Low Potassium Dextran Lung Preservation Solution Reduces Reactive Oxygen Species Production

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1 Low Potassium Dextran Lung Preservation Solution Reduces Reactive Oxygen Species Production Rosemary F. Kelly, MD, Jozef Murar, MD, Zhigang Hong, MD, PhD, Daniel P. Nelson, BS, Fangxiao Hong, MD, Anthony Varghese, PhD, and E. Kenneth Weir, MD Departments of Surgery and Medicine, Veterans Affairs Medical Center, and Departments of Surgery and Medicine, University of Minnesota, Minneapolis, Minnesota GENERAL THORACIC Background. Low potassium dextran lung preservation solution has reduced primary graft failure in animal and human studies. Though the mechanism of reducing primary graft failure is unknown, low potassium dextran differs most significantly from solutions such as Euro- Collins (EC) and University of Wisconsin in its potassium concentration. The aim of this study was to investigate the impact that potassium concentration in lung preservation solutions had on pulmonary arterial smooth muscle cell depolarization and production of reactive oxygen species. Methods. Using isolated pulmonary artery smooth muscle cells from Sprague-Dawley rats, the patch-clamp technique was used to measure resting cellular membrane potential and whole cell potassium current. Measurements were recorded at base line and after exposure to low potassium dextran, EC, and University of Wisconsin solutions. Pulmonary arteries from rats were isolated from the main pulmonary artery to the fourth segmental branch. Arteries were placed into vials containing low potassium dextran, EC, low potassium EC, Celsior, and University of Wisconsin solutions with reactive oxygen species measured by lucigenin-enhanced chemiluminescence. Accepted for publication Jan 12, Address reprint requests to Dr Kelly, One Veterans Drive, Cardiovascular Surgery (112), VA Medical Center, Minneapolis, MN 55417; Results. Pulmonary artery smooth muscle cell membrane potentials had a significant depolarization when placed in the University of Wisconsin or EC solutions, with changes probably related to inhibition of voltagegated potassium channels. Low potassium dextran solution did not alter the membrane potential. Production of reactive oxygen species as measured by chemiluminescence was significantly higher when pulmonary arteries were exposed to University of Wisconsin or EC solutions (51,289 5,615 and 35, counts/0.1 minute, respectively) compared with low potassium dextran, Celsior, and low potassium EC (12, , 13,717 3,844 and 15,187 3,792 counts/0.1 minute, respectively). Conclusions. Preservation solutions with high potassium concentration are clearly able to depolarize the pulmonary artery smooth muscle cells and increase pulmonary artery reactive oxygen species production. Low potassium preservations solutions may limit reactive oxygen species production and thus reduce the incidence of primary graft failure in lung transplantation. (Ann Thorac Surg 2003;75: ) 2003 by The Society of Thoracic Surgeons Lung transplantation is an effective treatment of endstage pulmonary diseases [1]. Yet primary graft failure, an apparent result of ischemia-reperfusion injury, continues to affect 20% to 30% of lung transplantations [2]. In an effort to reduce the incidence of primary graft failure, the composition of lung preservation solutions has been intensely studied both clinically and experimentally [3]. Solutions most commonly included are Euro-Collins (EC), University of Wisconsin (UW), low potassium dextran (LPD), and Wallwork [4, 5]. Various pharmacologic adjuncts to the preservation solution have been studied as well [5]. Frequently the distinction is made between extracellular (LPD, Celsior, Wallwork) and intracellular (EC, UW) perfusate composition, with the potassium concentration being a particularly important component. Recent clinical experience has supported experimental evidence that use of LPD has resulted in a reduction in primary graft failure [6, 7]. The mechanism by which potassium may affect lung preservation has not been elucidated. The role of potassium concentration in lung preservation solution is of interest as potassium channels serve many functions in the regulation of pulmonary vascular tone. Although there are many modulators of pulmonary circulation, the regulation of hypoxic vasoconstriction by voltage-gated potassium channels suggests that the potassium concentration of lung preservation solution may dynamically alter pulmonary vasculature tone. Hypoxic pulmonary vasoconstriction occurs when a hypoxia inhibits voltage-gated potassium channels in pulmonary artery smooth muscle cells leading to depolarization and entry of calcium through the L-type calcium channels [8]. It is possible that a similar mechanism may activate reactive oxygen species (ROS) production from the pulmonary vasculature. This study was performed to analyze pulmonary artery 2003 by The Society of Thoracic Surgeons /03/$30.00 Published by Elsevier Inc PII S (03)

