20

25

**Tie-2 (ug/L)**

30

35

**Figure 3.** Comparison of Tie-2 concentrations (median, interquartile range) in the patients with peripheral arterial dis‐ ease (PAD) and control subjects.

**PAD CONTROL**

**Figure 2.** Comparison of Ang-2 concentrations (median, interquartile range) in the patients with peripheral arterial

Triglyceride (mmol/L) 0,01 0,955 -0,07 0,489 0,02 0,861

Total cholesterol (mmol/L) -0,13 0,182 -0,02 0,839 0,08 0,424

HDL-cholesterol (mmol/L) -0,26 0,006 -0,14 0,134 0,03 0,735

LDL- cholesterol (mmol/L) -0,05 0,581 0,01 0,955 0,01 0,909

CRP (mg/L) 0,45 <0,001 0,36 <0,001 0,25 0,008

**Table 8.** Spearman coefficient of correlation between the lipid profile, CRP and biomarkers of angiogenesis in patients

r P r P r P

**Biochemical parameters (units) VEGF( ng/L) Ang-2 (ng/L) Tie-2 (μg/L)**

1000

disease (PAD) and control subjects.

with peripheral arterial disease (n=110).

1500

2000

2500

**Ang-2 (ng/L)**

3000

3500

4000

106 Current Trends in Atherogenesis

**Figure 4.** Correlation between serum concentrations of VEGF and CRP in patients with peripheral arterial disease. Sperman coefficient of correlation r= 0,45; P<0,001.

**Figure 5.** Correlation between serum concentrations of Ang-2 and CRP in patients with peripheral arterial disease. Sperman coefficient of correlation r= 0,36; P<0,001.

significant increase in the score compared with nondiabetic subjects (13.77 ± 6.67 compared

concentrations as a cardiovascular risk marker. Levels of CRP below 1mg/L are considered low; levels of 1 - 3 mg/L are considered moderate and levels greater than 3 mg/L are considered high risk. Data are given as median (interquartile

**Patients with PAD (N=110)**

The Evaluation of New Biomarkers of Inflammation and Angiogenesis in Peripheral Arterial Disease

**low risk moderate risk high risk**

1,97

5,55

1,10

3,45

197 (100-319)

1803 (1527-2216)

20,4 (17,7-23,4)

PAF-AH (U/L) 350 (294-458) 417 (355-468) 399 (322-480) 0,452

**Table 9.** Biochemical parameters in the patients with peripheral arterial disease (PAD) according to CRP

**P**

109

http://dx.doi.org/10.5772/53341

Triglyceride (mmol/L) -0,13 0,167 Total cholesterol (mmol/L) -0,14 0,156 HDL-cholesterol (mmol/L) 0,06 0,539 LDL- cholesterol (mmol/L) -0,12 0,208 CRP (mg/L) 0,07 0,461 VEGF (ng/L) 0,08 0,406 PAF-AH (U/L) -0,08 0,450 Ang-2 (ng/L) 0,04 0,684 Tie-2 (μg/L) 0,13 0,171

**Table 10.** Spearman coefficient of correlation between the biochemical parameters and angiographic score in

**Angiographic score r P**

(1,39-2,40) 0,87 (1,19-2,38) 0,674

(4,83-6,10) 5,40 (4,50-6,00) 0,732

(0,93-1,30) 1,10 (0,90-1,30) 0,017

(2,80-4,18) 3,20 (2,70-3,90) 0,549

332

2256

22,4

(170-504) 0,002

(1707-3185) 0,003

(19,5-25,2) 0,018

with 11.02 ± 5.50; P=0,023).

**Biochemical parameters (units)**

Triglyceride (mmol/L) 1,59

Total cholesterol (mmol/L) 5,60

HDL-cholesterol (mmol/L) 1,40

LDL- cholesterol (mmol/L) 3,40

Tie-2 (μg/L) 18,7

VEGF (ng/L)

Ang-2 (ng/L)

range).

