- Email: [email protected]
Hemoglobin-Based Oxygen Carrier Induces Heme Oxygenase-1 in the Heart and Lung but Not Brain
Hemoglobin-Based Oxygen Carrier Induces Heme Oxygenase-1 in the Heart and Lung but Not Brain Sagar S Damle, MD, Ernest E Moore, MD, FACS, Ashok N Babu, MD, Xianzhong Meng, PhD, David A Fullerton, MD, Anirban Banerjee, PhD The clinical sequel of ischemia and reperfusion remains a challenge in several clinical areas. Overexpression of heme oxygenase-1 (HO-1), using viral vectors, endotoxemia, and hypoxia, provides protection against ischemia and reperfusion injury. To date, however, no clinically viable therapy exists to safely induce HO-1. We have recently observed that administration of a hemoglobin-based oxygen carrier (HBOC) attenuates postinjury systemic inflammation. We have further demonstrated that an HBOC can induce HO-1 in vitro. We now explore the tissue-specific induction of heme oxygenase-1 after administration of an HBOC. STUDY DESIGN: Rats were infused with doses of HBOC or saline through femoral vein injection (n ⫽ 5 per group). Animals were sacrificed and organs were flushed. Heart, lung, and brain samples were taken for evaluation of total organ levels of HO-1 induction and for histologic localization of the cellular expression of the HO-1. Heat shock protein 72 levels were also analyzed to determine whether HO-1 induction was a generalized stress response. RESULTS: Both the heart and lung demonstrated a dose-dependent induction of total organ HO-1. Interestingly, brain tissue did not have any significant amount of HO-1, either at baseline or after HBOC therapy. The cellular localization of HO-1 between organs was also specific, predominantly occurring in the cardiac myocyte and alveolar macrophages. Heat shock protein 72 levels were not significantly changed in any group examined, suggesting the induction of HO-1 is specific. CONCLUSIONS: This study demonstrates that a clinically accessible product, HBOC, can specifically and selectively induce the expression of the protective enzyme HO-1 in vivo. These findings begin to characterize which organ systems may benefit by preischemic treatments with HBOC and further expand potential clinical applications of HBOCs. (J Am Coll Surg 2009;208:592–598. © 2009 by the American College of Surgeons) BACKGROUND:
mary graft dysfunction and failure.2 Developing strategies to attenuate the adverse effects of reperfusion injury may have direct clinical benefit. Heme oxygenase-1 (HO-1) induction has emerged as potential therapy to minimize the effects of ischemia and reperfusion.3 HO-1, also known as heat shock protein (HSP)32, is a member of the heme oxygenase family of proteins, which function primarily to metabolize heme to iron, biliverdin, and carbon monoxide. Experimentally, HO-1 induction has been shown to be protective in a variety of models of I/R injury in the heart4-8 and lung.9,10 But these promising investigations have used clinically inaccessible or unsafe methods, such as heat shock, arsenic, endotoxin, and hemin, to induce HO-1. The current generation of hemoglobin-based oxygen carriers (HBOCs) are solutions of tetrameric hemoglobin chemically polymerized to prevent endothelial extravasation and vasoconstriction, dissolved in an isotonic solution.11 Clinical investigation has documented the safety of
Ischemia and reperfusion (I/R) injury remains a major contributor to morbidity and mortality in diverse clinical scenarios. After cardiac bypass, ischemic time correlates with postoperative morbidity and mortality.1 In the setting of lung transplantation, prolonged I/R time can lead to priDisclosure Information: Nothing to disclose. The project described was supported by award numbers T32GM008315 and P50GM049222 from the National Institutes of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of General Medical Sciences or the National Institutes of Health. Abstract presented at the American College of Surgeons 93rd Annual Clinical Congress, Surgical Forum, New Orleans, LA, October 2007. Received November 26, 2008; Revised January 7, 2009; Accepted January 9, 2009. From the Departments of Surgery, University of Colorado at Denver and Health Sciences Center (Damle, Moore, Babu, Meng, Fullerton, Banerjee) and Denver Health Medical Center (Moore), Denver, CO. Correspondence address: Ernest E Moore, MD, Denver Health Medical Center, 777 Bannock St, Denver, CO 80204.
© 2009 by the American College of Surgeons Published by Elsevier Inc.
