gamma-Secretase inhibitor reduces diet-induced atherosclerosis in apolipoprotein E-deficient mice

Abstract

Atherosclerosis is a chronic inflammatory disease resulting from interactions between lipids, macrophages and arterial wall cells. The Notch signaling pathway is involved in the activation of macrophages in atherosclerotic lesions. This study examined whether pharmacological inhibition of Notch signaling using a gamma-secretase inhibitor (GSI) can reduce atherosclerotic lesion formation. Notch-related molecules were significantly increased in aortas from apolipoprotein E-deficient (ApoE(-/-)) mice. In particular, macrophages in the plaques showed strong expression of Notch1 and a downstream transcriptional factor, Hes-1. A GSI (LY411,575, 0.2, and 1.0mg/kg/day) or vehicle control was then administered to ApoE(-/-) mice fed Western diet for 8 weeks before measuring the expression of Notch-related molecules. Systemic administration of GSI suppressed Notch signaling in vivo and reduced total plaque areas and fatty streak content in the aortic sinus in a dose-dependent manner without serious adverse effects. The GSI also suppressed the migratory activity of macrophages and reduced the expression of intercellular adhesion molecule-1, resulting in significantly decreased macrophage infiltration in the atherosclerotic plaques. These results provided new insight into the anti-atherogenic properties of GSI in Apo E(-/-) mice fed Western diet.

Fig. 1

(A) Semiquantitative analysis of expression of Notch-related molecules (n = 10 from each group). Values were normalized to β-actin expression. Results are presented as mean ± SEM of 10 mice. ^*P < 0.05 compared with wild-type mice. ^†P < 0.05 compared with vehicle-treated ApoE^−/− mice. (B) Expression of Notch1 and Hes-1 in macrophages in atheromatous plaques. MOMA2-positive cells (Red) in plaques of control mice show highly expressed Notch1 and Hes-1 (green), unlike those of GSI-treated mice. Scale bar = 100 μm. C. Expression of cleaved Notch1 and β-actin (internal control) in aortas of ApoE^−/− mice after vehicle or GSI treatment was determined by immunoblot analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Systemic effects of GSI. (A) Representative images of intestine and thymus from vehicle- and GSI-treated ApoE^−/− mice. Intestine of GSI-treated mice showed increased goblet cells (dark violet cells) compared to vehicle-treated mice. Thymus of GSI-treated mice showed severe cortical atrophy. Co: cortex, Ma: marrow. (B) and (C) GSI treatment decreased the numbers of mature T cells (CD3⁺) (B) and B cells (CD19⁺) (C) Results are mean ± SEM. n = 10 for each group.^*P < 0.01, compared with vehicle-treated mice). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

GSI reduces plaque formation in ApoE^−/− mice. (A) Representative images showing aortic cusps stained with hematoxylin-eosin (top), Masson’s trichrome (middle), and oil red O (bottom). Left and right panels show aortic cusps from vehicle-treated and GSI-treated ApoE^−/− mice, respectively. Scale bar = 300 μm. (B) Plaque areas of aortic cusps (^*P < 0.03, ^#P < 0.01, n = 10, compared with vehicle-treated mice, respectively). (C) Lipid content in plaque areas of aortic cusps (oil red O-positive areas) (^*P < 0.03, ^#P < 0.01, n = 10, compared with vehicle-treated mice, respectively). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

GSI treatment decreases macrophage accumulation and activity. (A) Representative micrographs of immunohistochemistry for MOMA2 (left) expression. MOMA2- positive areas were significantly less in GSI-treated mice compared to control mice. Results are mean ± SEM. n = 10 for each group. (^*P < 0.01; right). (B) Immunohistochemistry for ICAM-1 and Hes-1 in primary-cultured peritoneal macrophages. (C) Expression of cleaved Notch1, ICAM-1, and β-actin (internal control) in RAW 264.7 cells after treatment with control or GSI was determined by immunoblot analysis. (D) Migration assay in presence or absence of GSI. Results are mean ± SEM. n = 8 for each group. (^*P < 0.005, ^#P < 0.001, compared with 0 nM with 10% serum, respectively).