(1,32-2,21)

(4,50-6,00)

(1,10-1,60)

(2,40-3,80)

167 (88-234)

1663 (1379-2279)

(17,5-22,3)

**Biochemical parameters (units)**

patients with peripheral arterial disease (n=110).

**Figure 6.** Correlation between serum concentrations of Tie-2 and CRP in patients with peripheral arterial disease. Sperman coefficient of correlation r= 0,25; P=0,008.

#### **3.5. The relationship between the biochemical parameters under study and the anatomical extent of peripheral arterial atherosclerotic changes**

None of the biochemical parameters investigated correlated with the angiographic score as a measure of the anatomic extent of atherosclerotic alterations in the peripheral arteries.

(Table 10). From among the traditional risk factors, only the subject age correlated significantly with the angiographic score (r=0.33; P<0,001). The patients with diabetes had a statistically


**Figure 5.** Correlation between serum concentrations of Ang-2 and CRP in patients with peripheral arterial disease.

**Figure 6.** Correlation between serum concentrations of Tie-2 and CRP in patients with peripheral arterial disease.

**3.5. The relationship between the biochemical parameters under study and the anatomical**

None of the biochemical parameters investigated correlated with the angiographic score as a

(Table 10). From among the traditional risk factors, only the subject age correlated significantly with the angiographic score (r=0.33; P<0,001). The patients with diabetes had a statistically

measure of the anatomic extent of atherosclerotic alterations in the peripheral arteries.

Sperman coefficient of correlation r= 0,36; P<0,001.

108 Current Trends in Atherogenesis

Sperman coefficient of correlation r= 0,25; P=0,008.

**extent of peripheral arterial atherosclerotic changes**

**Table 9.** Biochemical parameters in the patients with peripheral arterial disease (PAD) according to CRP concentrations as a cardiovascular risk marker. Levels of CRP below 1mg/L are considered low; levels of 1 - 3 mg/L are considered moderate and levels greater than 3 mg/L are considered high risk. Data are given as median (interquartile range).

significant increase in the score compared with nondiabetic subjects (13.77 ± 6.67 compared with 11.02 ± 5.50; P=0,023).


**Table 10.** Spearman coefficient of correlation between the biochemical parameters and angiographic score in patients with peripheral arterial disease (n=110).

## **4. Discussion**

Peripheral artery disease is a systemic manifestation of atherosclerosis with significant morbidity and mortality. Pathophysiological processes implicated in the development, progression, and complications of the disease are complex and interdependent and include interactions between genetic and environmental factors. Pathophysiological events associated with peripheral artery disease include tissue ischaemia, and the severity of clinical presentation is dependent of the site and extent of peripheral arterial stenotic-occlusive changes and the availability of collateral circulation. Ischaemia incites a cascade of biochemical reactions, leading directly or indirectly to endothelial homeostasis disturbance. Dysfunctional endothe‐ lium is incapable of maintaining adhesiveness coagulation neutrality within the circulating blood, or regulating tonic arterial activity. In addition to disturbing vessel movements and promoting atherosclerosis formation, endothelium actively modulates the architecture of already present atherosclerotic plaques and increases vulnerability of the lesions which thus become prone to rupture and lead directly to the development of thromboembolic incidents. The role of the new biomarkers of inflammation, thrombosis, lipoprotein metabolism and oxidative stress, which are involved in the regulation of vascular homeostasis, is under an intensive investigation aimed at earlier detection and better understanding of the aetiology and progression of peripheral artery disease, as well as development of new therapeutic possibilities.

a significant positive correlation with the concentrations of triglicerides, total and LDL cholesterol. Thus, changes in enzymatic acitivities may also result from the changed concen‐ trations of lipid parameters, particularly if standardized catalytic PAF-AH concentrations are

