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THE SYNTHETIC PENTASACCHARIDE FONDAPARINUX ATTENUATES MYOCARDIAL ISCHEMIA-REPERFUSION INJURY IN RATS VIA STAT-3 Laurent Macchi,*†‡ Walid Ben Moussa,*† Sophie Guillou,*† Sophie Tamareille,*† Delphine Lamon,*† Delphine Prunier,*§ and Fabrice Prunier*†|| *L’Universite´ Nantes Angers Le Mans; † Universite´ d’Angers, Laboratoire Cardioprotection, Remodelage et Thrombose; ‡ CHU Angers, Laboratoire d’He´matologie; §Universite´ d’Angers, INSERM U771, CNRS UMR 6214, CHU Angers, De´partement de Biochimie et Ge´ne´tique; and ||CHU Angers, Service de Cardiologie, Angers, France Received 8 Jul 2013; first review completed 23 Jul 2013; accepted in final form 13 Aug 2013 ABSTRACT—Acute myocardial infarction is a leading cause of mortality and morbidity worldwide. Although essential for successful recovery, myocardium reperfusion is associated with reperfusion injury. Two major cell survival signaling cascades are known to be protective against ischemia-reperfusion (I/R) injury: the reperfusion injury salvage kinase, including Akt, extracellular signalYregulated kinase 1/2, and the downstream target GSK-3", and the survivor activating factor enhancement, which involves STAT-3. Pharmacologic inhibition of factor Xa has been shown to attenuate I/R injury, but the cellular mechanism is poorly understood. Our aim was to determine the role of whole blood in fondaparinux (FDX)-induced cardioprotection and the involvement of reperfusion injury salvage kinase and survivor activating factor enhancement pathways. We investigated FDX ability to prevent in vivo I/R injury using a transient coronary ligation rat model and ex vivo using a model of crystalloid-perfused isolated rat heart. In both models, infarct size was assessed after 120 min of reperfusion. Myocardial tissues were collected after 15 and 30 min of reperfusion for Western blot analysis. In vivo, FDX decreased infarct size by 29% and induced significant STAT-3 and GSK-3" phosphorylation in comparison to controls. Adding AG490, an inhibitor of JAK/ STAT pathway, before I/R, prevented STAT-3 phosphorylation and abolished FDX-induced cardioprotection. On the contrary, FDX did not have an effect on infarct size or hemodynamic parameters in the isolated-heart model. Fondaparinux decreased I/R injury in vivo, but not in a crystalloid-perfused isolated heart. Under our experimental conditions, FDX required whole blood to be protective, and this beneficial effect was mediated through STAT-3 phosphorylation. KEYWORDS—Cardioprotection, fondaparinux, ischemia-reperfusion injury, SAFE pathway

INTRODUCTION

against I/R injury (6) and to ultimately protect against chronic allograft vasculopathy (7). Low-molecular-weight heparin and unfractionated heparin are commonly used anticoagulants that inhibit factor Xa (FXa) and thrombin in an antithrombin (AT)Ydependent manner (8). These agents have been shown to reduce inflammation in a rodent model of acute renal I/R injury (9). The synthetic pentasaccharide fondaparinux (FDX) is a newly developed anticoagulant that acts as a selective ATdependent inhibitor of FXa (10). This drug is currently used in the prevention and treatment of venous thromboembolism and as treatment of acute coronary syndrome (11). Fondaparinux has also been studied in I/R models. Frank et al. (12) demonstrated that FDX was protective in a lethal murine model of kidney I/R injury by reducing fibrin deposition, inflammation, and recruitment of neutrophils in the kidney. It has been hypothesized that FDX may exert its anti-inflammatory effects independently of its anticoagulant effect and AT binding (13, 14). Recently, FDX-induced inflammatory properties and cardioprotection have been associated with ERK1/2 phosphorylation, suggesting RISK pathway activation (14). The present study was designed to clarify blood’s role in FDX-induced cardioprotection. Therefore, we investigated the ability of FDX to prevent I/R injury, in vivo using a transient coronary ligation model and ex vivo using a model of crystalloid-perfused isolated heart. Furthermore, we explored the involvement of RISK and SAFE pathways, two major survival signaling cascades commonly associated with protection against I/R injury.