592
ISSN 1072-7515/09/$36.00 doi:10.1016/j.jamcollsurg.2009.01.015
Vol. 208, No. 4, April 2009
Abbreviations and Acronyms
HBOC HO-1 HSP I/R
⫽ ⫽ ⫽ ⫽
hemoglobin-based oxygen carrier heme oxygenase-1 heat shock protein ischemia and reperfusion
Damle et al
Blood Substitute Induces Heme Oxygenase-1
593
blood volume, normal saline (10% calculated blood volume), over 30 minutes, or received no fluid (sham). All animals were sacrificed 12 hours after infusion for tissue collection. Tissue protein isolation
these solutions in clinical trials, and in a Food and Drug Administration phase II trial, we observed a marked attenuation of circulating interleukin 6 (IL-6) and interleukin 8 (IL-8) in severely injured patients resuscitated with an HBOC.12 We have subsequently shown that HBOC induces the expression of HO-1 in isolated cells in vitro.13 We have also recently demonstrated that HBOC attenuates liver dysfunction after I/R injury (under review). This prompted us to explore the potential use of HBOC in other organs. In this study, we determined the organ- and cellspecific induction of HO-1 after administration of an HBOC.
METHODS Materials
HBOC (PolyHeme, pyridoxyilated, polymerized human hemoglobin) was donated as a generous gift by Northfield Laboratories, Inc. Saline was obtained from Sigma-Aldrich Corp. Antibodies against HO-1 and heat shock protein 72 (HSP72) were obtained from Assay Designs, Inc. All fluorescent and isotype control antibodies were obtained from Jackson ImmunoResearch Laboratories unless otherwise stated.
Animals were sacrificed by aortic exsanguination. The vascular space was then flushed with cold saline until the aortic effluent became clear. Organs (specifically, brain, heart, and lung) were immediately excised and samples were flash frozen for protein analysis. Frozen organs were homogenized in commercial homogenization buffer (1:10 w/v; Pierce Biotechnology, Inc). Samples were spun twice at 14,000 g for 10 minutes at 4°C. Supernatants were removed, aliquoted, and snap frozen. Protein concentrations were determined before freezing using the Bradford Colorimetric Assay kit (Pierce Chemical). Immunoblot
Equal protein amounts of tissue homogenates were loaded into 8% to 16% acrylamide gels (Pierce), fractioned with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc). Membranes were blocked with 5% nonfat milk solution overnight and immunoprobed for HO-1 and HSP72. Bound antibodies were detected with an enhanced chemoluminescence detection system (Pierce). Densitometry was performed using Kodak 1D software. All plots were normalized to protein load and results were expressed as a percent of control.
Animals
Animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee at Denver Health Medical Center, Denver, CO. Adult male Sprague-Dawley rats (Harlan Laboratories) weighing 320 to 350 g were housed under barrier-sustained conditions and allowed free access to chow and water before use. All animals were maintained in accordance with the recommendations of the NIH Guide for the Care and Use of Laboratory Animals. Experimental design
HBOC or saline was administered to animals through their femoral vein (5 animals per group). For time course studies, animals were sacrificed immediately, 12 hours, and 24 hours after infusion to determine HO-1 and HSP72 induction. HSP72 was measured as a marker of nonspecific stress induction. For dose-response studies, animals were first infused with either HBOC at 5% or 10% of calculated
Immunohistochemistry
Representative samples of organs were taken and frozen in embedding medium for immunohistochemistry. Tissue blocks were cut and sections were allowed to air dry at room temperature for 24 hours. Slides were fixed and permeabilized with 70:30 acetone:methanol solution at ⫺20°C. Nonspecific antigen sites were blocked with 10% donkey serum (Jackson Laboratories) in 1% BSA/PBS for 1 hour at room temperature. Slides were then probed with rabbit polyclonal anti-HO-1 antibody (AssayDesign) and mouse polyclonal antirat CD68 antibody (Santa Cruz), a specific marker of macrophages, in 1% BSA/PBS for 1 hour. Negative control slides were incubated with isotype controls. After three PBS washes, the slides were incubated for 1 hour at room temperature with donkey antirabbit Cy3-conjugated antibody, donkey antimouse Alexa488conjugated antibody (Molecular Probes), and bisbenzimide nuclear stain (Sigma-Aldrich).