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111

The development of vascular endothelial dysfunction is a key mechanism linking the risk factors and atherosclerosis, and it plays an important role in the pathophysiology of peripheral artery disease ( Brevetti et al., 2010). Vascular remodeling, as an adaptive response to haemo‐ dynamic and biochemical stressors, is characterized by progressive structural and functional alterations in blood vessel walls, preceding the development of a cardiovascular disease. Recent investigations suggest that a crucial role in the regulation of vascular homeostasis is played by the Tie ligand receptor system. Some smaller scale clinical trials have revealed that the concentrations of Ang-2, Tie-2, or both, are found in the patients with peripheral arterial disease (Findley et al., 2008), congestive heart failure (Chong et al., 2004), acute coronary syndrome (Lee et al., 2004), hypertension (Lim et al., 2004), and that they have a predictive

In our investigation, the serum concentrations of Ang-2 and its tyrosine kinase receptor, Tie-2, in the subjects analyzed were statistically significantly higher compared with those in the control subjects, which is in agreement with the results by Findley et al., (2008) (8). However, contrary to their results, the VEFG concentrations were not found to be statistically signifi‐ cantly different between our groups. The above mentioned differences in the results may be accounted for by the great biological variability observed for VEGF. In fact, it is well known that interindividual and intraindividual variability of VEFG differ significantly depending on the kind of material used. Analysis samples include serum, whole blood, and plasma. The intraindividual variation of VEGF in serum, plasma, and whole blood is 10.7%, 14.1%, and 14.1%, respectively, and the interindividual variation of VEGF in serum, whole blood, and plasma is 47.6%, 28.8%, and 18.1%, respectively (Meo et al., 2005). The greater intraindividual variability in the whole blood is impacted by the release of VEGF from lymphocytes, granu‐ locytes, monocytes, and megakariocytes, variability also being dependent on the process of leukocyte lysis, irrespective of the use of standardized methods (Meo et al., 2005) In light of the potential clinical utility of VEGF in the prognosis, patient selection, and follow-up of anti-VEFG therapeutic effects, Kong et al., (2008) (49) have constructed the reference intervals for VEFG in the serum and plasma of the population of the Republic of North Korea using the ELISA method with R&D Systems reagents. The reference intervals were calculated in 131 subjects, aged 20 to 78 years (68 males and 63 females). Reference intervals differ considerably in serum and plasma, whereat the values in serum are ten- to twenty eight- fold higher than

Moreover, plasma concentrations of VEGF depend on the kind of anticoagulant, with the values being considerably higher when determined by EDTA as an anticoagulant than when determined using heparin as an anticoagulant. In addition to VEGF, concentrations of Ang-2 and Tie-2 also statistically significantly differ according to gender and kind of material used

observed in relation to LDL cholesterol.

ability for myocardial infarction (Patel et al., 2005).

those in plasma.

(Lieb et al., 2010).

The catalytic concentrations of PAF-AH did not differ significantly between the subjects and the control group, contrary to their standardized catalytic concentrations (PAF-AH/LDL) which were statistically significantly higher (<0,001) in the subjects analyzed compared with the control group. The catalytic levels of PAF-AH were significantly different between the genders in the control group, females (n=36) having lower values than males (n=28), which is consistent with the literature data (Winkler et al., 2005; Iribarren, 2010). Moreover, females also had lower PAF-AH standardized catalytic concentrations in both groups studied. Changes in the PAF-AH catalytic levels depend on the concentrations of lipid status parameters, whereat the PAF-AH catalytic concentrations show a statistically significant positive correlation with the concentration of triglicerides, total and LDL cholesterol, the atherosclerosis index, and the total/HDL cholesterol ratio. Statistically significant negative correlation was found between the catalytic concentration of PAF-AH and the concentration of HDL cholesterol in the control group, which is consistent with literature data (Winkler et al., 2005; Flegar-Meštrić et al., 2003; Kamisako et al., 2003; Flegar-Meštrić et al., 2008; Flegar-Meštrić et al, 2012). PAF-AH catalytic levels did not correlate with the CRP concentration in either of the groups examined.

