The study was conducted in accordance to the Institutional "Guide for the Care and Use of Laboratory Animals" and was approved by the Institutional Animal Care and Use Committee of the West Greece Prefecture and the University of Patras. All experiments were performed in the Animal House of the Medical School of Patras University. Sixteen male New Zealand White rabbits, weighing 3-3.5 kg, were fed regular rabbit chow for one week, randomly divided into two treatment groups of eight animals and then fed either an atherogenic diet (Group A, 4% cholesterol plus regular rabbit chow, without additional atherogenic components; ELPEN pharmaceutical, Athens, Greece) or regular chow (Group B) for 8 weeks. Rabbits were housed individually at 20 ± 3°C with a 12-h:12-h light/dark cycle and with free access to water. Feeding was restricted to 120 g/day. Blood samples were collected every 4 weeks. The general appearance of the rabbits was observed daily. Body weights were measured every 4 weeks.

Rabbits were anesthetized with ketamine (50 mg/kg) plus xylazine (10 mg/kg) intramuscularly, and sacrificed by intravenous injection of a saturated KCl solution. The descending thoracic aorta was exposed, and a 50 mm segment was labeled in situ with two reference dots, cut at these points and excised. The marks guided us to prestrain the vessel segment in vitro so as to retain the above mentioned physiological length in situ [⁸]. External fatty layers were trimmed, and lateral branches were ligated. The specimens were stored in normal saline 0.9% at 4°C prior to mechanical testing. All mechanical tests were performed immediately after animal sacrifice.

The cut ends of each arterial segment were carefully secured in plastic hollow nozzles of proper size and connected to the "arterial branch" of a custom designed, closed loop mock circulatory system consisting of a left ventricular device (LVD) connected to hydraulic circuit of transparent, rigid plastic tubes. A diagram of the apparatus is presented in figure 1. The LVD was composed of a pneumatic silicon rubber sack enclosed in a tightened box, with mechanical disc valves at the inlet and outlet to produce unidirectional fluid flow in the closed circuit. A custom electronic controller (SC) stimulated two solenoid valves (S) working sequentially in opposite on-off mode. A pressurized air line connected to the mid-space between the rubber sack and the rigid box of the LVD was used to produce alternating compression and expansion of the LVD at time-periods 1/3 and 2/3 of the cycle, respectively. Windkessel elements (air-liquid interface in closed vessels connected with the hydraulic line) at the "proximal" (AC) and "distal" (VC) ends of the aortic segment, and flow resistance elements (PR) produced the corresponding vascular compliance and peripheral resistance. Arterial pressure was monitored invasively at the proximal end of the aortic segment using an electronic manometer (PM, CYQ 103 bridge transducer interface, Cybersience Inc. USA) and a Deltran DP-100 pressure transducer (PT, Utah Medical Products USA). External vessel diameter was measured using a 30-mm range laser micrometer (LSM, micro-epsilon GMBH, optocontrol type ODC 1201-30; Figure 2). Arterial flow was monitored using a cannulated medical electromagnetic flow meter (FM501) with a 300 A 1/2" flow probe (Karolina Medical Electronics, USA). All electronic analog voltage signals (pressure, diameter, and flow) were recorded electronically via an A/D converter card (DI 700, Data Q, USA). Raw data (in volts) were converted into mechanical units using appropriate calibration factors and used for mechanical analysis.

For mechanical testing, aortic segments were positioned into the hydraulic system and the whole circuit was filled with normal saline at room temperature. The aortic specimens, after physiological prestraining, were subjected to pulsatile flow of 400 ml/min (mean arterial flow), at 60 cycles/min, with peak systolic/diastolic pressure values of 180/0⁺ mmHg respectively. The diastolic pressure was non physiological low (approximately 0 mmHg), to maximize the pressure-dilation range of mechanical testing. Representative flow, pressure, and diameter traces for four consecutive cycles are shown in Figure 3.

