Evidence for markers of hypoxia and apoptosis in explanted human carotid atherosclerotic plaques Alessandra Bitto, MD, Giovanni De Caridi, MD, Francesca.

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Evidence for markers of hypoxia and apoptosis in explanted human carotid atherosclerotic plaques Alessandra Bitto, MD, Giovanni De Caridi, MD, Francesca Polito, PhD, Margherita Calò, PhD, Natasha Irrera, JD, Domenica Altavilla, PhD, Francesco Spinelli, MD, Francesco Squadrito, MD Journal of Vascular Surgery Volume 52, Issue 4, Pages 1015-1021 (October 2010) DOI: 10.1016/j.jvs.2010.05.116 Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 1 A, Macroscopic appearance of a normal carotid artery. Arrow head indicates an early fatty streak. B, H&E staining used for routine tissue preparation, nuclei stain blue, while connective tissue stains pink. The rectangle represents the area studied in C and D. Original magnification ×2. C, H&E staining, detail showing a normal arterial morphology. Original magnification ×20. D, Masson's trichrome staining, collagen fibers stain green, while muscle fibers stain red. Arrowhead indicates muscular fibers, green color indicates collagen tissue. Original magnification ×20. E, Macroscopic appearance of an atheromatous common carotid artery. F, H&E staining. The rectangle represents the area studied in H. Original magnification ×2. G, Masson's trichrome staining. The rectangle represents the area studied in I. Original magnification ×2. H, H&E staining, detail showing an altered morphological architecture with inflammatory infiltrate. Arrow head indicates adipose tissue deposition. Original magnification ×20. I, Masson's trichrome staining, arrowheads indicates calcification, and green color indicates fibrose tissue. Original magnification ×20. Journal of Vascular Surgery 2010 52, 1015-1021DOI: (10.1016/j.jvs.2010.05.116) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 2 A, Representative Western blot of p-eNOS in carotid arteries. Bars represents mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. B, Representative Western blot of p-eNOS in carotid arteries. Bars represents mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. C, Representative Western blot of p-ERK in carotid arteries. Bars represents mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. D, Representative Western blot of p-ASK-1 in carotid arteries. Bars represents mean ± SD. Controls n = 5. All the other groups n=10. *P < .05 vs controls. Journal of Vascular Surgery 2010 52, 1015-1021DOI: (10.1016/j.jvs.2010.05.116) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 3 A, HIF-1α immunostaining in fibrocalcific carotid artery. Arrow heads shows nuclear reactivity. Original magnification ×40. P < .01 vs controls. B, p-JNK immunostaining in fibrocalcific carotid artery. Arrow heads shows diffuse cytoplasmic reactivity. Original magnification ×40. P < .01 vs controls. C, Representative Western blot of HIF-1α in carotid arteries. Bars represent mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. D, Representative Western blot of p-JNK in carotid arteries. Bars represents mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. Journal of Vascular Surgery 2010 52, 1015-1021DOI: (10.1016/j.jvs.2010.05.116) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 4 A, Representative Western blot of p-p38 in carotid arteries. Bars represent mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. B, Representative Western blot of active caspase-9 in carotid arteries. Bars represent mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. C, Representative Western blot of OPG in carotid arteries. Bars represents mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. D, Representative Western blot of RANKL in carotid arteries. Bars represent mean ± SD. Controls n = 5. All the other groups n = 10. *P < .05 vs controls. Journal of Vascular Surgery 2010 52, 1015-1021DOI: (10.1016/j.jvs.2010.05.116) Copyright © 2010 Society for Vascular Surgery Terms and Conditions

Fig 5 Proposed cascade of events triggered by oxidative stress in atherosclerotic plaque. Journal of Vascular Surgery 2010 52, 1015-1021DOI: (10.1016/j.jvs.2010.05.116) Copyright © 2010 Society for Vascular Surgery Terms and Conditions