Acute myocardial infarction is a leading cause of morbidity and mortality worldwide (1). Although highly beneficial, prompt reperfusion of the ischemic myocardium can paradoxically have deleterious consequences and may lead to lethal myocardial ischemia-reperfusion (I/R) injury (2). Therefore, novel treatment strategies are required to attenuate I/R injury and improve clinical outcome (3). Of these strategies, some have been shown to produce their cardioprotective effects by activating intrinsic prosurvival signaling cascades, such as reperfusion injury salvage kinase (RISK) pathway and the recently described survivor activating factor enhancement (SAFE) pathway (4). Key components of RISK pathway are Akt, extracellular signalYregulated kinase 1/2 (ERK1/2), and the downstream target GSK-3", whereas SAFE pathway involves JAK and STAT-3. Thrombin is the central protease in the coagulation cascade. In addition to its recognized role in the coagulation cascade and hemostasis, thrombin is known to exhibit multiple pleiotropic effects, as it plays a role in inflammation and cellular proliferation, and it displays mitogenic activity on smooth muscle and endothelial cells, predominantly via angiogenesis activation (5). In the heart, thrombin inhibition has been shown to protect Address reprint requests to Fabrice Prunier, MD, PhD, Protection et Remodelage du Myocarde, EA 3860, Faculte´ de Me´decine, Rue Haute de Recule´e, FR-49045 Angers Cedex 1, France. E-mail: [email protected]. DOI: 10.1097/SHK.0000000000000072 Copyright Ó 2013 by the Shock Society

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SHOCK FEBRUARY 2014 MATERIALS AND METHODS Male Wistar rats, 8 to 10 weeks of age weighing 200 to 250 g, were used in this study. All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication 85[23], revised 1996). The protocol was approved by our regional ethic committee: Comite´ Re´gional d’Ethique pour l’Experimentation Animale (permit no. 2012.138). Rats were housed at 22-C with a 12:12-h lightdark cycle with water and food ad libitum.

Study 1: Langendorff-isolated heart Heart preparation—Rat hearts were isolated and perfused, as previously described (15). Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). The heart was rapidly excised and immediately immersed in ice-cold heparinized-modified Krebs-Henseleit buffer containing (in mmol/L) 118 NaCl, 5.6 KCl, 1.2 MgCl2, 1.2 Na2HPO4, 20 NaHCO3, 12 glucose, and 2.4 CaCl2 (pH 7.4). The heart was mounted on a Langendorffperfusion apparatus (EMKA Technologies, Paris, France) and retrogradely perfused through the aorta with nonrecirculating buffer saturated with 95% O2Y5% CO2 at 37-C. The heart was maintained in a thermostatic chamber at 37-C. Perfusion was maintained at a constant pressure of 75 mmHg. A fluid-filled latex balloon was inserted in the left ventricle (LV) via the left atrium for pressure recording. The balloon was connected to a pressure transducer (EMKA Technologies) and inflated to an initial LV end-diastolic pressure between 8 and 10 mmHg. After the global ischemia period, perfusate circulation was switched from an open circuit to a recirculation mode with a perfusion buffer volume of 50 mL. Isolated heart experimental protocol—All hearts were allowed to stabilize for 20 min. Hearts failing to develop LV systolic pressure over 70 mmHg when the end-diastolic pressure was set at 8 to 10 mmHg were excluded from analysis. After the stabilization period, hearts underwent 25 min of global noflow ischemia and 120 min of reperfusion. Hearts were randomly assigned to one of the following groups (Fig. 1A): (i) control (no intervention, n = 7) and (ii) FDX (0.1 mg/mL in recirculating perfusion buffer started at reperfusion onset, n = 7). The dose of 0.1 mg/mL used in our Langendorff study was chosen according to the study of Montaigne et al. (14), in which 0.1 mg/mL FDX was cardioprotective in a crystalloid-perfused isolated rat heart model. Hemodynamic parameter assessment—The following parameters of cardiac function were continuously monitored during the 30 min of reperfusion and simultaneously recorded using IOX 1.593 software (EMKA Technologies): coronary flow rate, heart rate, LV systolic pressure, LV end-diastolic pressure, and the maximal (dP/dtmax) and minimal (dP/dtmin) values of the first derivative of LV pressure. Left ventricleYdeveloped pressure was calculated as the difference between LV systolic and diastolic pressure. Infarct size measurement—After 2 h of reperfusion, the hearts were harvested, and the LVs were sectioned from apex to base into five to six 1-mm sections using a coronal heart slicer matrix (Braintree Scientific Inc, Braintree, Mass). Sections were incubated in 1% triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St Louis, MO) in phosphate-buffered solution (pH 7.4) at 37-C for 10 min and then fixed in 10% formalin. For each section, the area of necrosis (AN) was quantified by planimetry using Image J software (NIH, Bethesda, Md) and expressed as a percentage of the total LV area.