2 GENERAL THORACIC 1706 KELLY ET AL Ann Thorac Surg LPD REDUCES ROS PRODUCTION 2003;75: Table 1. Composition of Lung Preservation Solutions Component Modified Euro-Collins Low Potassium Dextran Low Potassium Euro-Collins University of Wisconsin Celsior Krebs Na (mmol/l) K (mmol/l) Cl (mmol/l) PO4 (mmol/l) HCO3 (mmol/l) SO4 (mmol/l) Glucose (g/l) Dextran-40 (g/l) Raffinose (mmol/l) Lactobionate (mmol/l) Glutathione (mmol/l) Adenosine (mmol/l) Allopurinol (mmol/l) Penta-fraction (g/l) Histidine (mmol/l) Mannitol (mmol/l) Glutamate (mmol/l) Osmolality ph smooth muscle cell membrane potential responses to the lung preservation solutions EC, UW, and LPD (Table 1). As these solutions have complex compositions, the impact of potassium concentration on membrane depolarization and, in particular, inhibition of whole-cell potassium current was studied by the patch-clamp technique. We hypothesized that membrane depolarization from intracellular preservation solutions may result in increased production of ROS by pulmonary arteries. Increased ROS production during the procurement phase of transplantation may predispose the lung graft to primary graft failure. Material and Methods Materials Sprague-Dawley Rats (200 to 250 gm) were obtained from Harlan (Madison, WI), lucigenin from Sigma (St. Louis, MO), LPD (Perfadex) from Vitrolife (Göttborg, Sweden), EC from Baxter Healthcare Corp (Deerfield, IL), UW solution from Du Pont Pharmaceuticals (Wilmington, DE), monosodium lactobionate gift from Reliable Biopharmaceutical Corp (St. Louis, MO), and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Arterial Isolation and Cell Dispersion for Patch Clamping The Institutional Review Board of the Veterans Affairs Medical Center (Minneapolis, MN) reviewed and approved the protocol for this study. All animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication No , revised 1985). Adult male Sprague-Dawley rats were euthanized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) overdose. The lungs were removed entirely and the main pulmonary artery was isolated. The left and right pulmonary arteries were then dissected to the fourth segmental branch and removed. The arteries from the second to fourth segmental branches were opened longitudinally and placed in calcium-free Hank s solution (140 mmol/l NaCl, 4.2 mmol/l KCl, 1.2 mmol/l KH 2 PO 4, 1.5 mmol/l MgCl 2, 10 mmol/l HEPES, 0.1 mmol/l ethylene glycol bis(-aininoethyl ether)- N, N, N 1, N 1, -tetraacetic acid, ph 7.4) for 20 minutes. The pulmonary arteries were then placed in Hanks solution that contained papain (1 mg/ml), dithiothreitol (0.75 mg/ml), collagenase (0.8 mg/ml), and bovine serum (0.8 mg/ml) at 4 C for 15 minutes, then heated to 37 C for 10 minutes. The arteries were transferred to iced Hanks solution and trituration with a Pasteur pipette that produced a suspension of single cells. Dispersed pulmonary artery smooth muscle cells were transferred to the perfusion chamber on the stage of an inverted microscope for patch clamp studies. Three animals were used to supply cells for each study group. Patch Clamping Whole cell patch clamp recordings were made as previously described [9]. Briefly, the microelectrodes had a resistance of approximately 3 ohms. The pipette solution contained 140 mmol/l KCl, 1.0 mmol/l MgCl 2, 10 mmol/l HEPES, and 5 mmol/l ethylene glycol bis (-aininoethyl ether)-n, N, N 1,N 1, - tetraacetic acid, ph 7.2. The chamber containing the cells was perfused (at 2 ml/min) with a solution containing 115 mmol/l NaCl, 4.5 mmol/l KCl, 0.5 mmol/l MgCl 2, 1.5 mmol/l CaCl 2, 10 mmol/l HEPES, 26 mmol/l NaHCO 3, and 10 mmol/l glucose, ph 7.4 with baseline readings obtained as the control. The solution was first changed to Krebs solution as the

3 Ann Thorac Surg KELLY ET AL 2003;75: LPD REDUCES ROS PRODUCTION control, and then it was changed to EC, UW, or LPD at a similar rate (2 ml/min). For current-voltage plots, cells were voltage-clamped at a potential of 70 mv and currents were evoked by 20 mv increments to 50 mv with test pulses of 200 ms duration at a rate of 0.1 Hz. Currents were filtered at 1 khz and sampled at 2 khz. For membrane potential recordings, cells were held at resting membrane potential in clamp-current mode. The microelectrode was connected to a CV203 headstage and an Axopatch 200B amplifier (Axon Instruments, Inc., Burlingame, CA). Data were recorded and analyzed with pclamp 6.02 software (Axon Instruments, Inc., Burlingame, CA). Pulmonary Artery Harvesting Procedure The pulmonary arteries were obtained as described above, opened longitudinally to expose the endothelium, and placed in Krebs solution at 4 C for 5 minutes. The right and left arteries were studied separately, and each artery was placed into a vial containing 10 ml of EC, UW, LPD, Celsior, low-potassium EC, or Krebs (control) solution GENERAL THORACIC Chemiluminescence We examined the