Different expressions of arterial wall elasticity have been introduced, using complicated constitutive equations of axial and peripheral stress and strain, so as to describe mechanical properties of arterial wall [¹⁷]. Pressure-strain elastic modulus (Peterson's modulus; E_P) is an expression of arterial wall stiffness, according the following relationship:

where D₀ is the outer vessel diameter at a certain P and ΔP and ΔD are the changes in pressure and diameter [¹⁸]. For small increments of P and D, E_P is represented by the slope of the tangential line to the loading portion of the P- (D/D₀) diagrams at a certain (P, D/D₀) point of the pressure-dilatation curve.

In present study, experimental data of pressure and external diameter were further analyzed to calculate a modified arterial stiffness E_Pm. External diameter (D) measurements, ranged from the minimum diameter D_min at minimum internal pressure (0⁺ mmHg) to that at maximum applied pressure, were divided by a reference pressure, (D_S), so as to compute normalized radial arterial dilatation (RAD) as follows:

where D is the external diameter at minimum or maximum internal pressures, and D_S is the external diameter at a reference pressure of 100 mmHg [⁵]. Hence, E_Pm at each pressure was computed as:

Total serum cholesterol (TC), triglyceride (TG), and high density lipoprotein (HDL) levels were used to measure hyperlipidemia, and renal and liver function were monitored using urine, creatinine and serum hepatic enzymes (SGPT and g-GT) levels, respectively.

After mechanical testing, vessels were fixed by immersion in neutrally buffered 10% formalin, followed by dehydration and embedding in paraffin wax using standard procedures. Four-micrometer sections were obtained from each vessel at 5-mm intervals and stained with hematoxylin and eosin for histopathologic analysis. Masson's trichrome aniline blue and Weigert Van Gieson's elastic stains (Histoline Laboratories, Italy) were used to assay collagen components and the thickness of the intima, respectively.

Consecutive 4-μm-thick sections from each aortic specimen were collected on Superfrost plus glass slides, deparaffinized, and rehydrated in graded alcohols. Endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide for 15 min, followed by incubation with protein blocking solution to eliminate nonspecific binding. Immunohistochemistry was performed using monoclonal antibodies to detect macrophages (RAM 11, 1:200 dilution; Dako Corp, CA, USA), and α-actin in SMCs (HHF-35, 1:100 dilution; DAKO A/S). The Envision Plus Detection System kit (DakoCytomation, USA) and 3, 3'-diaminobenzidine (DAB) were used to visualize antibody binding, according to manufacturer's instructions. Sections were counterstained with Harris' hematoxylin, dehydrated, and mounted permanently. For each antibody, all tissues from the different study animals were immunostained concurrently. Negative controls were performed in all cases by omitting the primary antibodies.

SPSS for Windows (release 17. 0. 0 SPSS Inc, Chicago IL, USA) was used for continuous data analysis. All data are expressed as the mean ± standard deviation (SD). Statistical significance was determined using a two-tailed Student t test. A p value < 0.05 denotes statistical significance.

In rabbits of group A fed the atherogenic diet, increases in mean TC, TG, and HDL levels (mg/dl) from 4 to 8 weeks were as follows: TC increased from 4121 ± 415 to 4423 ± 493.40, TG from 381 ± 54.60 to 502 ± 96.24, and HDL from 383 ± 40.50 to 412 ± 15.40 (Table 1). In control animals (group B), TC and TG increased slightly after 8 weeks but remained within the normal range, revealing a statistical significant difference (p < 0.001) between the two groups (Table 1). At the end of the 8-week period, there was no statistically significant change in body weight between the hypercholesterolemic and control rabbits (3600 ± 92.58 g and 3706 ± 152.20 g, respectively), and the animals on the atherogenic diet had normal renal and liver function, confirming that the 4% cholesterol diet was not overtly toxic.

