Prevention of atherosclerosis by the mTOR inhibitor everolimus in LDLR−/− mice despite severe hypercholesterolemia  Marc A. Mueller, Frank Beutner, Daniel Teupser, Uta Ceglarek, Joachim Thiery  Atherosclerosis  Volume 198, Issue 1, Pages 39-48 (May 2008) DOI: 10.1016/j.atherosclerosis.2007.09.019 Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Atherosclerotic lesion area in the aortic root (a) and the brachiocephalic artery (b) of male LDL-receptor−/− mice fed a 0.1% cholesterol diet for 14 weeks and receiving solvent (group A), everolimus 0.05mg (group B) or 1.5mg/kg/bodyweight per day (group C). Atheroma development was quantified morphometrically in five equidistant sections of the aortic root and in three equidistant sections of the brachiocephalic artery by computerized oil red O stained sections. Each datapoint represents the mean of the lesion size from one individual animal. The lines represent means of the individual data points in group A, B and C. Atherosclerosis 2008 198, 39-48DOI: (10.1016/j.atherosclerosis.2007.09.019) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Effect of everolimus on the complexity of atherosclerotic lesions of the brachiocephalic artery in LDL-receptor knockout mice fed a 0.1% cholesterol diet over 14 weeks. The animals received either solvent (group A, n=19) or everolimus (0.05mg/kg body weight per day group B, n=15; or 1.5mg/kg/bw per day group C, n=13) over a period of 12 weeks. χ2-test group A vs. group B, p<0.01, group A vs. group C, p<0.001; group B vs. group C, p<0.01. The morphological results are based on sections from the same region of the artery. Atherosclerosis 2008 198, 39-48DOI: (10.1016/j.atherosclerosis.2007.09.019) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Effect of everolimus on the lesion development in LDLR−/− mice. Animals were placed on a 0.1% cholesterol-rich diet and solvent (group A), everolimus 0.05mg (group B) or 1.5mg/kg/bodyweight per day (group C) was administered over a period of 12 weeks. The mice were sacrificed and the aortic root (magnification ×10, Fig. 3a) and the brachiocephalic artery (magnification ×10, Fig. 3b) were fixed, sectioned, stained with oil red O (ORO), Movat's pentachrome (MOVAT) or immunostained with CD68-antibodys (CD68) and used for morphometric analyses. The photographs reflect one representative animal of each investigated group and the morphological findings for the three different staining methods were taken from the same region of the artery in all three groups. Atherosclerosis 2008 198, 39-48DOI: (10.1016/j.atherosclerosis.2007.09.019) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Total cholesterol and cholesterol levels of plasma lipoproteins (LDL-, VLDL, and HDL) isolated from EDTA-plasma at 20 weeks of age in male LDLR−/− C57Bl/6 mice on a 0.1% cholesterol diet, receiving solvent (group A), everolimus 0.05mg (group B) or 1.5mg/kg/bw per day (group C). Atherosclerosis 2008 198, 39-48DOI: (10.1016/j.atherosclerosis.2007.09.019) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Effect of everolimus on circulating cell mediators in plasma. Plasma cytokines, growth factors and chemokines were measured in individual plasma samples of each animal. Everolimus treatment resulted in a significant reduction of plasma interleukin-1a [IL-1a], interleukin-5 [IL-5], granulocyte-monocyte colony–stimulating factor [GM-CSF] and dose dependant for interleukin-12p40 [IL-12p40]. An increase of keratinocyte-derived chemokine [KC] was observed by treatment with everolimus in group B (34.8±28.8; p=n.s.) and in group C (59.0±36.1pg/ml; p<0.001). For all other investigated cytokines, growth factors and chemokines, everolimus treatment did not change the circulating plasma levels of cell mediators. Atherosclerosis 2008 198, 39-48DOI: (10.1016/j.atherosclerosis.2007.09.019) Copyright © 2007 Elsevier Ireland Ltd Terms and Conditions