generation of ROS by pulmonary resistance arteries from Sprague-Dawley rats using chemiluminescence with lucigenin (bis-nmethylacridinium nitrate) enhancement. The right or left pulmonary artery was placed in a glass vial containing EC, UW, LPD, Celsior, low-potassium EC, or Krebs solution after a base line reading of the solution had been obtained. Reactive oxygen species measurement was obtained by lucigenin-enhanced chemiluminescence recorded by a liquid scintillation counter at 4 C (Packard 1900CA, Meriden, CT). Results are expressed as a change in chemiluminescence in counts per 0.1 minute compared with base line. Statistical Analysis To evaluate differences among groups regarding the production of ROS measured by lucigenin-enhanced chemiluminescence, two-way analysis of variance was performed. A p value of less than 0.05 was considered statistically significant. SPSS Release 9.0 (SPSS Inc, Chicago, IL) and Sigmastat Version 1.0 (Jandel Scientific, San Rafael, CA) were used for statistical analysis. Results Patch Clamping Resting membrane potential of the pulmonary artery smooth muscle cells was measured when the cell was first exposed to Hanks solution and then measured over time as the preservation or control solution was added. At base line, the membrane potential was mv for the Krebs solution. Each preservation solution was added to the perfusion chamber with the results illustrated in Figure 1. EC solution had the greatest alteration in membrane potential with a depolarization to 22 mv. This was sustained for more than 6 minutes and Fig 1. Depolarization effect of University of Wisconsin (UW) and Euro-Collins (EC) on isolated pulmonary arterial smooth muscle cells. Resting membrane potential changes were elicited by a control perfusate solution (Krebs) followed by application of preservation solution low potassium dextran (LPD), EC, or UW, and then washout with Krebs solution. (A) Euro-Collins and UW cause initial membrane potential hyperpolarization followed by depolarization that never returned to baseline. (B) Summary of data showing resting membrane potential changes in control (Krebs) solution followed by application of preservative solutions. Values are means standard error n 3 cells per solution. **p less than did not return to base line membrane potential. A similar response was noted with UW solution but with depolarization to 24 mv. This too was sustained for more than 6 minutes. The extracellular solutions (LPD and Krebs) did not alter the membrane potential of the cell. Potassium channel opening was then evaluated as the preservation solutions were introduced. Figure 2 illustrates depolarization of the cell as it is exposed to EC or UW solution. Figure 2A illustrates the potassium channel response to LPD, which remains unchanged from the baseline. Note that the control in Figure 2B is similar to that of Figures 2A and 2C, but that there is substantial inward current upon stepping to 50 mv in the presence of UW preservation solution. This indicates that the reversal potential for potassium has shifted to the positive end caused by the high levels of extracellular potassium. The response to the EC solution is similar in that an

4 GENERAL THORACIC 1708 KELLY ET AL Ann Thorac Surg LPD REDUCES ROS PRODUCTION 2003;75: Fig 2. Changes in current-voltage relationship of pulmonary artery smooth muscle cell K channels. Currents elicited by 200 ms voltage steps from 70 to 50 mv in 20 mv increments before and after application of (A) low potassium dextran (LPD); (B) University of Wisconsin (UW); and (C) Euro-Collins (EC) perfusate solutions. The average current during the last 50 ms of each pulse versus the voltage step (I-V relationship) for the various preservative solutions is shown on the right. initial hyperpolarization occurs followed by a depolarization. As the initial reading for EC was below base line, which may have indicated cell injury, washout with Krebs solution was performed. This demonstrated viability of the cell and the ability to recover normal function. Chemiluminescence and ROS Production The pulmonary arteries immersed in UW solution produced the highest increase in chemiluminescence counts (51,289 5,615 counts/0.1 minute). The EC solution produced a similar response with change of chemiluminescence (35,702 4,353 counts/0.1 minute). The chemiluminescence of the pulmonary arteries stored in low potassium solutions was significantly lower (LPD, 12,537 3,623 counts/0.1 minute; Celsior, 13,717 3,844 counts/ 0.1 minute; and low potassium EC, 15,187 3,792 counts/ 0.1 minute). The chemiluminescence of the Krebs solution was 5, counts/0.1 minute. Figure 3 illustrates the difference in ROS production between the preservation and control solutions. Comment One of the most important limitations in lung transplantation is the poor tolerance of lung grafts to ischemia. The unfortunate clinical manifestation of ischemiareperfusion injury in the lung is primary graft failure. Over the past 15 years there has been extensive labora-

