Protection by 20-5,14-HEDGE Against Surgically Induced Ischemia Reperfusion Lung Injury in Rats Irshad Ali, PhD, Stephanie Gruenloh, BS, Ying Gao, MS,

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Protection by 20-5,14-HEDGE Against Surgically Induced Ischemia Reperfusion Lung Injury in Rats Irshad Ali, PhD, Stephanie Gruenloh, BS, Ying Gao, MS, Anne Clough, PhD, John R. Falck, PhD, Meetha Medhora, PhD, Elizabeth R. Jacobs, MD, MBA The Annals of Thoracic Surgery Volume 93, Issue 1, Pages 282-288 (January 2012) DOI: 10.1016/j.athoracsur.2011.08.074 Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 (A) Chemical structure of 20-HETE and 20-5,14-HEDGE. (B, C) Ischemia-reperfusion (IR) lungs from vehicle-treated and 20-5,14-HEDGE–treated rats. The IR resulted in gross hemorrhage in the left (L) lung, and to a lesser extent, right (R) lung. Treatment with 20-5,14-HEDGE decreased visible hemorrhage. (D, E) Hematoxylin and eosin sections revealed hemorrhage (arrows point to red blood cells in intraalveolar spaces) in ischemic left lungs, which exceeded that in right lungs. (20-5, 14-HEDGE = N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine.) The Annals of Thoracic Surgery 2012 93, 282-288DOI: (10.1016/j.athoracsur.2011.08.074) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Representative immunohistochemical analyses demonstrate the presence of myeloperoxidase-positive cells (see arrow in A) in both left and right lungs of vehicle-treated and 20-5,14-HEDGE–treated animals. The myeloperoxidase expression was quantitated as brown color in photomicrographic images analyzed by an investigator masked to treatment groups. (A) Vehicle ischemia-reperfusion (IR) left lung; (B) vehicle IR right lung; (C) 20-5,14 HEDGE IR left lung; (D) 20-5,14 HEDGE IR right lung. (IR = ischemia reperfusion; 20-5, 14-HEDGE = N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine.) The Annals of Thoracic Surgery 2012 93, 282-288DOI: (10.1016/j.athoracsur.2011.08.074) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Western blots for toll-like receptor 4(TLR4). (A) Representative images of Western blots probed with a primary antibody to TLR4 show increased band density in samples from vehicle-treated left lungs compared with right lungs. Treatment with 20-5,14-HEDGE partially blunted IR-induced increases in TLR4 protein. (B) Average densities and standard error bars of TLR4 normalized for β-actin. *Indicates p ≤ critical level relative to right lung vehicle. #Indicates p ≤ critical level relative to left lung vehicle. Numbers (n) for paired studies appear in the bar for right lung values. (Black bars = left lung; white bars = right lung.) (IR = ischemia reperfusion; 20-5, 14-HEDGE = N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine.) The Annals of Thoracic Surgery 2012 93, 282-288DOI: (10.1016/j.athoracsur.2011.08.074) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Western blots for high mobility group box 1(HMGB1). (A) Representative images of Western blots probed for HMGB1. Immunospecific band density was increased in samples from ischemia-reperfusion (IR) lungs of vehicle-treated hosts. Treatment with 20-5,14-HEDGE blunted the increase in HMGB1. Lungs from sham-operated animals showed no difference in HMGB1 levels. (B) Graph depicts average densities and SEM of HMGB1 normalized for β-actin. *Indicates p ≤ critical level relative to right lung vehicle. Numbers (n) for paired studies appear in the bar for right lung values. (Black bars = left lung; white bars = right lung.) (20-5, 14-HEDGE = N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine.) The Annals of Thoracic Surgery 2012 93, 282-288DOI: (10.1016/j.athoracsur.2011.08.074) Copyright © 2012 The Society of Thoracic Surgeons Terms and Conditions