The results of the present study are consistent with our previous results obtained for the patients with lesions of the cerebral arteries (Flegar-Meštrić et al., 2003; Flegar-Meštrić et al., 2008; Flegar-Meštrić et al., 2012). However, in this investigation, we failed to confirm our previous results in 182 patients with peripheral arterial disease in whom PAF-AH catalytic concentrations were significantly higher compared with the control group (Perkov et al., 2010). The differences in the results obtained can be explained by the differences in the number of patients included in the analysis. Furthermore, the PAF-AH catalytic concentrations are in a significant positive correlation with the concentrations of triglicerides, total and LDL cholesterol. Thus, changes in enzymatic acitivities may also result from the changed concen‐ trations of lipid parameters, particularly if standardized catalytic PAF-AH concentrations are observed in relation to LDL cholesterol.

**4. Discussion**

110 Current Trends in Atherogenesis

possibilities.

Peripheral artery disease is a systemic manifestation of atherosclerosis with significant morbidity and mortality. Pathophysiological processes implicated in the development, progression, and complications of the disease are complex and interdependent and include interactions between genetic and environmental factors. Pathophysiological events associated with peripheral artery disease include tissue ischaemia, and the severity of clinical presentation is dependent of the site and extent of peripheral arterial stenotic-occlusive changes and the availability of collateral circulation. Ischaemia incites a cascade of biochemical reactions, leading directly or indirectly to endothelial homeostasis disturbance. Dysfunctional endothe‐ lium is incapable of maintaining adhesiveness coagulation neutrality within the circulating blood, or regulating tonic arterial activity. In addition to disturbing vessel movements and promoting atherosclerosis formation, endothelium actively modulates the architecture of already present atherosclerotic plaques and increases vulnerability of the lesions which thus become prone to rupture and lead directly to the development of thromboembolic incidents. The role of the new biomarkers of inflammation, thrombosis, lipoprotein metabolism and oxidative stress, which are involved in the regulation of vascular homeostasis, is under an intensive investigation aimed at earlier detection and better understanding of the aetiology and progression of peripheral artery disease, as well as development of new therapeutic

The catalytic concentrations of PAF-AH did not differ significantly between the subjects and the control group, contrary to their standardized catalytic concentrations (PAF-AH/LDL) which were statistically significantly higher (<0,001) in the subjects analyzed compared with the control group. The catalytic levels of PAF-AH were significantly different between the genders in the control group, females (n=36) having lower values than males (n=28), which is consistent with the literature data (Winkler et al., 2005; Iribarren, 2010). Moreover, females also had lower PAF-AH standardized catalytic concentrations in both groups studied. Changes in the PAF-AH catalytic levels depend on the concentrations of lipid status parameters, whereat the PAF-AH catalytic concentrations show a statistically significant positive correlation with the concentration of triglicerides, total and LDL cholesterol, the atherosclerosis index, and the total/HDL cholesterol ratio. Statistically significant negative correlation was found between the catalytic concentration of PAF-AH and the concentration of HDL cholesterol in the control group, which is consistent with literature data (Winkler et al., 2005; Flegar-Meštrić et al., 2003; Kamisako et al., 2003; Flegar-Meštrić et al., 2008; Flegar-Meštrić et al, 2012). PAF-AH catalytic levels did not correlate with the CRP concentration in either of the groups examined.

The results of the present study are consistent with our previous results obtained for the patients with lesions of the cerebral arteries (Flegar-Meštrić et al., 2003; Flegar-Meštrić et al., 2008; Flegar-Meštrić et al., 2012). However, in this investigation, we failed to confirm our previous results in 182 patients with peripheral arterial disease in whom PAF-AH catalytic concentrations were significantly higher compared with the control group (Perkov et al., 2010). The differences in the results obtained can be explained by the differences in the number of patients included in the analysis. Furthermore, the PAF-AH catalytic concentrations are in

The development of vascular endothelial dysfunction is a key mechanism linking the risk factors and atherosclerosis, and it plays an important role in the pathophysiology of peripheral artery disease ( Brevetti et al., 2010). Vascular remodeling, as an adaptive response to haemo‐ dynamic and biochemical stressors, is characterized by progressive structural and functional alterations in blood vessel walls, preceding the development of a cardiovascular disease. Recent investigations suggest that a crucial role in the regulation of vascular homeostasis is played by the Tie ligand receptor system. Some smaller scale clinical trials have revealed that the concentrations of Ang-2, Tie-2, or both, are found in the patients with peripheral arterial disease (Findley et al., 2008), congestive heart failure (Chong et al., 2004), acute coronary syndrome (Lee et al., 2004), hypertension (Lim et al., 2004), and that they have a predictive ability for myocardial infarction (Patel et al., 2005).

