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Xianyao Xu, MS, Jennifer L. Philip, MD, Md

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1 High-molecular-weight polyethylene glycol inhibits myocardial ischemia–reperfusion injury in vivo 
Xianyao Xu, MS, Jennifer L. Philip, MD, Md. Abdur Razzaque, PhD, James W. Lloyd, BS, Charlie M. Muller, MD, Shahab A. Akhter, MD  The Journal of Thoracic and Cardiovascular Surgery  Volume 149, Issue 2, Pages (February 2015) DOI: /j.jtcvs Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

2 Figure 1 PEG is present in the myocardium following intracoronary or intravenous delivery. Fluorescein-labeled PEG or PBS (control) was delivered via direct injection into the left ventricular outflow tract following transient aortic crossclamping (intracoronary delivery) or by intravenous delivery via the right internal jugular vein. Both delivery techniques resulted in robust uptake of PEG in the heart, as demonstrated by the green fluorescein labeling. PEG, Polyethylene glycol; PBS, phosphate-buffered saline. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

3 Figure 2 PEG treatment improves cardiac function following I-R injury. Cardiac function was studied by echocardiography in rats 4 weeks following I-R injury and treatment with either intravenous PBS or PEG. A, Fractional shortening is significantly higher in the PEG compared to the PBS group at 2 to 4 weeks. n = 6 in each group; *P < .03 versus PBS. B, Ejection fraction in PBS- versus PEG-treated rats up to 4 weeks post I-R injury. n = 6 in each group; *P < .04 versus PBS. C, End-systolic diameter is significantly smaller in PEG versus PBS groups at 3 and 4 weeks post I-R injury. n = 6 in each group; *P < .03 versus PBS, #P = .05 versus PBS. D, Mortality rate at 48 hours following 1 hour ischemia in PBS- and PEG-treated animals. n = 14 for PBS and 11 for PEG. P = .34 using Fisher exact test. PEG, Polyethylene glycol; PBS, phosphate-buffered saline; I-R, ischemia-reperfusion. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

4 Figure 3 PEG treatment decreases fibrosis at 4 weeks following I-R injury. A, Representative photomicrographs of Picro-Sirius Red–stained left ventricles of control rats and PBS- or PEG-treated rats harvested 4 weeks post I-R injury. B, Quantitation of fibrotic area (red staining area) of left ventricle. n = 5 to 6 per group; *P = .001 versus Control, #P < .01 versus PBS. C, Representative photomicrographs of Masson trichrome–stained left ventricles from control, PBS-treated, and PEG-treated rats harvested 4 weeks post–I-R injury. D, Quantitation of left ventricular fibrotic area (purple/blue staining area). n = 5 to 6 per group; *P < .001 versus Control, #P < .001 versus PBS. PEG, Polyethylene glycol; PBS, phosphate-buffered saline. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

5 Figure 4 PEG treatment decreases apoptosis following I-R injury. A, Representative photomicrographs of TUNEL staining in left ventricles of control rats and PBS- or PEG-treated rats harvested 48 hours post–I-R injury. Black arrows show examples of TUNEL-positive cells in each image. B, Quantitation of TUNEL-positive cells/hpf. n = 3 to 5 per group; *P = .01 versus Control, #P < .02 versus PBS. C, Representative immunoblot shows cleaved caspase 3 expression in left ventricles at 48 hours following I-R in rats treated with PBS or PEG. α-Tubulin was used as a loading control. D, Densitometric analysis of cleaved caspase 3/α-tubulin expression. n = 5 per group; *P < .003 versus PBS. PBS, Phosphate-buffered saline; PEG, polyethylene glycol; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling; hpf, high-powered field; CTRL, control. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

6 Figure 5 Increased left ventricular survival signaling with PEG treatment at 48 hours post–I-R injury. A, Representative immunoblot shows phosphorylated Akt (p-Akt) and total Akt (t-Akt) expression in left ventricles at 48 hours following I-R in rats treated with PBS or PEG and densitometric analysis of p-Akt/t-Akt expression. n = 4 to 5 per group; *P < .002 versus PBS. B, Representative immunoblot of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) expression and densitometric analysis of p-ERK1/2:t-ERK1/2 expression. n = 4 per group; #P < .05 versus PBS. C, Representative immunoblot shows phosphorylated GSK 3β (p-GSK 3β) and total GSK 3β (t-GSK 3β) expression and densitometric analysis of p-GSK 3β/t-GSK 3β expression. n = 4 to 5 per group; *P < .05 versus PBS. D, Representative immunoblot shows phosphorylated eNOS (p-eNOS) and total eNOS (t-eNOS) expression at 48 hours following I-R in rats treated with PBS or PEG and densitometric analysis of p-eNOS/t-eNOS expression. n = 5 per group; #P < .02 versus PBS. PBS, Phosphate-buffered saline; PEG, polyethylene glycol; eNOS, endothelial nitric oxide synthase. The Journal of Thoracic and Cardiovascular Surgery  , DOI: ( /j.jtcvs ) Copyright © 2015 The American Association for Thoracic Surgery Terms and Conditions

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