Arterioscler Thromb Vasc Biol Role of the Intrinsic Coagulation Pathway in Atherogenesis Assessed in Hemophilic Apolipoprotein E Knockout Mice by J. Khallou-Laschet, G. Caligiuri, E. Tupin, A.-T. Gaston, B. Poirier, E. Groyer, D. Urbain, S. Maisnier-Patin, R. Sarkar, S.V. Kaveri, S. Lacroix-Desmazes, and A. Nicoletti Arterioscler Thromb Vasc Biol Volume 25(8):e123-e126 August 1, 2005 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Immunohistochemistry and lesion morphology analysis were performed on aortic sections from E° and E°/FVIII° mice included in the 8-week and 16-week protocols. Figure 2. Immunohistochemistry and lesion morphology analysis were performed on aortic sections from E° and E°/FVIII° mice included in the 8-week and 16-week protocols. Consecutive sections cut between 500 and 700 μm from the cusp origin were used. Primary antibodies used for immunohistochemistry were rabbit anti-human fibrin(ogen) (Dako), rat anti-mouse GP Iα+Iβ (Xia.C3 and Xia.G5, Emfret), VCAM-1, and MAC-3 (BD Biosciences). Fluorescent secondary antibodies were from Molecular Probes (Alexa 488 goat anti-rabbit and Alexa 546 goat anti-rat). Nuclei were stained with the 33342 Hoechst dye (Sigma). Periodic acid–Schiff/Alcian blue, Perl’s iron, and Picrosirius red/polarization light microscopy were used to respectively detect neutral and acid proteoglycans, iron deposits, and collagen. Perl’s iron staining was negative in all lesions. Original magnification ×40 for overviews of aortas and ×200 for detailed views. Frames in the ×40 images indicate the sites where the magnifications were made. J. Khallou-Laschet et al. Arterioscler Thromb Vasc Biol. 2005;25:e123-e126 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. A, FVIII activity, TC, and weight were measured from plasma collected at death on E°/FVIII°, E°, and FVIII° mice maintained for 8 or 16 weeks on ND or WD. FVIII activity was measured by a chromogenic assay in which thrombin-activated FVIII acts as a cofactor for Factor IXa in the conversion of Factor X to Factor Xa, using plasma from wild-type C57BL/6 mice as a standard. Figure 1. A, FVIII activity, TC, and weight were measured from plasma collected at death on E°/FVIII°, E°, and FVIII° mice maintained for 8 or 16 weeks on ND or WD. FVIII activity was measured by a chromogenic assay in which thrombin-activated FVIII acts as a cofactor for Factor IXa in the conversion of Factor X to Factor Xa, using plasma from wild-type C57BL/6 mice as a standard. Results are expressed as arbitrary units (au). TC level was measured using a Boehringer Mannheim kit (France-méthode “CHOD-PAP”). B, Extent of lesion development was analyzed by computer-assisted morphometry on oil red–O stained and hematoxylin counterstained sections of the aortic root from E°/FVIII°, E°, and FVIII° mice maintained for 8 or 16 weeks on ND or WD. Serial cryostat sections were cut from the proximal 1 mm of the aortic root. The lesion density (surface area of lesions/surface area of vessel; %) was determined in each aortic root on 4 hematoxylin/oil red O–stained sections cut at 200, 400, 600, and 800 μm from the appearance of the first cusp. (Results are expressed as means±SEM.) Nonparametric Mann–Whitney tests were performed using Statview 5.0 software (SAS Institute Inc.). Differences between groups were considered significant if P<0.05, and significant differences shown for lesion densities refer to differences compiled from all sections. †E°/FVIII° vs E°; §E°/FVIII° vs FVIII°; ¶E° vs FVIII°. Four symbols P<0.0001; 3 symbols P<0.001; 2 symbols P<0.01; 1 symbol P<0.05. J. Khallou-Laschet et al. Arterioscler Thromb Vasc Biol. 2005;25:e123-e126 Copyright © American Heart Association, Inc. All rights reserved.