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Study 2: in vivo I/R Myocardial infarction model—All animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg) and endotracheally intubated with a 16-gauge tube. Rats were ventilated using a small animal ventilator (SAR-830 A/P; CWE, Ardmore, Pa). Body core temperature was continuously monitored throughout the surgical procedure and maintained at 36-C to 38-C using a homeothermic blanket set that was connected to a temperature control unit (HB101/2 RS; Bioseb, Vitrolles, France). A left thoracotomy was performed in the fifth intercostal space. The pericardium was removed, the heart was exposed, and a 7-0 monofilament suture (Premio 7.0; Peters Surgical, Bobigny, France) was placed around the proximal portion of the left anterior descending coronary artery (LAD) and passed through a short piece of tubing (PE50) to create a reversible snare. Following heart stabilization, coronary occlusion was initiated by clamping the snare onto the epicardial surface directly above the coronary artery. Ischemia was confirmed by epicardial cyanosis below the suture and by dyskinesis of the ischemic region. After 40 min of occlusion, reperfusion was achieved by loosening the snare, which was confirmed by a marked hyperemic response at reperfusion. Study groups and experimental protocol—Rats were subjected to 40 min of LAD occlusion followed by 2 h of reperfusion for myocardial infarct size assessment and 15 and 30 min of reperfusion for Western blot analysis. Rats were randomly assigned to one of four groups (Fig. 1B): (i) control (no intervention; n = 7); (ii) FDX (10 mg/kg FDX administered intraperitoneally 10 min before reperfusion; n = 7); (iii) control + AG490 [JAK/STAT pathway inhibitor 3 mg/kg (16) intravenous bolus injection 15 min before reperfusion; n = 7]; and (iv) FDX + AG490 (n = 7). An additional group of sham-operated rats was performed (n = 4). Sham-operated rats were submitted to the same procedure as other groups without clamping the snare. The dose of 10 mg/kg FDX used in our in vivo study was chosen according to the study of Frank et al. (12), in which 10 mg/kg FDX reduced coagulation activation, inflammation, and neutrophil accumulation in a kidney I/R mice model (12).

Area at risk and infarct size determination At the end of the 2-h reperfusion period, the heart was removed quickly, and the LAD was reoccluded using the monofilament suture kept in place as previously described (15, 17). The heart was then retrogradely perfused ex vivo with Evans blue (1%) to delineate the area at risk (AAR). The LVs were then cut into five to six slices from apex to base, which was followed by incubation at 37-C in a 1% solution of phosphate-buffered 2,3,5-TTC, as described in study 1, to delineate infarcted myocardium. Slices were then fixed in formalin 10%, and infarct size was quantified by computerized planimetry using Image J software. Area of necrosis was expressed as a percentage of AAR, and AAR as a percentage of total LV area.

Western blot analysis Western blots were performed on myocardium from the AAR. The ventricular tissue was excised and freeze clamped in liquid nitrogen before being stored at j80-C. Frozen myocardial tissue samples were powdered using a mortar and pestle precooled to the temperature of liquid nitrogen. Approximately 200 mg of powdered ventricular tissue was used for protein extraction.

FIG. 1. Experimental protocols. A, Langendorff-isolated heart model: all groups were subjected to 25-min ischemia followed by 120-min reperfusion. Fondaparinux was diluted at a concentration of 0.1 mg/mL in 50 mL of perfusion buffer. B, In vivo I/R model: all groups were subjected to 40 min of coronary artery occlusion followed by either 120 min of reperfusion for infarct size measurement or 30 min of reperfusion for protein phosphorylation analysis. Fondaparinux (10 mg/kg) was injected intraperitoneally 10 min before reperfusion. Pharmacologic inhibitor AG490 (3 mg/kg) was injected intravenously 15 min before reperfusion.