In our investigation, the serum concentrations of Ang-2 and its tyrosine kinase receptor, Tie-2, in the subjects analyzed were statistically significantly higher compared with those in the control subjects, which is in agreement with the results by Findley et al., (2008) (8). However, contrary to their results, the VEFG concentrations were not found to be statistically signifi‐ cantly different between our groups. The above mentioned differences in the results may be accounted for by the great biological variability observed for VEGF. In fact, it is well known that interindividual and intraindividual variability of VEFG differ significantly depending on the kind of material used. Analysis samples include serum, whole blood, and plasma. The intraindividual variation of VEGF in serum, plasma, and whole blood is 10.7%, 14.1%, and 14.1%, respectively, and the interindividual variation of VEGF in serum, whole blood, and plasma is 47.6%, 28.8%, and 18.1%, respectively (Meo et al., 2005). The greater intraindividual variability in the whole blood is impacted by the release of VEGF from lymphocytes, granu‐ locytes, monocytes, and megakariocytes, variability also being dependent on the process of leukocyte lysis, irrespective of the use of standardized methods (Meo et al., 2005) In light of the potential clinical utility of VEGF in the prognosis, patient selection, and follow-up of anti-VEFG therapeutic effects, Kong et al., (2008) (49) have constructed the reference intervals for VEFG in the serum and plasma of the population of the Republic of North Korea using the ELISA method with R&D Systems reagents. The reference intervals were calculated in 131 subjects, aged 20 to 78 years (68 males and 63 females). Reference intervals differ considerably in serum and plasma, whereat the values in serum are ten- to twenty eight- fold higher than those in plasma.

Moreover, plasma concentrations of VEGF depend on the kind of anticoagulant, with the values being considerably higher when determined by EDTA as an anticoagulant than when determined using heparin as an anticoagulant. In addition to VEGF, concentrations of Ang-2 and Tie-2 also statistically significantly differ according to gender and kind of material used (Lieb et al., 2010).

From among the parameters analyzed, only VEGF showed a statistically significant negative relationship with age in the control subjects. The Ang-2 concentrations were statistically significantly higher in the control group females. Other parameters were not statistically significantly different between male and female subjects of the groups studied.