Hematoxylin and eosin-stained sections of aorta were examined for signs of atheroma. Whereas animals on the normal diet had no visible atheroma, the atherogenic diet induced a statistically significant incidence of atherosclerotic lesions in all group A animals (p < 0.001). Lesions were classified according to the guidelines of the American Heart Association [¹⁹] as intermediate (type III, n = 4 animals) or advanced (mostly type IV; n = 4 animals). Type III lesions consisted of intimal hyperplasia, foam cells, SMCs with visible lipid inclusions, and extracellular deposits of lipid. Type IV lesions consisted of nuclear lipid droplets, lack of any marked fibrous tissue, extracellular matrix rich in proteoglycans, and abundant infiltrating macrophages, lymphocytes, and foam cells [¹⁹]. Notably, type IV is the first lesion considered advanced in this classification because of the severe intimal disorganization caused by the lipid core. The characteristic core appears to develop from an increase and the consequent confluence of the small isolated pools of extracellular lipid that characterize type III lesions. The increase in lipid is believed to result from continued insudation from the plasma [¹⁹]. Masson and Van Gieson staining revealed a slight deposition of collagen tissue in tunica media and intima, as well as disruption of elastic fibers in internal elastic lamina of atherosclerotic aortas, whereas no such changes were observed in aortas from group B (Figures 4 and 5). The severity of the aortic atherosclerotic lesions was positively correlated with the high total cholesterol serum level.

Immunohistochemical staining was graded on a scale of 0 to 3 based on the percentage of immunopositive cells as follows: 0, <10% positive cells; 1 (mildly positive), 10-35% positive cells; 2 (moderately positive), 35-70% positive cells; and 3 (strongly positive), >70% positive cells. The high cholesterol diet was associated with a significant increase in lipid deposition and foam cell formation, indicated by the increase in RAM-11 immunoreactivity in animals of group A (Table 2). The aortas from all animals of group A were strongly positive for RAM-11 staining, whereas no staining was observed in aortas of control animals (Figure 6). We also observed that the number of RAM-11 positive cells correlated with the severity of the atherosclerotic lesions.

HHF-35 immunostaining for α-actin revealed that the aortic atherosclerotic lesions of all hypercholesterolemic animals contained either mild (n = 4 animals) or moderate (n = 4) numbers of SMCs, compared with 0% in control animals (Figure 7). Moreover, the percentage of SMCs correlated with the severity of atherosclerosis.

The aorta segments were subjected to alternating cycles of physiological systole and diastole, and the changes in external vessel wall diameter were measured. During each pressure cycle we observed a >30% increase in external vessel diameter at peak pressure (Dmax) in comparison with the diameter at minimum pressure (Dmin). Plotting pressure vs. RAD demonstrated a nonlinear relationship between 0-200 mmHg (Figure 8), similar to stress-strain curves of other soft tissues. During each pressure cycle, the pressure loading curve followed a different pathway compared to pressure unloading. This loop relationship indicates energy dissipation during each pressure cycle and demonstrates the viscoelastic characteristics of the arterial wall [²⁰,²¹]. In contrast to the nonlinear curve observed over in the entire pressure range, the relationship between pressure and RAD was close to linear at physiological pressures 50-150 mmHg (correlation coefficient R2 of the linear fitting curve equation was computed with values > 0.8). Rather than calculate E_Pm values at particular pressures [¹⁸], we determined average E_Pm throughout the linear range. Diameter measurements were performed for 50 sequential pressure cycles at the proximal, center and distal portions of each vessel. Sets of at least five consecutive stable cycles were identified, and the slope value in each position was computed from a single pressure cycle from each set. The mean stiffness of the local slope values at all three locations were derived for each specimen.

These results demonstrated that the atherogenic diet resulted in a significant decrease of mean stiffness (51.52 ± 8.76 kPa; p = 0.005) compared to control animals (68.98 ± 11.98 kPa; Figure 9). Comparing the mechanical properties and histological changes, we found that the decrease in wall stiffness was negatively correlated with the severity of the atherosclerosis after the 8-week feeding period, as atherosclerotic lesions are mainly fatty, composed of foam cells and high extracellular lipid accumulation, without any marked fibrous tissue formation, as determined by Masson staining, that might increase aortic stiffness (type III and IV lesions).