Structural and biomechanical alterations in rabbit thoracic aortas are associated with the progression of atherosclerosis
© Koniari et al; licensee BioMed Central Ltd. 2011
Received: 27 May 2011
Accepted: 26 July 2011
Published: 26 July 2011
Atherosclerosis is a diffuse and highly variable disease of arteries that alters the mechanical properties of the vessel wall through highly variable changes in its cellular composition and histological structure. We have analyzed the effects of acute atherosclerotic changes on the mechanical properties of the descending thoracic aorta of rabbits fed a 4% cholesterol diet.
Two groups of eight male New Zealand White rabbits were randomly selected and fed for 8 weeks either an atherogenic diet (4% cholesterol plus regular rabbit chow), or regular chow. Animals were sacrificed after 8 weeks, and the descending thoracic aortas were excised for pressure-diameter tests and histological evaluation to examine the relationship between aortic elastic properties and atherosclerotic lesions.
All rabbits fed the high-cholesterol diet developed either intermediate or advanced atherosclerotic lesions, particularly American Heart Association-type III and IV, which were fatty and contained abundant lipid-filled foam cells (RAM 11-positive cells) and fewer SMCs with solid-like actin staining (HHF-35-positive cells). In contrast, rabbits fed a normal diet had no visible atherosclerotic changes. The atherosclerotic lesions correlated with a statistically significant decrease in mean vessel wall stiffness in the cholesterol-fed rabbits (51.52 ± 8.76 kPa) compared to the control animals (68.98 ± 11.98 kPa), especially in rabbits with more progressive disease.
Notably, stiffness appears to decrease with the progression of atherosclerosis after the 8-week period.
Atherosclerosis is a progressive inflammatory process involving complex interactions between elevated plasma cholesterol and low-density lipoprotein (LDL) levels, arterial endothelial and smooth muscle cells (ECs and SMCs, respectively), migratory inflammatory cells (monocytes, T lymphocytes, platelets), and the pro-inflammatory cytokines that they release [1, 2]. According to the response to injury hypothesis, atherosclerosis is triggered by factors, such as hyperlipidemia or mechanical injury, that stimulate endothelial dysfunction, the migration and proliferation of monocytes and SMCs into subendothelial spaces, followed by neointimal hyperplasia, plaque fibrosis and calcification [3, 4]. Atherosclerotic lesions are highly variable in morphology at different stages of disease progression and are structurally heterogeneous, anisotropic, and incompressible [5–7]. These pathophysiologic changes alter the morphology, material properties, and mechanical behavior of the arterial wall [8–10], which becomes non-linearly viscoelastic and subject to large strain deformations that induce additional rheological abnormalities .
Many in vitro and in vivo studies have demonstrated changes in the biomechanical properties of the aortic wall due to pathological conditions such as hypertension and atherosclerosis . High resolution echo tracking technology [12–14] has shown reduced elasticity (stiffening) of arterial walls in vivo, particularly at sites of plaque formation. Mechanical testing has been used in vitro to study strips of arterial wall under uniaxial tension [15, 16] or whole arterial segments dilated by pressurized fluid [7, 8]. Typically these studies used animals with advanced disease and the aortic wall was found to be stiffer than normal, possibly due to the fibrous change and calcification found in the advanced lesions.
Here we used a quasi-physiological method to measure elasticity of pressurized segments of the thoracic aorta from New Zealand rabbits fed a high cholesterol diet. Using this method, we identified decreases in arterial wall stiffness associated with the progression and histological features of atherosclerotic lesions.
Animal model and specimens
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 . 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.
Experimental system and mechanical tests
where D0 is the outer vessel diameter at a certain P and ΔP and ΔD are the changes in pressure and diameter . For small increments of P and D, EP 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.
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.
