Increased vascularization of shoulder regions of carotid atherosclerotic plaques from patients with diabetes Fredrik J. Olson, PhD, Sofia Strömberg,

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Increased vascularization of shoulder regions of carotid atherosclerotic plaques from patients with diabetes Fredrik J. Olson, PhD, Sofia Strömberg, MD, Ola Hjelmgren, MD, Josefin Kjelldahl, BS, Björn Fagerberg, MD, PhD, Göran M.L. Bergström, MD, PhD Journal of Vascular Surgery Volume 54, Issue 5, Pages 1324-1331.e5 (November 2011) DOI: 10.1016/j.jvs.2011.04.061 Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 1 Box plot shows the density of neovascularization in shoulder regions of plaques from patients with (DM) and without (non-DM) diabetes mellitus. Differences between the patient groups were assessed with the Mann-Whitney U test. The top border of the box shows the 75th percentile, the whisker shows the range of the data, and the horizontal line in the box shows the median. Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 2 Histogram shows the frequency distribution (%) of neovascularization density in the plaque shoulder regions in patients with and without diabetes mellitus (non-DM). The number on top of each bar indicates the exact number of plaques within each frequency domain. The χ2 test was used to assess the difference between the plaques of patients with and without DM. Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 3 Extent of immunohistochemical staining is shown in the shoulder regions of carotid atherosclerotic plaques in patients with (DM) and without (non-DM) diabetes mellitus. The box plots show the areas stained with antibodies relative to the total shoulder area. The borders of the box show the 75th and 25th percentiles, the whiskers shows the range of the data, and the horizontal line in the box shows the median. A, Vascular endothelial growth factor (VEGF) staining is shown in DM and non-DM patients. B, VEGF receptor-2 (VEGFR-2) staining is shown in DM and non-DM patients. C, VEGFR-2 staining is shown in vascularized and nonvascularized plaque shoulders. D, CD68 staining is shown in DM and non-DM patients. The Mann-Whitney U test was used to assess differences between the patient groups. Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Fig 4 The proportion of patients with plaque shoulder neovascularization in patients with (DM) and without (non-DM) is shown in relation to diabetes mellitus and time from the clinical event to surgery. Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Appendix Fig 1 (online only) A significant Spearman correlation was found between vascular density in plaque shoulders and the time since the last clinical event. This suggests that there is an association between the clinical event and increased vascularization. Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Appendix Fig 2 (online only) A, Schematic illustration shows the location of the analyzed section in the plaque. B-D, The arrow indicates from which side cross-sectional 4-μm slices were taken for histology in examples of the tissue sections with definition of shoulder and cap regions. C, Lipid core; F, fibrous cap; L, blood lumen; S, shoulder region. Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Appendix Fig 3 (online only) Illustration shows a plaque that (A, B) scored positive for vascularization in the shoulder region and a plaque that (C, D) scored negative for shoulder vascularization in (A, C) normal and (B, D) high-magnification (original magnification ×10) views. The arrows show CD34+ cells in vessels in the (B) shoulder area and on the (D) lumen of the vessel. Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Appendix Fig 4 (online only) Schematic illustration of immunohistochemical staining of (A, B) smooth muscle cells (anti-SMA) and (C, D) macrophages (anti-CD68) in (A, C) normal and (B, D) high-magnification (original magnification ×10) views. Immune complexes were visualized using the LSAB System HRP Kit (Dako). Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions

Appendix Fig 5 (online only) Schematic illustration shows immunohistochemical staining of (A, B) vascular endothelial growth factor (VEGF) and (C, D) VEGF receptor 2 in (A, C) normal and (B, D) high-magnification (original magnification ×10) views. Immune complexes were visualized using the MACH3 Mouse-Probe Alk Phos Polymer Kit and the Vulcan Fast Red Chromogen Kit (BioCare Medical). Journal of Vascular Surgery 2011 54, 1324-1331.e5DOI: (10.1016/j.jvs.2011.04.061) Copyright © 2011 Society for Vascular Surgery Terms and Conditions