Angiopoietin-2 confers Atheroprotection in apoE-/- mice by inhibiting LDL oxidation via nitric oxide

Abstract

Atherosclerosis is promoted by a combination of hypercholesterolemia and vascular inflammation. The function of Angiopoietin (Ang)-2, a key regulator of angiogenesis, in the maintenance of large vessels is unknown. A single systemic administration of Ang-2 adenovirus (AdAng-2) to apoE(-/-) mice fed a Western diet significantly reduced atherosclerotic lesion size ( approximately 40%) and oxidized LDL and macrophage content of the plaques. These beneficial effects were abolished by the inhibition of nitric oxide synthase (NOS). In endothelial cells, endothelial NOS activation per se inhibited LDL oxidation and Ang-2 stimulated NO release in a Tie2-dependent manner to decrease LDL oxidation. These findings demonstrate a novel atheroprotective role for Ang-2 when endothelial cell function is compromised and suggest that growth factors, which stimulate NO release without inducing inflammation, could offer atheroprotection.

Figure 1. Ang-2 reduces atherosclerotic plaque formation, LDL oxidation and macrophage accumulation in apoE^-/- mice

ApoE^-/- mice maintained on a Western diet were administered AdAng-2 or control empty virus (AdEV). A, Atherosclerotic lesions in the aortic valves were stained with oil red O and the results expressed as the mean plaque area ± SEM. Ang-2 significantly reduced the mean plaque area (*P <0.01) compared with AdEV-treated apoE^-/- mice. B, Immunohistochemical analysis showed that CD31-positive endothelium (EC) remained intact, and CD11b-positive macrophages (Mø) and malondialdehyde–lysine/MDA2 (ox-LDL) staining was reduced in Ang-2 treated mice.

Figure 2. Ang-2 suppresses LDL oxidation and stimulates NO release via Tie2 activation

A, Ang-2-mediated NO release in HUVEC was inhibited by 0.5 mM NG-nitro-L-arginine (L-NNA). B, HUVEC were pretreated with either Tie2 (anti-Tie2; 5 μg/ml), or Tie1 (anti-Tie2; 5 μg/ml) neutralizing antibodies or Tie2 blocking peptide (Tie2 peptide; 0.5 mM) prior to incubation with Ang-2 (400 ng/ml) for 1 hour and NO release quantified. Results are the mean (±SEM) of three independent experiments (n = 9). C and D, HUVEC were incubated in serum-free medium containing 100 μg/ml LDL, 500 ng/ml of Ang-2 and/or 100 μM L-NAME for 16 hours. Oxidative modification of LDL was assessed using: C, TBARS assay (data represents the mean ± SEM; *P <0.01 vs. control, ^#P<0.05 vs. HUVEC+Ang-2 without L-NAME) and D, the relative electrophoretic mobility of LDL.

Figure 3. Ang-2-mediated reduction in atherosclerotic plaque formation requires NO

One day after administration of adenovirus, apoE^-/- mice were treated with L-NAME. A, Representative images of plaques stained for neutral lipids (oil red O) and macrophage (MOMA-2) content. Quantification of B, mean plaque and C, MOMA-2 positive areas show that the atheroprotective effect of AdAng-2 is abolished following L-NAME treatment. Data are the mean area ± SEM; *P <0.01 vs. AdEV without L-NAME, ^§P<0.05 and ^#P<0.01 vs. AdAng-2 without L-NAME.