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TABLE 1. Hemodynamic parameters in the Langendorff-isolated heart model Parameters

Group

LV systolic pressure, mmHg

LV diastolic pressure, mmHg

LV developed pressure, mmHg

LV dP/dtmax, mmHg/s

Baseline

Coronary flow, mL/min

Heart rate, beats/min

30-min Reperfusion

82.9 T 12.6

75.9 T 6.4

Control (n = 7)

98.9 T 3.6

FDX (n = 7)

102.0 T 3.4

101.3 T 7.4

92.7 T 5.0

Control

1.6 T 1.2

60.0 T 7.5

48.6 T 3.5

FDX

3.0 T 1.0

71.7 T 6.3

61.3 T 6.0

Control

98.6 T 3.3

29.6 T 3.7

31.4 T 6.5

FDX

85.9 T 3.7

27.3 T 6.2

38.5 T 8.1

2524 T 117

507 T 58

2,244 T 79

491 T 112

Control FDX

LV dP/dtmin, mmHg/s

5-min Reperfusion

749 T 164 1,090 T 220

Control

j1,639 T 60

j327 T 81

j500 T 103

FDX

j1,592 T 76

j361 T 47

j588 T 101

Control

18.2 T 0.1

11.9 T 0.5

12.4 T 0.5

FDX

17.2 T 0.8

11.6 T 0.6

12.0 T 0.7

Control

289 T 23

336 T 96

343 T 59

FDX

272 T 14

171 T 14

296 T 122

Values are mean T SEM. Frozen myocardial tissue samples were homogenized on ice in 1 mL ice-cold lysis buffer containing 30 mM HEPES, 20 mM KCl, 2.5 mM EGTA, 2.5 mM EDTA, 40 mM sodium fluoride, 4 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10% glycerol, and 1% Nonidet P-40, a phosphatase inhibitor cocktail (Sigma-Aldritch), and a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Mannheim, Germany). The homogenates were centrifuged at 13,000 revolutions/min at 4-C for 1 h, and the resulting supernatant was collected. Protein concentration was determined using Bio-Rad DC protein assay kit (Bio-Rad, Hercules, Calif) according to the manufacturer’s instructions. Aliquots of the supernatant containing equal amounts of proteins (40 Hg) were heated to 95-C for 5 min in a sample loading buffer. Proteins were separated on a 10% sodium dodecyl sulfateYpolyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane (Amersham Bioscience, Amersham, UK). After the nonspecific binding sites were blocked with 5% nonfat milk for 1 h in Tris-buffered saline Tween containing 20 mM Tris-HCl, 137 mM NaCl (pH 7.6), and 0.1% Tween-20, the membranes were incubated overnight at 4-C with rabbit antibodies against 473Ser-phospho-Akt, total Akt, phospho-ERK1/2, total ERK1/2, 9Ser-phospho-GSK-3", total GSK-3", 705Tyr-phosphor-STAT-3,

and total STAT-3 (1/1,000; Cell Signaling, Danvers, Mass). GAPDH (SigmaAldrich) was used as a loading control. After being washed in Tris-buffered saline Tween, the membranes were incubated for 1 h at room temperature with horseradish peroxidaseYconjugated antiYrabbit immunoglobulin G secondary antibodies (1/2,000; Santa Cruz Biotechnologies, Santa Cruz, Calif) and washed, and bound antibodies were detected using an enhanced chemiluminescence Western blotting kit (Santa Cruz Biotechnologies). The densities of the bands with appropriated molecular mass (60 kd for Akt, 42/44 kd for ERK1/2, 46 kd for GSK-3", and 80 kd for STAT-3) were determined semiquantitatively using a lumino-image analyzer, LAS-3000 mini (Fujifilm, Tokyo, Japan).

Measurement of anti-FXa activity The anti-FXa chromogenic assay was performed using the Biophen Heparin 6 kit (HYPHEN BioMed, Neuville-sur-Oise, France) on an automated analyzer (STA-R; STAGO, Asnie`res-sur-Seine, France) according to the manufacturer’s protocol. We determined anti-FXa activity in blood samples collected before injection and also at 10, 30, and 40 min after injection of FDX 10 mg/kg IP (n = 3).