5 Ann Thorac Surg KELLY ET AL 2003;75: LPD REDUCES ROS PRODUCTION Fig 3. High-potassium solutions compared with low potassium solutions increase reactive oxygen species production by pulmonary arteries as measured by lucigenin-enhanced chemiluminescence. Results are expressed as averages standard error of the mean. *p less than 0.05; n 6 in each group. Krebs solution is the control. (CL chemiluminescence; EC Euro-Collins; K potassium; LPD low potassium dextran; UW University of Wisconsin.) tory research done on lung preservation solutions and techniques in an effort to eliminate or at least minimize ischemia-reperfusion injury during lung transplantation. A large amount of this research has suggested a central role of ROS in lung primary graft failure, similar to the mechanism of ischemia-reperfusion injury in other organ systems. It is possible that the use of intracellular solutions such as EC and UW may contribute to cellular injury during lung procurement. Studies have demonstrated increased pulmonary vascular resistance in lungs preserved with EC [10]. Vasodilators such as calcium-channel blockers and prostacyclin have been used in conjunction with EC to improve graft tolerance to ischemia reperfusion by reducing this pulmonary vasoconstriction in experimental models [11 13]. However, these additives may be unnecessary if a low potassium solution is used instead. The pulmonary vasculature is known to respond to high serum potassium concentration with membrane depolarization and subsequent vasoconstriction [14]. What is unclear is whether vasoconstriction alone, with poor distribution of perfusate and cooling, is detrimental to lung preservation or if injury is occurring through another mechanism. Previous studies have demonstrated the benefits of LPD in decreasing the incidence of primary graft failure after transplantation in the clinical setting [6, 7]. The physiologic factors document less tissue edema, reduced pulmonary vascular resistance, and better oxygenation compared with historical controls with lungs preserved using EC or UW solutions. Experimentally, in addition to the physiologic factors, the indirect evidence of ROS production in the transplanted lung has been measured with less lipid peroxidation occurring in lungs preserved with LPD. However, the mechanism by which a low potassium solution reduces lung ischemia-reperfusion injury has not been elucidated. Our data suggest that the perfusion solutions directly 1709 impact the lung vasculature. The potassium concentration of the solution is able to alter the membrane potential of the pulmonary artery smooth muscle cells. In the pulmonary artery smooth muscle cells, membrane potential is primarily regulated by potassium current, whereas the other ion components of the solutions do not appear to be contributing to the alteration in membrane potential. Exposing the pulmonary artery smooth muscle cells to preservation solutions with a high potassium concentration (EC, UW) results in a sustained membrane depolarization that reflects a decrease in outward alteration in potassium current. The current is affected by both the steep concentration gradient and the inhibition of voltage-gated potassium channels. The membrane depolarization should return to normal after washout as suggested in Figure 2C, however the sustained depolarization demonstrated in Figure 1 suggests significant cellular injury may be occurring. Rapid, sustained depolarization of the membrane results in vasoconstriction in the intact vessel. In contrast with the high potassium solutions, the LPD solution did not cause membrane depolarization, as the cells flushed with LPD solution remained at or near resting membrane potential throughout the study. The lack of membrane depolarization means that the stimulus for vasoconstriction has not occurred and explains the relatively normal pulmonary vascular resistance noted in physiologic studies. This indicates stability of the resting potential of the pulmonary artery smooth muscle cells in the presence of a low potassium concentrated preservation solution. This solution may provide a more appropriate ionic milieu that minimizes cell injury or activation as indicated by vasoconstriction or ROS production. In order to evaluate the pulmonary vascular response to the preservation solutions, lucigenin-enhanced chemiluminescence was used to measure ROS production in isolated rat pulmonary arteries placed in the solutions. Of note, in the presence of a physiologic solution such as Krebs, there is a basal production of ROS by the pulmonary artery. There is a slight increase in ROS production when the pulmonary arteries are exposed to LPD. Similarly, other low-potassium solutions, low-potassium EC and Celsior, were also studied and found to have low ROS production. However, in the presence of high potassium concentration solutions (EC and UW), there is a statistically