594
Damle et al
Blood Substitute Induces Heme Oxygenase-1
J Am Coll Surg
Images were acquired with a Zeiss Axiovert fitted with a Cooke CCD SensiCam using Chroma Sedat cubes with single excitation and emission filters and a multiple bandpass dichroic and narrow motorized Sutter filter control. The 3 channel images were analyzed with Intelligent Imaging Innovations Slidebook 4.1 software (Intelligent Imaging Innovations, Inc). Statistical analysis
Data were analyzed by ANOVA for statistical significance followed by Tukey’s multiple comparison adjustment test. All ANOVA models were adjusted for the effect of daily variability and different gels. Statistical significance was accepted as p ⬍ 0.05. For the purpose of cleaner graphic presentation of the data, after confirmation of statistical significance, experimental data were normalized by dividing individual values by the average of the control values for that specific experiment or gel. The values represented in the graphs are the average of these fold changes over the average control values.
RESULTS HBOC induces a dose-dependent increase in HO-1 expression in the heart and lung but not in the brain
In the heart, there were 1.5- and 3-fold increases in HO-1 levels over baseline after 5% and 10% v/v administration of HBOC, respectively (Fig. 1). By contrast, saline administered at 10% v/v did not significantly increase levels of HO-1 over baseline. In addition, HSP72 levels did not change in any group over baseline levels, suggesting the HO-1 induction is not from a generalized stress response. Similar results are noted in the lung, with 2- and 3.7-fold increases in tissue HO-1 levels over baseline after 5% and 10% v/v administration, respectively (Fig. 2). Saline administration at 10% v/v did not result in a significant increase in HO-1. HSP72 levels were also unchanged in all groups, compared with baseline. In contrast, in the brain there was minimal HO-1 at baseline and no significant induction after 5% or 10% v/v administration of HBOC (data not shown). HSP72 levels also remained unchanged. The time-dependent induction of HO-1 by HBOC was organ specific
Heart HO-1 induction was measured at 3 time intervals during the initial 24 hours after induction (Fig. 3). There was minimal HO-1 expression at the time of infusion. Thereafter, HO-1 increased 2.5- and 3.5-fold over these baseline levels at 12 and 24 hours, respectively, after HBOC administration. In contrast, HO-1 expression in
Figure 1. Hemoglobin-based oxygen carrier (HBOC) induces a dosedependent increase in heme oxygenase (HO)-1 in the heart. Animals were injected with normal saline (10% v/v) or HBOC (5% and 10% v/v) and then sacrificed 12 hours after injection. After flushing the organs with cold saline, organs were homogenized and then analyzed by Western blot for tissue levels of HO-1. HBOC induced a dose-dependent increase in tissue levels of HO-1: 1.5- and 3-fold increases in HO-1 expression over that in sham animals after infusion of 5% and 10% HBOC, respectively. HSP72 (heat shock protein 72, generalized marker for stress) levels did not change in any group.
the lung was maximal 12 hours after HBOC infusion, with a 3-fold increase, and was approaching baseline at 24 hours (Fig. 4). Brain tissue did not have evidence of HO-1 induction at the 3 time points examined (data not shown). HSP72 levels did not change significantly in any organ at any of the times examined. Cellular localization of HO-1 was tissue specific and predominated in the cardiac myocyte and alveolar macrophages, respectively
In the heart, immunohistochemistry was used to localize tissue expression of HO-1. HO-1 expression occurred in most cells of the heart, suggesting that the predominant cell type to express the HO-1 is the cardiac myocyte (Fig. 5). In the lung, cellular localization of HO-1 appeared to be concentrated in a subset of cells. Counterstaining sections with HO-1 and CD68, a macrophage-specific cellular stain, indicated that the predominant cell expressing HO-1 in the lung was the alveolar macrophage (Fig. 6).
DISCUSSION Tissue I/R injury remains a major contributor to organ dysfunction in a variety of clinical settings. HO-1 activity has been shown to provide protection against I/R injury in an array of tissues, but clinically safe methods to induce HO-1 have been lacking.3 Our ongoing work with an
Vol. 208, No. 4, April 2009
Figure 2. Hemoglobin-based oxygen carrier (HBOC) induces a dosedependent increase in heme oxygenase (HO)-1 in the lung. Animals were injected with normal saline (10% v/v) or HBOC (5% and 10% v/v) and then sacrificed 12 hours after injection. After flushing the organs with cold saline, organs were homogenized and analyzed by Western blot for tissue levels of HO-1. Saline alone did not increase tissue levels of HO-1 over levels in sham animals. There were 2- and 4-fold increases in HO-1 expression over that in sham animals after infusion of 5% and 10% HBOC, respectively. Heat shock protein (HSP) 72 levels did not change in any group, suggesting the HO-1 induction was specific and not from a generalized stress response.
