Angiopoietin-2 blocking antibodies reduce early atherosclerotic plaque development in mice

Abstract

Objective: Angiopoietin-2 (Ang-2) blocking agents are currently undergoing clinical trials for use in cancer treatment. Ang-2 has also been associated with rupture-prone atherosclerotic plaques in humans, suggesting a role for Ang-2 in plaque stability. Despite the availability of Ang-2 blocking agents, their clinical use is still lacking. Our aim was to establish if Ang-2 has a role in atheroma development and in the transition of subclinical to clinically relevant atherosclerosis. We investigated the effect of antibody-mediated Ang-2 blockage on atherogenesis after in a mouse model of atherosclerosis.

Methods: Hypercholesterolemic (low-density lipoprotein receptor(-/-) apolipoprotein B(100/100)) mice were subjected to high-cholesterol diet for eight weeks, one group with and one group without Ang-2 blocking antibody treatment during weeks 4-8.To enhance plaque development, a peri-adventitial collar was placed around the carotid arteries at the start of antibody treatment. Aortic root, carotid arteries and brachiocephalic arteries were analyzed to evaluate the effect of Ang-2 blockage on atherosclerotic plaque size and stable plaque characteristics.

Results: Anti-Ang-2 treatment reduced the size of fatty streaks in the brachiocephalic artery (-72%, p < 0.05). In addition, antibody-mediated Ang-2 blockage reduced plasma triglycerides (-27%, p < 0.05). In contrast, Ang-2 blockage did not have any effect on the size or composition (collagen content, macrophage percentage, adventitial microvessel density) of pre-existing plaques in the aortic root or collar-induced plaques in the carotid artery.

Conclusions: Ang-2 blockage was beneficial as it decreased fatty streak formation and plasma triglyceride levels, but had no adverse effect on pre-existing atherosclerosis in hypercholesterolemic mice.

Keywords: Angiogenesis; Angiopoietin-2; Atherosclerosis; Microvascular leakage; Plaque stability.

Fig. 1

Anti Ang-2 antibody treatment increases plasma Ang-2 levels and decreases plasma triglycerides. Plasma Ang-2 levels in anti-Ang and IgG treated mice (A). During the five weeks of administration, the antibody treatment did not affect the general health status, body weight (B), cardiac function (E–J) or shear stress in carotid artery after collar implantation (K + L). However, the treatment led to a decrease in plasma triglycerides (D) without effects on plasma cholesterol (C). IgG n = 12; anti-Ang-2 n = 14; *p < 0.05; **p < 0.01.

Fig. 2

Antibody mediated Ang-2 blockage delays fatty streak formation in the brachiocephalic artery. Representative images of HE-stained brachiocephalic arteries (A + B) show a decrease in fatty streak size in animals treated with anti Ang-2 antibody (C). There is no difference in lumen area or medial thickness (D + E) but a reduction in stenosis after anti-Ang-2 treatment (F). Plaque collagen content (percentage of Sirius Red positive area/total plaque area, G–I), percentage of macrophage area (Mac3+ area/total plaque area, J–L) and adventitial microvessel density (microvessels/mm2) (MVD) (M–O). Representative images: left panels IgG, right panels anti-Ang-2 antibody. IgG n = 12; anti-Ang-2 n = 14; *p < 0.05.

Fig. 3

Anti Ang-2 antibody treatment does not alter pre-existing atherosclerosis in the aortic root. Representative images of HE-stained plaques in the aortic root (A + B) show no difference in plaque size (C) or necrotic core (D) in pre-existing plaques of the aortic root after antibody treatment. Also, collagen (percentage of Sirius Red positive area/total plaque, E–G), macrophage content (Mac3+ area/total plaque area, H–J) and adventitial microvessel density (microvessels/mm2, K–M) were not altered by the anti-Ang-2 antibody treatment. Representative pictures: left panels control IgG, right panels anti-Ang-2. IgG n = 12; anti-Ang-2 n = 14.

Fig. 4

Advanced lesions in the carotid arteries are not affected by Ang-2 blockage. Representative images of HE-stained collar induced advanced plaques (A + B) in the right common carotid artery. Plaque size (C) necrotic core (D). Representative images and quantification of (E–G) collagen, (H–J) macrophages, (K–M) microvessels: left control IgG, right anti-Ang-2. IgG n = 12; anti-Ang-2 n = 14.