FIG. 2. Infarct size in the in vivo I/R and Langendorff-isolated heart models. A, Representative sections of TTC-stained heart following 25 min of global ischemia and 120 min of reperfusion from the isolated heart experiments in control and FDX-treated rats. Area of necrosis (AN) is expressed as percentage of LV area. NS indicates not statistically significant. B, Representative sections of TTC-stained heart following 40 min of ischemia and 120 min of reperfusion from the in vivo I/R model in control and FDX-treated rats. Bar graph shows AN expressed as percentage of AAR and AAR as percentage of total LV area (LV). All data are expressed as mean T SEM. *P G0.05 vs. control group; n = 7 in each group.

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Drugs and chemicals Pentobarbital was obtained from Ce´va Sante´ Animale. Fondaparinux was purchased from GlaxoSmithKline Laboratories (Marly le roi, France), and AG490 from Tocris Bioscience (Minneapolis, Minn). AG490 was dissolved in dimethyl sulfoxide and diluted into saline in order for the vehicle to constitute less than 1% of the total injected volume.

Statistic analyses All values were expressed as mean TSEM. Statistical analyses were performed using SPSS Statistics 17.0 (SPSS Inc, Chicago, Ill). Differences between groups were evaluated using Mann-Whitney U test for unpaired samples or analysis of variance followed by post hoc Fisher least significant differenceYcorrected multiple-comparisons test, when applicable. Analysis of variance for repeated measures was used for hemodynamic data analysis in study 1. P G 0.05 was considered to be statistically significant.

RESULTS FDX decreased postYischemic cardiac injury in vivo only

Study 1: Langendorff-isolated heart—There was no significant difference in heart rate and coronary flow levels at baseline and throughout the reperfusion period between the control and FDX groups (Table 1). None of the LV systolic and diastolic function parameters were significantly different between FDXtreated hearts and control. As illustrated in Fig. 2A, FDX-treated hearts exhibited similar infarct size as control (AN/LV = 29.7% T 3.5% in FDX vs. 25.4% T 3.4% in control, P = 0.17). Study 2: in vivo I/R—As shown in Fig. 2B, the ischemic area induced by LAD ligation did not differ between FDX-treated rats and control (AAR/LV = 38.2% T 2.4% vs. 36.6% T 2.4% in FDX and control, respectively, P = 0.52). To the contrary of results observed in study 1, FDX injection significantly decreased necrosis with comparison to control (AN/AAR = 44.3% T 2.3% vs. 62.0% T 2.5% in FDX and control, respectively, P G0.05) (Fig. 2B). The measurement of anti-Xa activity showed that 10 mg/kg of FDX increased anti-Xa activity in plasma. The anti-Xa activity was less than 0,01 Hg/mL just before the injection, 8.2 T 3.3 Hg/mL 10 min after the injection, 10.4 T 3.4 Hg/mL 30 min after the injection, and 11.3 T 2.7 Hg/mL 40 min after the injection. FDX-induced cardioprotection involved SAFE pathway

To explain the cardioprotection afforded by FDX in vivo, we evaluated the RISK pathway (Akt, ERK1/2), its downstream target GSK-3", and SAFE pathway (STAT-3) phosphorylation levels after 15 and 30 min of reperfusion. No significant dif-

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ference in the phosphorylation of these kinases was observed after 15 min of reperfusion (data not shown). Conversely, after 30 min of reperfusion, myocardial GSK-3" and STAT-3 phosphorylations were significantly increased by 27% and 128%, respectively, as compared with control (Fig. 3). STAT-3 inhibition abolished FDX-induced cardioprotection in vivo