Atherogenic diet resulted in hypercholesterolemia
Blood assays of cholesterol-fed and normal rabbits
4121 ± 415
4423 ± 493.40
35 ± 7.25
55.5 ± 11.82
381 ± 54.60
502 ± 96.24
38 ± 2
43.8 ± 9.66
383 ± 40.50
412 ± 15.40
21.8 ± 3.60
34.5 ± 2.88
46.5 ± 5.53
53.75 ± 5.57
32.5 ± 8
39.7 ± 6
6.25 ± 2.05
7.25 ± 1.58
4.25 ± 0.89
5.25 ± 0.89
38.75 ± 2.87
39.25 ± 2.43
42.25 ± 2.87
44.25 ± 7.10
0.79 ± 0.11
0.89 ± 0.13
0.78 ± 0.46
0.93 ± 0.18
Hypercholesterolemia was associated with intermediate or advanced atheroma formation
Immunostaining scores for RAM-11 and HHF-35 in hyperlipidemic and control animals
Atherosclerosis reduced the wall stiffness of the aorta
We used quasi-physiologic conditions and a closed loop flow system to measure the pressure-diameter relationship in segments of thoracic aortas from a New Zealand rabbit model of induced atherosclerosis. After 8 weeks of a 4% high-cholesterol diet without additional atherogenic components, we found that the aortic elastic modulus decreased as the atherosclerosis became more advanced, specifically with the appearance of intermediate (type III) and early advanced (type IV) atherosclerotic lesions. Histologically, these lesions were fatty, composed mainly of foam cells filled with lipid (RAM 11-positive cells), and with a lesser number of SMCs rich in solid-like actin (HHF-35 positive cells); while fibrous change was notably absent. Especially, in type IV atherosclerotic lesions, the usual intimal smooth muscle cells and the intercellular matrix of the deep intima are dispersed and replaced by accumulated particles of extracellular lipid. Between the lipid core and the endothelial surface, the intima contains macrophages and smooth muscle cells with and without lipid droplet inclusions. Much of the tissue between the core and the surface endothelium corresponds to the proteoglycan-rich layer of the intima, although infiltrated with the cells as macrophages, foam cells, and lymphocytes are more densely concentrated in the lesion periphery . Taken together, these factors could account for the observed decreases in stiffness. The potential clinical significance of decreased stiffness can be defined considering the histological basis of atherosclerotic lesions. Taking into account, that the region between the lipid core and the lesion surface contains proteoglycans and macrophage foam cells and only isolated smooth muscle cells and minimal collagen (potential mechanism of decreased stiffness), it may be susceptible to formation of fissures, intramural hematomas, aortic dilatation or even dissection. Thus, the periphery of advanced lesions, particularly type IV, may be vulnerable to rupture because macrophages are generally abundant in this location.
Our results are in accordance with previous studies of the mechanical properties of arterial segments with atherosclerosis. Firstly, Newman et al  reported that structural stiffness and wall elasticity of abdominal aorta was decreased during the earliest stages of atherosclerosis in cockerels fed an atherogenic diet. The decreased stiffness accompanying early atherosclerosis was attributed to the weakening of medial interlamellar elastic tissue-collagen network, probably due to lipid infiltration, whereas in advanced disease with calcification and fibrosis, aortic structural stiffness was higher than the control aortas. Nichol  reported that atherosclerosis reduced aortic stiffness at pressures below 70 mmHg, due to the destruction of elastic tissues. In a recent study designed to represent early and intermediate atherosclerosis, Hayashi and Imai  demonstrated that the force-deformation characteristics of atherosclerotic specimens of denuded thoracic rabbit aorta were less stiff than controls. Moreover, Hamilton et al  used intravascular ultrasound (IVUS) imaging, 3D reconstruction, and finite element analysis (FEA) to examine alterations in denuded femoral artery wall of Yucatan pigs fed a high cholesterol diet. The elastic modulus of the non-denuded femoral arteries decreased significantly on the high cholesterol diet, in agreement with our results. The decrease in elastic modulus with early/intermediate (fatty and fibrofatty lesions) atheroma was reversed and then increased as the lesions became fibrotic. Similarly, Vonesh et al  used IVUS image data with FEA to perform 3D reconstruction of human atherosclerotic segments of iliac and femoral arteries respectively, and found that the elastic modulus of non-atherosclerotic tissue regions was greater than early lipidous atherosclerotic regions for transmural pressure load at normal and hypertensive pressures (80-160 mmHg). De Korte et al  also used IVUS elastography of diseased human femoral and coronary arteries at 80 and 100 mmHg, and then compared the differences in strain between normal and diseased tissue. They reported that the pressure-strain modulus of fibrous tissue was double the modulus of fatty tissue, indicating the vulnerability of fatty lesions with only a thin fibrous cap. A recent study  used IVUS elastography to analyze denuded iliac and femoral arteries of Yacatan pigs on an atherogenic diet for 9-10 months. The mean strain value of arteries with early fat lesions (0.46) was greater that non diseased arteries (0.21) or diseased arteries with fibrous lesions (0.24), in agreement with our findings. Finally, Matsumoto et al  used pipette aspiration to measure the local elastic modulus of rabbit thoracic aortas fed a 1% cholesterol diet for 8-28 weeks. Similar to our results, the local elastic modulus of vessels with early lesions after 8 weeks of the atherogenic diet were significantly lower than normal tissue, although the modulus significantly increased after 24-28 weeks. The local elastic modulus appeared to decrease concurrently with the formation of early atherosclerotic lesions (intimal hyperplasia filled with foam cells), and increased gradually, coincident with the appearance of SMCs and calcification.
