Turbulent blood flow plays an essential localizing role in the development of atherosclerotic lesions in experimentally induced hypercholesterolaemia in rats

Abstract

Taking into account that atherosclerosis is a focal disease and high levels of plasma cholesterol are closely correlated with its pathogenesis, it is a challenge to explain how equal concentrations of cholesterol bathing the endothelium can produce local, rather than global, effects on arteries. The focal distribution of atherosclerotic lesions has been considered to be dependent, at least in part, on hydrodynamic factors. The present study was carried out to further test the hypothesis that these forces are an important localizing factor in rats feeding a hypercholesterolaemic diet and submitted to infra-diaphragmatic aortic constriction. These animals develop a normotensive prestenotic region with laminar blood flow that serves as control for a normotensive poststenotic region with turbulent blood flow. Our findings clearly demonstrated that the combination of turbulent blood flow and low wall shear stress (WSS) in the presence of hypercholesterolaemia and oxidative stress creates conditions to the formation of focally distributed incipient atherosclerotic lesions observed in the poststenotic segment. In contrast, only diffuse fatty streaks could be observed in the normotensive prestenotic segment with laminar blood flow and normal WSS in the presence of hypercholesterolaemia and oxidative stress. Although haemodynamic forces are not by themselves responsible for the pathogenesis of atherosclerosis, they prime the local vascular wall in which the lesion develop. Further studies are required to establish how haemodynamic forces are detected and transduced into chemical signalling by the cells of the artery wall and then converted into pathophysiologically relevant phenotypic changes.

Figure 1

Mean carotid and femoral blood pressure of animals (n = 7 each day) submitted to surgical abdominal aorta stenosis and feeding hypercholesterolaemic diet during the 28-day period of study. ***P < 0.001.

Figure 2

(a) Blood flow rate (ml/min). Box and whisker plot graph shows the batches of data in prestenotic and poststenotic aortas at day 28 of the experiment. (b) Wall shear stress mean values (dyne/cm²) in prestenotic and poststenotic aortas at day 28 of the experiment (n = 6). (c) Color Doppler shows a laminar flow in the prestenotic segment characterized by orange-red colour. In the poststenotic segment, a mixed of orange-red and blue colour was seen characterizing turbulent blood flow.

Figure 3

Panels A and B show representative aspects of the prestenotic and poststenotic aorta with high resolution light microscopy (a) (n = 9) and transmission electron microscopy (b) (n = 4). The appearance did not differ from that reported for mammalian aorta. Panels C and D show representative views of the prestenotic small flat lesions corresponding to fatty streaks characterized by intimal foam cells accumulation with high resolution light microscopy (c) and a few vacuolated mononuclear and smooth muscle cells with transmission electron microscopy (d). Panels E and F show representative views of the poststenotic raised incipient atherosclerotic lesions composed of mononuclear and smooth muscle cells, many of them vacuolated, with high resolution light microscopy (e) and vacuolated mononuclear cells and a great number of vacuolated smooth muscle cells surrounded by extracellular matrix with transmission electron microscopy (f). End, endothelial cell; iel, internal elastic lamina; smc, smooth muscle cell; mc, mononuclear cell; arrow, migration of smooth muscle cell from the media to the intima. In a, c and e, bar magnification=20 μm; in b, d and f, bar magnification=5 μm.

Figure 4

Percentile frequency distribution of intimal thickness. When the percentile frequency distribution of intima thickness in the prestenotic and poststenotic segments was plotted, the small flat lesions can be evidenced. The occurrence of marked intimal thickening, corresponding to the raised incipient atherosclerotic lesions, absent in the prestenotic segment, can be clearly demonstrated.

Figure 5

Immunohistochemistry (n = 6). The analysis revealed an increased expression (brown stained features) of 3-nitrotyrosine in endothelial and smooth muscle cells in the prestenotic (panel c) and more markedly in the poststenotic (panel d) segments of aortas from animals feeding hypercholesterolaemic diet. Aortas of animals feeding normal chow diet (panel a) show expression of 3-nitrotyrosine mainly in endothelial cells. Non-constricted aortas from rats given the hypercholesterolaemic diet also showed an increased expression of 3-nitrotyrosine (panel b) in endothelial and smooth muscle cells, comparable to that of the aorta prestenotic segment. Bar magnification=50 μm. The graph (panel e) shows the results of the semi-quantitative evaluation of 3-nitrotyrosine immunoreactivity intensity grade.