factor in the development and progression of atherosclerosis, is extensively investigated. Tzoulaki et al., (2005), in a large prospective trial nested within the Edinburgh Artery Study confirmed the role of CRP, interleukin-6 (IL-6), and intercellular adhesion molecule (ICAM) in the progression of peripheral artery disease in the general population. The trial included 1582 individuals, ranging in age 55 to 75 years, and atherosclerotic progression was defined as reduction in the ankle brachial index (ABI) over the period of 5 and 12 years. In the investigation of the patients with peripheral arterial disease who had ABI<0.90, the CRP levels greater than 3.0 mg/L had an additive predictive value to risk assessment for adverse cardio‐ vascular events (Khawaja & Kullo, 2009). Although in the clinical practice, ABI measurement is considered a simple method of assessing peripheral artery disease progression, and the ABI values correlate well with the degree of peripheral arterial atherosclerotic changes as measured using the digital subtraction angiography method, these two methods represent different aspects of severity assessment of peripheral arterial disease, and cannot be directly compared ( Nylaende et al., 2006). Nylaende et al., (2006) evaluated the relationship between inflamma‐ tory markers and the severity of peripheral artery disease assessed on the basis of the angio‐ graphic score and ABI determined with and without Treadmill test. The study was conducted in 127 patients, range 45-79 years, with the simptoms of intermittent claudication in whom the angiographic score was determined based on the angiographic criteria for haemodynamically significant stenosis. The results of their study demonstrated significant associations of MCP-1, CD40L, IL-6, and TNF-alpha with the angiographic score contrary to the concentrations of CRP, IL-10, E-selectin, P-selectin, ICAM-1, and VCAM-1 for which no significant associations with the angiographic score were observed. ICAM-1 and IL-6 showed a statistically significant correlation with the maximum walking distance on the treadmill, and neither of the markers under study correlated with the ABI. Based on the available data, this is the only investigation into the association between the inflammation marker and the extent of angiographically detected atherosclerotic alterations in the patients with peripheral aterosclerosis. A substantial number of studies have investigated the correlation between the marker of inflammation and the degree of angiographically demonstrated atherosclerotic changes in cerebral and coronary atherosclerosis. Flegar-Meštrić et al., ( 2007), in a study of 119 patients, age range between 43 and 80 years, with stenosis of extracranial cerebral arteries found a significant association between the CRP level and stenotic extent greater than 70% compared with the control group with normal-appearing cerebral arteries on ultrasonography. The association with CRP of angiographically confirmed coronary atherosclerosis is controversial. Coronary disease and CRP are considered to independently and additively contribute to the risk for adverse cardiovascular events. Angiographic imaging seems to detect stable and instable plaques, and the value of CRP lies in its ability to predict myocardial infarction or fatal outcome independ‐ ently of the result of angiography (Niccoli et al., 2008; Geluk et al., 2008). Niccoli et al., (2008), in an investigation of 97 patients with unstable angina, failed to demonstrate any correlation between the basal CRP values and the severity of angiographic changes. In a prospective study within the Prevention of Renal and Vascular Endstage Disease (PREVEND) trial, including 8,139 individuals with no presence of coronary artery disease, Geluk et al., (2008) found weak correlations between the basal CRP concentrations and the degree of alterations demonstrated

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113

on angiogram in 216 patients who developed coronary disease over a 5-year period.

The levels of VEGF, Ang-2, and Tie-2 determined in the serum of the control group were within the value range for healthy individuals set out by the manufacturer and other au‐ thors using the same method and reagent from the same manufacturer (Lieb et al., 2010; Nylaende et al., 2006).

A significant difference in the concentrations of VEGF was found between diabetic and nondiabetic subjects, with the median concentration of VEGF in diabetics being higher than that observed in nondiabetic subjects. There was no difference in concentrations of VEGF, Ang-2, and Tie-2 receptor between smokers and non-smokers, nor between the subjects on lipolythic and antihypertensive therapy and subjects off therapy.

In the patients with peripheral arterial disease, VEGF significantly correlated with CRP (r=0,45, P<0.001) and HDL cholesterol (r= - 0,26, P=0,006). Angiopoietin-2 significantly correlated with CRP (r=0,36, P<0,001), as well as Tie-2 which showed a weak but significant association with CRP (r=0,25, P=0,008).

Because all three markers of angiogesis correlated with the CRP concentration in the group studied, compared with the controls, and a correlation between the concentrations of VEGF and HDL cholesterol was found, we examined whether the concentrations of the biochemical parameters under study differed depending on the CRP concentration as a cardiovascular risk factor. The concentrations of HDL- cholesterol, VEGF, Ang-2, and Tie-2 were statistically significantly different among the subjects with various cardiovascular risk profiles, with the HDL -cholesterol values being significantly higher in the low risk subjects (CRP<1,0 mg/L) compared with the moderate (CRP between 1,0-3,0 mg/L) (P=0,004) and high risk (P=0,011) subjects (CRP >3,0 mg/L). The subject groups of moderate and high cardiovascular risk did not differ significantly in the HDL cholesterol concentration (P=0,666). Statistically significant difference was found in the concentrations of VEGF (P=0,011), Ang-2 (P<0,001), and Tie-2 receptor (P=0,005) between low and high risk subjects, as well as in the concentrations of VEGF (P=0,012), Ang-2 (P<0,001), and Tie-2 receptor (P=0,02) between the moderate and high cardiovascular risk subjects, whereas there were no statistically significant differences in the concentrations of VEGF (P=0,377), Ang-2 (P=0,438), and Tie-2 receptor (P=0,673) between the groups of low and moderate cardiovascular risk subjects. The results are suggestive of an association between inflammation and angiogenesis in peripheral arterial disease.