To further explore the role of the SAFE pathway in FDXinduced cardioprotection, we used AG490, a pharmacologic inhibitor of JAK/STAT pathway. As shown in Fig. 4A, FDXinduced cardioprotection was completely abolished in the presence of AG490 (3 mg/kg) injected 5 min before FDX injection. Fondaparinux-induced phosphorylation of STAT-3 was also inhibited by AG490 (Fig. 4B). DISCUSSION In this study, we examined the protective effect of FDX, a highly specific AT-dependent inhibitor of the coagulation protease FXa (10), using two models of myocardial I/R. In vivo, intraperitoneal FDX injection 10 min before reperfusion decreased infarct size by 29%. The protective effect of FDX has been previously evaluated in vivo in both renal and intestinal I/R injuries (12, 18). In a lethal murine model of kidney I/R injury, FDX injected intraperitoneally 5 min before reperfusion reduced plasma creatinine levels and increased survival from 0% to 44% compared with saline-treated control mice (12). In their model, the authors analyzed the effects of ancrod (used to deplete fibrinogen) on the survival of I/R-injured mice. Ancrod slightly prolonged the survival time of these mice compared with controls but did not protect them from death. This suggests that FDX was protective not only via reducing fibrin deposition. Using the same animal model, a nonanticoagulant pentasaccharide, which cannot bind AT, was shown to significantly reduce plasma creatinine levels 48 h after reperfusion (13). In these experiments, FDX was protective mainly through an antiinflammatory effect (12, 13). To evaluate the cardioprotective effect of FDX independently of its circulating anticoagulant properties, we used a crystalloidperfused heart model in which hearts were reperfused in the presence of FDX without circulating blood. Neither hemodynamic parameters nor infarct size was improved by FDX treatment in

FIG. 3. Reperfusion injury salvage kinase Reperfusion injury salvage kinase and SAFE pathways after 30 min of reperfusion in the in vivo model. Western blot analysis of Akt (A), ERK1/2 (B) GSK-3" (C), and STAT-3 (D) phosphorylation. Top, Representative bands from Western blots of phosphorylated Akt (p-Akt) and total Akt (A), phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (B), phosphorylated GSK-3" (p-GSK-3") and total GSK-3" (C), and phosphorylated STAT-3 (p-STAT-3) and total STAT-3, in LV homogenates from hearts subjected to I/R. Bottom, Bar graphs show mean T SEM of the densitometry of p-AktYtoYAkt ratio (A), p-ERK1/2YtoYERK1/2 ratio (B), p-GSK-3"YtoYGSK-3" ratio (C), and p-STAT-3YtoYSTAT-3 ratio (D). All data are expressed as mean T SEM. *P G 0.05 vs. control; †P G 0.05 vs. sham; control group n = 7; FDX group n = 7; sham group n = 4.

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FIG. 4. Effect of AG490 on infarct size and protein phosphorylation in the in vivo model. A, Top, Representative sections of rat hearts stained with TTC after 40 min of coronary artery occlusion and 120 min of reperfusion. Bottom, Bar graph shows AN expressed as percentage of AAR for each group in the absence or presence of AG490. *P G 0.05 vs. control; n = 7 in each group. B, Top, Representative bands from Western blots of p-GSK-3", total GSK-3", p-STAT3, and total STAT-3 in the absence or presence of AG490. Bottom, Bar graph shows the densitometry of p-GSK-3"YtoYGSK-3" ratio and p-STAT-3YtoYSTAT-3 ratio. *P G 0.05 vs. control; n = 7 in each group. All data are expressed as mean T SEM.

this model, which suggests that FDX requires whole blood to be cardioprotective. Montaigne et al. (14) previously published contradictory results. In a crystalloid-perfused heart model, FDX improved postYischemic myocardial contractile performance (14). The reasons for these discrepancies are unclear. Whereas FDX dose, timing, and route were similar in both studies, the Langendorff-isolated heart model was slightly different. Montaigne et al. (14) used a constant coronary flow rate, whereas we used a constant perfusion pressure. Nevertheless, we were previously able to show the cardioprotective effects of erythropoietin in the constant perfusion pressure model (15). In addition, we used Wistar rats, whereas Montaigne et al. (14) used Sprague-Dawley rats. It has already been demonstrated in a model of experimental stroke that ischemic lesion volume and edema formation can differ between Wistar and Sprague-Dawley rats (19). Apoptotic cell death during the reperfusion phase is a major contributor to lethal reperfusion-induced injury (20). Therefore, targeting antiapoptotic mechanisms of cellular protection at the time of reperfusion may offer a potential approach to attenuating reperfusion-induced cell death. In this regard, activating RISK and SAFE survival cascades at the time of reperfusion has been demonstrated to confer protection against reperfusioninduced injury, along with antiapoptotic effects (16). Furthermore, it has been suggested that FDX decreases myocardial damage through ERK1/2 phosphorylation (14). In the present study, we demonstrated that FDX-induced cardioprotection involved STAT-3 and GSK-3" phosphorylation after 30 min of reperfusion, but not after 15 min of reperfusion. These two

