Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice

Abstract

This study investigated whether endothelin-1 (ET-1), a potent vasoconstrictor, which also stimulates cell proliferation, contributes to endothelial dysfunction and atherosclerosis. Apolipoprotein E (apoE)-deficient mice and C57BL/6 control mice were treated with a Western-type diet to accelerate atherosclerosis with or without ETA receptor antagonist LU135252 (50 mg/kg/d) for 30 wk. Systolic blood pressure, plasma lipid profile, and plasma nitrate levels were determined. In the aorta, NO-mediated endothelium-dependent relaxation, atheroma formation, ET receptor-binding capacity, and vascular ET-1 protein content were assessed. In apoE-deficient but not C57BL/6 mice, severe atherosclerosis developed within 30 wk. Aortic ET-1 protein content (P < 0.0001) and binding capacity for ETA receptors was increased as compared with C57BL/6 mice. In contrast, NO-mediated, endothelium-dependent relaxation to acetylcholine (56 +/- 3 vs. 99 +/- 2%, P < 0.0001) and plasma nitrate were reduced (57.9 +/- 4 vs. 93 +/- 10 micromol/liter, P < 0.01). Treatment with the ETA receptor antagonist LU135252 for 30 wk had no effect on the lipid profile or systolic blood pressure in apoE-deficient mice, but increased NO-mediated endothelium-dependent relaxation (from 56 +/- 3 to 93 +/- 2%, P < 0.0001 vs. untreated) as well as circulating nitrate levels (from 57.9 +/- 4 to 80 +/- 8.3 micromol/liter, P < 0.05). Chronic ETA receptor blockade reduced elevated tissue ET-1 levels comparable with those found in C57BL/6 mice and inhibited atherosclerosis in the aorta by 31% without affecting plaque morphology or ET receptor-binding capacity. Thus, chronic ETA receptor blockade normalizes NO-mediated endothelial dysfunction and reduces atheroma formation independent of plasma cholesterol and blood pressure in a mouse model of human atherosclerosis. ETA receptor blockade may have therapeutic potential in patients with atherosclerosis.

Figure 1

Quantification of atherosclerosis in the aorta of control apoE-deficient mice (white) and mice treated with LU135252 for 30 wk (black). Intimal area of the aortic surface area covered with atheromatous plaques (a) and lesion size in histologic sections of the thoracic aorta (b) were reduced by chronic ET_A receptor blockade. No macroscopic or microscopic lesions were observed in C57BL/6 mice (unpublished observation and ref. 34). P < 0.001 (a) and P < 0.05 (b) for control vs. LU135252.

Figure 2

Photomicrographs of atherosclerotic lesion in the aortic arch of apoE-deficient mice. Advanced intimal lesion at the root of the carotid artery in an untreated apoE-deficient mouse exhibiting necrosis, calcification, and cholesterol crystals (a). Lesion at the same location in an apoE-deficient mouse after 30 wk of ET_A receptor blockade (b). Cellular composition and morphology of the plaques was similar to that seen in untreated animals. (Elastica van Giesson stain, original magnification X60).

Figure 3

Photomicrographs and autoradiographs of the aorta from C57BL/6 (Left) and apoE-deficient mice (Right). Representative histologic sections from the aorta of a C57BL/6 control mouse (a) and an atherosclerotic apoE-deficient mouse (b). Note the large plaque occluding the lumen in the aorta of the apoE-deficient mouse (b). In these animals, endothelin binding by using [¹²⁵I]ET-1 was markedly increased as compared with C57BL/6 mice (c) and was localized both in the vascular wall and in the atheromatous plaque (d). (Hematoxilin/eosin stain, original magnification X10. Bar: 1,000 μm).

Figure 4

Effects of chronic ET_A receptor blockade (black) on aortic ET-1 content and vascular function in isolated aortic rings of apoE-deficient mice compared with untreated animals (white). Endothelin blockade reduced aortic ET-1 protein content comparable to levels of C57BL/6 mice (a). NO-mediated endothelium-dependent relaxation to acetylcholine was normalized by treatment with the ET_A antagonist LU135252 (b) and inversely correlated with vascular ET-1 protein content. Sensitivity to exogenous NO as assessed by relaxations to sodium nitroprusside was slightly enhanced (c). Analyses were performed on n = 8–10 (a) and n = 8 (b and c) from each group. P < 0.0001 control vs. untreated.