In this investigation, no association was found of the biochemical parameters under study, namely, triglycerides, total, HDL-, LDL-cholesterol, CRP, and novel biomarkers of inflamma‐ tion (PAF-AH) and angiogenesis (VEGF, Ang-2, and Tie-2 receptor) with the angiographic score as a measure of the anatomic extent of atherosclerotic alterations in the peripheral arteries.

It has been well documented that inflammation is implicated in all stages of the atherosclerotic process. The role of CRP, as a nonspecific marker of inflammation and cardiovascular risk From among the parameters analyzed, only VEGF showed a statistically significant negative relationship with age in the control subjects. The Ang-2 concentrations were statistically significantly higher in the control group females. Other parameters were not statistically

The levels of VEGF, Ang-2, and Tie-2 determined in the serum of the control group were within the value range for healthy individuals set out by the manufacturer and other au‐ thors using the same method and reagent from the same manufacturer (Lieb et al., 2010;

A significant difference in the concentrations of VEGF was found between diabetic and nondiabetic subjects, with the median concentration of VEGF in diabetics being higher than that observed in nondiabetic subjects. There was no difference in concentrations of VEGF, Ang-2, and Tie-2 receptor between smokers and non-smokers, nor between the subjects on

In the patients with peripheral arterial disease, VEGF significantly correlated with CRP (r=0,45, P<0.001) and HDL cholesterol (r= - 0,26, P=0,006). Angiopoietin-2 significantly correlated with CRP (r=0,36, P<0,001), as well as Tie-2 which showed a weak but significant association with

Because all three markers of angiogesis correlated with the CRP concentration in the group studied, compared with the controls, and a correlation between the concentrations of VEGF and HDL cholesterol was found, we examined whether the concentrations of the biochemical parameters under study differed depending on the CRP concentration as a cardiovascular risk factor. The concentrations of HDL- cholesterol, VEGF, Ang-2, and Tie-2 were statistically significantly different among the subjects with various cardiovascular risk profiles, with the HDL -cholesterol values being significantly higher in the low risk subjects (CRP<1,0 mg/L) compared with the moderate (CRP between 1,0-3,0 mg/L) (P=0,004) and high risk (P=0,011) subjects (CRP >3,0 mg/L). The subject groups of moderate and high cardiovascular risk did not differ significantly in the HDL cholesterol concentration (P=0,666). Statistically significant difference was found in the concentrations of VEGF (P=0,011), Ang-2 (P<0,001), and Tie-2 receptor (P=0,005) between low and high risk subjects, as well as in the concentrations of VEGF (P=0,012), Ang-2 (P<0,001), and Tie-2 receptor (P=0,02) between the moderate and high cardiovascular risk subjects, whereas there were no statistically significant differences in the concentrations of VEGF (P=0,377), Ang-2 (P=0,438), and Tie-2 receptor (P=0,673) between the groups of low and moderate cardiovascular risk subjects. The results are suggestive of an

association between inflammation and angiogenesis in peripheral arterial disease.

In this investigation, no association was found of the biochemical parameters under study, namely, triglycerides, total, HDL-, LDL-cholesterol, CRP, and novel biomarkers of inflamma‐ tion (PAF-AH) and angiogenesis (VEGF, Ang-2, and Tie-2 receptor) with the angiographic score as a measure of the anatomic extent of atherosclerotic alterations in the peripheral

It has been well documented that inflammation is implicated in all stages of the atherosclerotic process. The role of CRP, as a nonspecific marker of inflammation and cardiovascular risk

significantly different between male and female subjects of the groups studied.

lipolythic and antihypertensive therapy and subjects off therapy.

