Loss of lymphatic vessels and regional lipid accumulation is associated with great saphenous vein incompetence Hiroki Tanaka, MD, Nobuhiro Zaima, PhD,

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Loss of lymphatic vessels and regional lipid accumulation is associated with great saphenous vein incompetence Hiroki Tanaka, MD, Nobuhiro Zaima, PhD, Takeshi Sasaki, PhD, Naoto Yamamoto, MD, Masaki Sano, MD, Hiroyuki Konno, MD, Mitsutoshi Setou, MD, Naoki Unno, MD Journal of Vascular Surgery Volume 55, Issue 5, Pages 1440-1448 (May 2012) DOI: 10.1016/j.jvs.2011.09.064 Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 1 Thickness of the great saphenous vein wall. The measured thickness of the (left) intima, (middle) media, and (right) adventitia layers is compared among the control vein (CV) and varicose veins from the CEAP C2-3 and C4-5 groups. *P < .05 indicates a statistically significant difference. The error bars show the standard deviation. Journal of Vascular Surgery 2012 55, 1440-1448DOI: (10.1016/j.jvs.2011.09.064) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 2 Lipid staining and quantification in vein walls. A, Oil red O staining (bar = 20 μm). B, Lipid content in vein tissue. The phospholipid (PL) and triglyceride (TG) content of vein walls were compared among the control vein (CV) and varicose veins from the CEAP C2-3 and C4-5 groups. *P < .05 indicates a statistically significant difference. The error bars show the standard deviation. Journal of Vascular Surgery 2012 55, 1440-1448DOI: (10.1016/j.jvs.2011.09.064) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 3 Lipid content analyses by mass spectrometry. A, Representative mass spectrum pattern and spectrum of assigned molecule. B, Imaging mass spectrometry of vein tissue (bar = 200 μm). C, The ratio of intensity in vein tissues was compared among control veins (CV) and varicose veins from the CEAP C2-3 and C4-5 groups by one-way analysis of variance, followed by the Tukey test. *P < .05 indicates a statistically significant difference. †P < .05 indicates a statistically significant difference between the C2-3 and C4-5 varicose vein groups. The error bars show the standard deviation. Journal of Vascular Surgery 2012 55, 1440-1448DOI: (10.1016/j.jvs.2011.09.064) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 3 Lipid content analyses by mass spectrometry. A, Representative mass spectrum pattern and spectrum of assigned molecule. B, Imaging mass spectrometry of vein tissue (bar = 200 μm). C, The ratio of intensity in vein tissues was compared among control veins (CV) and varicose veins from the CEAP C2-3 and C4-5 groups by one-way analysis of variance, followed by the Tukey test. *P < .05 indicates a statistically significant difference. †P < .05 indicates a statistically significant difference between the C2-3 and C4-5 varicose vein groups. The error bars show the standard deviation. Journal of Vascular Surgery 2012 55, 1440-1448DOI: (10.1016/j.jvs.2011.09.064) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 4 Lymphatic vessel in vein wall. A, Immunofluorescent staining for von Willebrand Factor (green) and D2-40 (red; bar = 20 μm). B, Number of lymphatic vessels in complete circumferential sections from each group. *P < .05 indicates a statistically significant difference. The error bars show the standard deviation. Journal of Vascular Surgery 2012 55, 1440-1448DOI: (10.1016/j.jvs.2011.09.064) Copyright © 2012 Society for Vascular Surgery Terms and Conditions