Preoperative dietary restriction reduces intimal hyperplasia and protects from ischemia- reperfusion injury Christine R. Mauro, MD, Ming Tao, MD, Peng.

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Preoperative dietary restriction reduces intimal hyperplasia and protects from ischemia- reperfusion injury Christine R. Mauro, MD, Ming Tao, MD, Peng Yu, MD, PhD, J. Humberto Treviño-Villerreal, MD, PhD, Alban Longchamp, Bruce S. Kristal, PhD, C. Keith Ozaki, MD, James R. Mitchell, PhD Journal of Vascular Surgery Volume 63, Issue 2, Pages 500-509.e1 (February 2016) DOI: 10.1016/j.jvs.2014.07.004 Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 1 Influence of short-term high-fat diet (HFD) on outcome of ischemia-reperfusion (IR) injury. Two-week exposure to HFD did not worsen outcome of IR injury in male B6D2F1 mice. A, Experimental design. Mice were fed a low-fat control diet (C) or HFD for 2 weeks before onset of renal or hepatic IR injury and then returned to the low-fat control diet after surgery. Blood was drawn before injury or at the indicated times after reperfusion and analyzed for markers of renal function (urea) or liver injury (alanine transaminase, ALT) as indicated. B, Outcome of 25 minutes of bilateral renal IR injury as measured by changes in urea in serum before (time = 0) and on the indicated day after IR injury (n = 5/group). C, Outcome of 30 minutes of hepatic IR injury as measured by the amount of the liver enzyme ALT in serum before (time = 0) and at the indicated time after reperfusion (n = 5/group). Journal of Vascular Surgery 2016 63, 500-509.e1DOI: (10.1016/j.jvs.2014.07.004) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 2 Influence of short-term protein-free (PF) dietary restriction (DR) after high-fat diet (HFD) on outcome of ischemia-reperfusion (IR) injury. A, Experimental design. Male B6D2F1 mice were fed HFD for 2 weeks followed by 1 week of low-fat control (C) vs low-fat PF diet before onset of renal or hepatic IR injury. All animals were returned to the low-fat control diet after surgery. B, Outcome of 30 minutes of bilateral renal IR injury as measured by changes in serum urea and creatinine on the indicated day after injury; n = 5/group. The percentage of tubular epithelial cell necrosis in the cortex and at the cortical-medullary junction in serial sections 100 μm apart from each kidney is quantitated at right; n = 3/group. Below, Representative images of hematoxylin and eosin (HE)-stained kidney sections from the indicated groups harvested 3 days after injury. The arrows point to tubular structures in which cell death and disruption of normal structure are evident. G, Glomerulus; scale bar = 20 μm. C, Outcome of 30 minutes of hepatic IR injury as measured by the amount of the liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST) in serum 3 and 24 hours after reperfusion; n = 15/group. The average percentage of necrosis in 10 microscopic fields per animal is quantitated at right; n = 6/group. The asterisks indicate the significance of the difference between groups at the indicated time point according to an unpaired, two-tailed Student t-test; *P < .05, ***P < .001. Below, Representative images of HE-stained liver sections from the indicated group harvested 24 hours after injury. The asterisk indicates the central vein associated with portal triads; eosinophilic regions are characteristic of cell necrosis. Scale bar = 100 μm. Journal of Vascular Surgery 2016 63, 500-509.e1DOI: (10.1016/j.jvs.2014.07.004) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 3 Influence of short-term high-fat diet (HFD) or fasting on intimal hyperplasia (IH). A, Experimental design. Male C57BL/6J mice were exposed for 2 weeks to HFD or low-fat control diet (C) for the indicated period with or without a 3-day water-only fast immediately before focal stenosis. All mice were then returned to the control diet. Four weeks later, IH was measured by histology. B and C, Intimal area, medial area, and intimal/medial area ratio at the indicated distance from the focal stenosis (n = 10 mice/per group). P value indicates the significance of the difference between intimal areas of the indicated groups according to a two-way analysis of variance. Note the absence of differences between HFD and control diets alone on intimal area. D, Representative Masson trichrome-stained images under each dietary condition taken at 0.6- to 3.0-mm distance proximal to a focal stenosis. The yellow line depicts internal elastic lamina. Scale bars = 50 μm. Journal of Vascular Surgery 2016 63, 500-509.e1DOI: (10.1016/j.jvs.2014.07.004) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Fig 4 Influence of short-term protein-free (PF) dietary restriction (DR) after high-fat diet (HFD) on intimal hyperplasia (IH). A, Experimental design. After 2 weeks on HFD, male B6D2F1 animals were exposed for 1 week to a control diet with 18% calories from protein (C, n = 10) or an isocaloric diet lacking protein (PF, n = 10) before focal stenosis. All mice were then returned to control chow. Four weeks later, IH was measured by histology. B, Intimal area, medial area, and intimal/medial area ratio at the indicated distance from the focal stenosis. P value indicates the significance of the difference between intimal areas of the indicated groups according to a two-way analysis of variance. Journal of Vascular Surgery 2016 63, 500-509.e1DOI: (10.1016/j.jvs.2014.07.004) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 1 (online only) Characteristics of animals on short-term exposure to high-fat diet (HFD) vs control low-fat diet (C). A, Food intake during a 2-week period (n = 10/group) presented as average grams of food eaten per gram of mouse. B, Calorie intake corrected for energy density of diet. C, Weight gain expressed as a percentage of starting weight. D, Adiposity expressed as a percentage of total body weight. E, Blood glucose in the fed state before harvest. F, Serum levels of creatinine at the indicated time after renal reperfusion (n = 5/group). G, Serum levels of aspartate transaminase (AST) at the indicated time after hepatic reperfusion (n = 5/group). The asterisks indicate the significance of the difference between groups according to an unpaired, two-tailed Student t-test; **P < .01, ***P < .001. IR, Ischemia-reperfusion. Journal of Vascular Surgery 2016 63, 500-509.e1DOI: (10.1016/j.jvs.2014.07.004) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

Supplementary Fig 2 (online only) Characteristics of animals on short-term protein-free (PF) dietary restriction (DR) after high-fat diet (HFD) exposure for 2 weeks. A, Daily food intake for 1 week before ischemia-reperfusion (IR) after the switch from HFD to the indicated control (C) or PF diet; n = 4 cages/group (20 mice/group). B, Total food intake 1 week before IR. C, DR calculated as a percentage of average food eaten by animals on the control diet. D, E, Blood glucose (D) and body weight (E) after 1 week on C or PF diet as indicated. The asterisks indicate the significance of the difference between groups according to an unpaired, two-tailed Student t-test; ***P < .001. Journal of Vascular Surgery 2016 63, 500-509.e1DOI: (10.1016/j.jvs.2014.07.004) Copyright © 2016 Society for Vascular Surgery Terms and Conditions