Nylaende et al., 2006).

112 Current Trends in Atherogenesis

CRP (r=0,25, P=0,008).

arteries.

factor in the development and progression of atherosclerosis, is extensively investigated. Tzoulaki et al., (2005), in a large prospective trial nested within the Edinburgh Artery Study confirmed the role of CRP, interleukin-6 (IL-6), and intercellular adhesion molecule (ICAM) in the progression of peripheral artery disease in the general population. The trial included 1582 individuals, ranging in age 55 to 75 years, and atherosclerotic progression was defined as reduction in the ankle brachial index (ABI) over the period of 5 and 12 years. In the investigation of the patients with peripheral arterial disease who had ABI<0.90, the CRP levels greater than 3.0 mg/L had an additive predictive value to risk assessment for adverse cardio‐ vascular events (Khawaja & Kullo, 2009). Although in the clinical practice, ABI measurement is considered a simple method of assessing peripheral artery disease progression, and the ABI values correlate well with the degree of peripheral arterial atherosclerotic changes as measured using the digital subtraction angiography method, these two methods represent different aspects of severity assessment of peripheral arterial disease, and cannot be directly compared ( Nylaende et al., 2006). Nylaende et al., (2006) evaluated the relationship between inflamma‐ tory markers and the severity of peripheral artery disease assessed on the basis of the angio‐ graphic score and ABI determined with and without Treadmill test. The study was conducted in 127 patients, range 45-79 years, with the simptoms of intermittent claudication in whom the angiographic score was determined based on the angiographic criteria for haemodynamically significant stenosis. The results of their study demonstrated significant associations of MCP-1, CD40L, IL-6, and TNF-alpha with the angiographic score contrary to the concentrations of CRP, IL-10, E-selectin, P-selectin, ICAM-1, and VCAM-1 for which no significant associations with the angiographic score were observed. ICAM-1 and IL-6 showed a statistically significant correlation with the maximum walking distance on the treadmill, and neither of the markers under study correlated with the ABI. Based on the available data, this is the only investigation into the association between the inflammation marker and the extent of angiographically detected atherosclerotic alterations in the patients with peripheral aterosclerosis. A substantial number of studies have investigated the correlation between the marker of inflammation and the degree of angiographically demonstrated atherosclerotic changes in cerebral and coronary atherosclerosis. Flegar-Meštrić et al., ( 2007), in a study of 119 patients, age range between 43 and 80 years, with stenosis of extracranial cerebral arteries found a significant association between the CRP level and stenotic extent greater than 70% compared with the control group with normal-appearing cerebral arteries on ultrasonography. The association with CRP of angiographically confirmed coronary atherosclerosis is controversial. Coronary disease and CRP are considered to independently and additively contribute to the risk for adverse cardiovascular events. Angiographic imaging seems to detect stable and instable plaques, and the value of CRP lies in its ability to predict myocardial infarction or fatal outcome independ‐ ently of the result of angiography (Niccoli et al., 2008; Geluk et al., 2008). Niccoli et al., (2008), in an investigation of 97 patients with unstable angina, failed to demonstrate any correlation between the basal CRP values and the severity of angiographic changes. In a prospective study within the Prevention of Renal and Vascular Endstage Disease (PREVEND) trial, including 8,139 individuals with no presence of coronary artery disease, Geluk et al., (2008) found weak correlations between the basal CRP concentrations and the degree of alterations demonstrated on angiogram in 216 patients who developed coronary disease over a 5-year period.

In our investigation, we found no evidence of associations between the CRP level and the extent of peripheral arterial changes on angiography, which is consistent with the results by Nylaende M. et al. (2006). It is also possible that some of the biomarkers for which a difference in concentrations between the groups studied has been found are involved in other mecha‐ nisms of vascular homeostasis regulation, and that they have importance in earlier phases of development of peripheral arterial atherosclerotic changes, which evade detection by the digital subtraction angiography method.

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