Our results conflict with a large number of studies that have used a variety of methods to examine the elastic properties of atherosclerotic artery walls in monkeys on high cholesterol diets for between 18-38 months. However, the contradiction with our findings is reasonable, as the duration of high cholesterol diet for atherosclerosis induction was much longer. Contrary to our findings, these studies found that arterial wall stiffness increases and decreases with the progression or regression of the atherosclerosis [28–30], respectively. It is possible that the increased stiffness after years of high cholesterol diet was due to the presence of highly fibrotic and calcified atherosclerotic lesions that would have an elastic modulus comparable to that of bone . Others have found similar changes in short duration studies of rabbits or rats [31–34]. Interestingly, Hayashi et al.  found that arterial stiffness in rabbits fed a high cholesterol diet for 4-32 weeks did not increase unless there was also considerable calcification and wall thickening, even if the atherosclerosis was highly advanced. Finally, Richter and Mittermayer  observed that the modulus of volume elasticity of autopsied human aortas was higher in more advanced stages of atherosclerosis. It is possible that the tangential strips of aorta used in some studies [31, 32] may behave differently in tests of elasticity than intact cylindrical arterial segments, such as we have used here, since it is difficult to set up isolated strips of tissue in a mechanical state that is comparable to the physiological loading conditions in vivo.
These conflicting results might also be attributed to different definitions or measurement of stiffness, testing methodologies, or stage of atherosclerosis examined. Although stiffness is generally described as a reflection of the arterial wall tissue rigidity, there are significant differences in how it is defined. While wall elasticity involves direct or indirect measurement of wall thickness, expressing the material properties of wall; stiffness, expressed as more or less similar to Peterson modulus EP, treats the vessel as a whole geometric-material structure. Thus, the different in vitro methods of physiological loading such as closed- or open-ended tube, with or without restriction of axial movement may also contribute to variability in the mechanical characteristics of the arterial wall. Determination of wall stiffness instead of wall elasticity in conjunction with the fixed ends of the aortic specimens during mechanical testing, that constrict the axial deformation occurred in vivo, could be regarded as limitations of our in vitro model.
In conclusion, we found that the material properties of the aortic wall altered with the degree of progression of atherosclerotic disease and the corresponding changes in arterial wall morphology. The presence of early atherosclerotic lesions, characterized by abundant fatty cells, few SMCs and the partial loss of integrity in the elastin-collagen network, led to the histological reorganization of the aortic wall and the initial decrease of wall stiffness. Thus, it will be interesting to study the potential impact of agents such as statins on the structural and biomechanical properties of atherosclerotic aortic wall, and their potential contribution to plaque stabilization or even regression.
List of abbreviations
external diameter at a reference pressure
modified arterial stiffness
high- density lipoprotein
left ventricular device
radial arterial dilatation
smooth muscle cells
We thank ELPEN Pharmaceuticals who provided the hypercholesterolemic diet.
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