time points have been determined considering (i) the study performed by Montaigne et al. (14), who showed an increase in ERK1/2 phosphorylation at 30 min after reperfusion, and (ii) the results obtained in our laboratory, in which remote ischemic conditioning, in combination with local ischemic postconditioning, induced RISK and SAFE pathway activation 15 min after reperfusion (16). To test the role of STAT-3 (SAFE pathway) in the FDX-induced cardioprotection mechanism, we injected a JAK/STAT pathway inhibitor (AG490) before the FDX injection. AG490 completely abolished FDX’s protective effects, attesting that FDX involved SAFE pathway to be effective. The beneficial effects of SAFE pathway activation on I/R injury would be mediated via the inhibition of mitochondrial permeability transition pore opening (4). Limitations

The mechanism by which FDX activates the SAFE pathway and the specific role of FXa inhibition in this cardioprotection remain unknown. Further studies are required to determine whether selective new anticoagulants directed against FXa could become attractive strategies in I/R injury management of the heart or other organs. Another limitation of the study lies in the choice of rat strain (19), which is known to greatly influence the results of experimental research and therefore might affect the applicability of results to human. In these conditions, our results must be further confirmed in other species. In conclusion, FDX decreased I/R injury in vivo but not in a crystalloid-perfused isolated heart. The protective effect was

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mediated through STAT-3 phosphorylation. Under our experimental conditions, FDX required whole blood to be protective. REFERENCES 1. Murray CJ, Lopez AD: Alternative projections of mortality and disability by cause 1990Y2020: Global Burden of Disease Study. Lancet 349(9064): 1498Y1504, 1997. 2. Piper HM, Garcn˜a-Dorado D, Ovize M: A fresh look at reperfusion injury. Cardiovasc Res 38(2):291Y300, 1998. 3. Hausenloy DJ, Baxter G, Bell R, Bøtker HE, Davidson SM, Downey J, Heusch G, Kitakaze M, Lecour S, Mentzer R, et al.: Translating novel strategies for cardioprotection: the Hatter Workshop Recommendations. Basic Res Cardiol 105(6):677Y686, 2010. 4. Lacerda L, Somers S, Opie LH, Lecour S: Ischaemic postconditioning protects against reperfusion injury via the SAFE pathway. Cardiovasc Res 84(2): 201Y208, 2009. 5. Siller-Matula JM, Schwameis M, Blann A, Mannhalter C, Jilma B: Thrombin as a multi-functional enzyme. Thromb Haemost 106(4):705Y711, 2011. 6. Erlich JH, Boyle EM, Labriola J, Kovacich JC, Santucci RA, Fearns C, Morgan EN, Yun W, Luther T, Kojikawa O, et al.: Inhibition of the tissue factorYthrombin pathway limits infarct size after myocardial ischemia-reperfusion injury by reducing inflammation. Am J Pathol 157(6):1849Y1862, 2000. 7. Ho¨lschermann H, Bohle RM, Schmidt H, Zeller H, Fink L, Stahl U, Grimm H, Tillmanns H, Haberbosch W: Hirudin reduces tissue factor expression and attenuates graft arteriosclerosis in rat cardiac allografts. Circulation 102(3): 357Y363, 2000. 8. Gross PL, Weitz JI: New anticoagulants for treatment of venous thromboembolism. Arterioscler Thromb Vasc Biol 28(3):380Y386, 2008. 9. Tyrrell DJ, Horne AP, Holme KR, Preuss JM, Page CP: Heparin in inflammation: potential therapeutic applications beyond anticoagulation. Adv Pharmacol San Diego Calif 46:151Y208, 1999. 10. Turpie AGG, Eriksson BI, Lassen MR, Bauer KA: Fondaparinux, the first selective factor Xa inhibitor. Curr Opin Hematol 10(5):327Y332, 2003. 11. Mehta SR, Boden WE, Eikelboom JW, Flather M, Steg PG, Avezum A, Afzal R, Piegas LS, Faxon DP, Widimsky P, et al.: Antithrombotic therapy with fondaparinux in relation to interventional management strategy in patients with

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The synthetic pentasaccharide fondaparinux attenuates myocardial ischemia-reperfusion injury in rats via STAT-3.

Acute myocardial infarction is a leading cause of mortality and morbidity worldwide. Although essential for successful recovery, myocardium reperfusio...
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