significant increase in ROS production. Although there are slight differences between EC compared with UW, the similarities in membrane depolarization and ROS production appear to be modulated by the potassium concentration in the solution. The mechanism by which the ROS production is activated in lung transplantation remains unknown. During lung transplantation, ROS produced during ischemia reperfusion, or ischemia alone, are potential causes of primary graft failure. Pathways of free radical production during lung transplantation include xanthine/xanthine oxidase activation, mitochondrial respiration, and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. Xanthine oxidase activation is considered a major pathway of ROS production in most organ GENERAL THORACIC

6 GENERAL THORACIC 1710 KELLY ET AL Ann Thorac Surg LPD REDUCES ROS PRODUCTION 2003;75: systems. However, the lung is less likely to sustain anoxic injury during procurement and storage, because it has the capacity to remain oxygenated. NADPH oxidase activation is a potentially important pathway, because it is an enzyme that may be activated by membrane depolarization that could link the membrane depolarization to the increase in ROS production [15], and it is known to be present in pulmonary vasculature as well as other organ systems, although its exact role has yet to be elucidated [16]. In conclusion, high potassium preservation solutions used in lung transplantation increase the production of ROS from the pulmonary arteries. It is possible that high potassium concentrations that inhibit potassium current and depolarize the pulmonary artery smooth muscle cell signal the production of ROS. NADPH oxidase activation through membrane depolarization is a potential pathway of ROS production. The lower ROS production from pulmonary arteries exposed to LPD, Celsior, and lowpotassium EC solutions parallel the improved physiologic factors noted in experimental and clinical lung transplantation [6, 7]. Therefore, although the signaling or pathway of ROS production is not yet determined, the use of a low potassium concentration in lung preservation solution seems to decrease the incidence of primary graft failure through reduction in ROS production from the pulmonary vasculature. This material is based on work supported in part by the Minnesota Medical Foundation (Minneapolis, MN) and the result of work supported with resources and the use of facilities at the Veterans Affairs Medical Center (Minneapolis, MN). E. Kenneth Weir is supported by VA Merit Review and National Institutes of Health ROI HL References 1. Patterson GA. Indications. Unilateral, bilateral, heart-lung, and lobar transplant procedures. Clin Chest Med 1997;18: Hosenpud JD, Bennett LE, Keck BM, Fiol B, Boucek MM, Novick RJ. The registry of the international society for heart and lung transplantation: sixteenth official report J Heart Lung Transplant 1999;18: Kelly RF. Current strategies in lung preservation. J Lab Clin Med 2000;136: Kirk AJ, Colquhoun IW, Dark JH. Lung preservation: a review of current practice and future directions. Ann Thorac Surg 1993;56: Hopkinson DN, Bhabra MS, Hooper TL. Pulmonary graft preservation: a worldwide survey of current clinical practice. J Heart Lung Transplant 1998;17: Fischer S, Matte-Martyn A, De Perrot M, et al. Lowpotassium dextran preservation solution improves lung function after human lung transplantation. J Thorac Cardiovasc Surg 2001;121: Struber M, Wilhelmi M, Harringer W, et al. Flush perfusion with low potassium dextran solution improves early graft function in clinical lung transplantation. Eur J Cardiothorac Surg 2001;19: Weir EK, Archer SL. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB J 1995;9(2): Hamel OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high resolution current recoding from cells and cell-free membrane patches. Pflugers Arch 1981;391: Kawahara K, Ikari H, Hisano H, et al. Twenty-four hour canine lung preservation using UW solution. Transplantation 1991;51(3): Sasaki S, Yasuda K, McCully JD, LoCicero J. Does PGE1 attenuate potassium-induced vasoconstriction in initial pulmonary artery flush on lung preservation? J Heart Lung Transplant 1999;18: Sasaki S, Yasuda K, McCully JD, LoCicero J 3rd. Calcium channel blocker enhances lung preservation. J Heart Lung Transplant 1999;18: Hachida M, Morton DL. The protection of ischemic lung with verapamil and hydralazine. J Thorac Cardiovasc Surg 1988;95: Unruh H, Hoppensack M, Oppenheimer L. Vascular properties of canine lungs perfused with Euro-Collins solution and prostacyclin. Ann Thorac Surg 1990;49: Sohn H, Keller M, Gloe T, Morawietz H, Rueckschloss, Pohl U. The small G-protein RAC mediates depolarizationinduced superoxide formation in human endothelial cells. J Biol Chem 2000;25: Hohler B, Holzapfel B, Kummer W. NADPH oxidase subunits and superoxide production in porcine pulmonary artery endothelial cells. Histochem Cell Biol 2000;114(1):29 37.