S. Shaikh, J. Brittenden, R. Lahiri, P. A. J. Brown, F. Thies, H. M

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Macrophage Subtypes in Symptomatic Carotid Artery and Femoral Artery Plaques S. Shaikh, J. Brittenden, R. Lahiri, P.A.J. Brown, F. Thies, H.M. Wilson European Journal of Vascular and Endovascular Surgery Volume 44, Issue 5, Pages 491-497 (November 2012) DOI: 10.1016/j.ejvs.2012.08.005 Copyright © 2012 European Society for Vascular Surgery Terms and Conditions

Figure 1 Schematic presentation of segmentation of the endarterectomy specimen. Plaques were cut into 7 segments of 2–3 mm in thickness depending on the size of plaque. For carotid plaques, segment 0 was taken as the last complete transaction of the common carotid artery at its bifurcation. For femoral plaques segment 0 represents the area of maximum stenosis, taken as the center of the plaque. Segments −3, 0 and +2 was further sectioned for histological and immunohistochemical analysis. European Journal of Vascular and Endovascular Surgery 2012 44, 491-497DOI: (10.1016/j.ejvs.2012.08.005) Copyright © 2012 European Society for Vascular Surgery Terms and Conditions

Figure 2 Morphological characterization of plaques was defined by four categories. Lipid – obvious confluent areas of lipid deposition whether intra- or extra-cellular. Calcification – obvious measurable areas of confluent calcification. Fibroconnective tissue – obvious confluent areas of fibroconnective tissue deposition with no confluent areas of calcification, lipid or lymphocytic infiltration. Fibroconnective tissue with obvious lymphocytic/macrophage cellular infiltrate – obvious confluent areas of fibrointimal change with obvious lymphocytic or lymphocytic and macrophage inflammation throughout the area. Outlines in dashed lines encircle confluent zones of lipid (A), calcification (B), lymphocytic and macrophage infiltration (C), and fibroconnective tissue present in A, B and C as highlighted by asterisks. These were measured by tracing around the perimeter of confluent abnormality and subsequent image analysis. In Fig. 1B, an example of spotty non-confluent calcification (black arrow) is also present. This spotty calcification would be designated within the “fibroconnective tissue” category and not included within areas designated as confluent calcification. Original magnification (A) ×200, (B & C) ×400. European Journal of Vascular and Endovascular Surgery 2012 44, 491-497DOI: (10.1016/j.ejvs.2012.08.005) Copyright © 2012 European Society for Vascular Surgery Terms and Conditions

Figure 3 Carotid artery plaques show significantly higher numbers of total CD3-positive T cells (A) and total CD68-positive macrophage (B) per unit area than that of femoral artery plaques (denoted by arrows). Box plots show the median numbers of cells per area (mm2) of carotid (n = 32) and femoral (n = 26) plaques. The box represents the inter-quartile range (IQR) and the whiskers represent the range (95% confidence interval). The circles are outliers which are 1.5 IQR from the end of the box and the stars are outliers which are 3IQRs from the end of a box. The three bars within carotid or femoral plaque groups represent stained section 0 at the bifurcation, section −3 representing 2 sections distal to bifurcation and section +2 representing 1 section proximal to the bifurcation, as described in Materials and methods. Original magnification for Figures ×200. European Journal of Vascular and Endovascular Surgery 2012 44, 491-497DOI: (10.1016/j.ejvs.2012.08.005) Copyright © 2012 European Society for Vascular Surgery Terms and Conditions

Figure 4 Double immunostaining of endarterectomy sections showing a greater predominance of M1-activated macrophages in carotid plaques and of M2-activated macrophages in femoral plaques. Total macrophages were detected by anti-CD68 and liquid permanent red staining (red) while macrophage activation markers were identified by the relevant antibodies and DAB staining (brown). Macrophage-rich areas were analyzed as outlined in Methods. Examples of double stained macrophages are highlighted by arrows in (A). Box plots show median values for numbers of M1-macrophages iNOS (B), MHC class II (C), SOCS3 (D) or M2-macrophages, CD163 (E), Dectin1 (F), and SOCS1 (G) in femoral or carotid plaques. The box represents the inter-quartile range (IQR) and the whiskers represent the range (95% confidence interval). The circles are outliers which are 1.5 IQR from the end of the box and the stars are outliers which are 3IQRs from the end of a box. The three bars grouped within carotid or femoral plaques represent stained sections 0, section −3 and section +2 representing 1 section proximal to the bifurcation, as described in Materials and methods. European Journal of Vascular and Endovascular Surgery 2012 44, 491-497DOI: (10.1016/j.ejvs.2012.08.005) Copyright © 2012 European Society for Vascular Surgery Terms and Conditions

Supplementary Figure 1 Double immunostaining of endarterectomy sections showing a greater predominance of M1-activated macrophages (MHC class II, iNOS, SOCS3 positive) in carotid plaques and of M2-activated macrophages (CD163, Dectin I, SOCS1 positive) in femoral plaques. Total macrophages were detected by anti-CD68 and liquid permanent red staining (red) while macrophage activation markers were identified by the relevant antibodies and DAB staining (brown). Original magnification ×200. European Journal of Vascular and Endovascular Surgery 2012 44, 491-497DOI: (10.1016/j.ejvs.2012.08.005) Copyright © 2012 European Society for Vascular Surgery Terms and Conditions