HBOC prompted us to examine the use of this solution to selectively induce HO-1 expression in tissue beds. The major findings of this investigation are: circulating HBOC can profoundly induce HO-1 levels in the heart and lung but has no induction of HO-1 in the brain; and the predominant cellular expression of HO-1 depends on the organ examined: the cardiac myocyte in the heart or the pulmonary alveolar macrophage in the lung. Heme oxygenase was originally described in the 1960s to be the enzyme responsible for metabolizing heme to biliverdin, carbon monoxide, and free iron.14 The first two of these products are believed to be the active cytoprotective agents, but their mechanisms remain to be elucidated. Once the HO-1 isoform was discovered and found to be highly inducible,15 subsequent studies showed HO-1 to have potent antiinflammatory properties. The concept that HO-1 induction can be immunomodulatory and protective has been established in a number of experimental models,4,6,7,10,16-18 including models of I/R.8,19-22 Although there are extensive data documenting the protective effects of HO-1 and its byproducts, there have been no safe clinical therapies to increase this protein’s expression in tissue. Methods such as hemin, adenoviral transfection, ischemic preconditioning, and protoporphyrins are useful tools in animal models but are not clinically applicable. Given our previous experience with HBOC, we hypothesized that HBOC would induce in vivo expression of
Damle et al
Blood Substitute Induces Heme Oxygenase-1
595
Figure 3. Hemoglobin-based oxygen carrier (HBOC) infusion stimulates progressive induction of heme oxygenase (HO)-1 in the myocardium over 24 hours. Animals infused with 10% HBOC were sacrificed at varying time intervals and homogenized samples of myocardium were analyzed for tissue expression of HO-1 by Western blot. There were 2.5- and 3.5-fold increases in tissue levels of HO-1 over baseline at 12 and 24 hours, respectively. Heat shock protein (HSP) 72 levels did not change significantly over baseline levels.
HO-1. We began our investigations in isolated cell lines. We found that HO-1 can be induced by HBOC in isolated pulmonary endothelial cells.13 In addition, we noted that there was a significant reduction in the expression of intercellular adhesion molecule (ICAM)-1 after stimulation with lipopolysaccharide.13 More recently, we showed that this HBOC induces HO-1 in isolated peritoneal macrophages, which results in attenuated cytokine production in response to lipopolysaccharide.23 This study extends our previous work by demonstrated that this HBOC can be exploited to induce HO-1 in vivo. Hemoglobin-based oxygen carriers are currently under investigation for trauma resuscitation in Food and Drug Administration-approved phase III clinical trials.24 The current generation of HBOC, consisting of polymerized tetrameric hemoglobin, eliminates the vasoconstrictive problems encountered with earlier-generation HBOCs. This is accomplished by polymerizing several hemoglobin tetramers with glutaraldehyde and thoroughly removing all native a2b2 tetramers such that circulating hemoglobin does not enter the subendothelial spaces.11,24 The differences in hemoglobin configuration between the earliergeneration HBOCs and the HBOC used in our work may
596
Damle et al
Blood Substitute Induces Heme Oxygenase-1
Figure 4. Hemoglobin-based oxygen carrier (HBOC) infusion demonstrated a maximal induction of heme oxygenase (HO)-1 12 hours after infusion in the lung. Animals infused with 10% HBOC were sacrificed at varying time intervals, and homogenized samples of myocardium were analyzed for tissue expression of HO-1 by Western blot. Maximal expression of HO-1 occurred at 12 hours after infusion, with a 3-fold increase in levels of HO-1 over baseline levels. By 24 hours, levels of HO-1 were returning to baseline levels but remained elevated compared with baseline. Heat shock protein (HSP) 72 levels did not change in any group.
explain conflicting data from previous similar studies. Kubulus and colleagues25 reported that although the diaspirin-cross-linked hemoglobin (modified tetramer) induced HO-1 modestly in rodent models of hemorrhagic shock, it also increased the release of the hepatic enzymes alanine transaminase and aspartate transaminase. These authors also found that although hemin bolused at high doses was the best inducer of HO-1 in this model, it was associated with high mortality. On the other end, the smallest dose of hemin preparation that was not hepatotoxic induced HO-1 minimally compared with the smallest doses of tetrameric hemoglobin. In sum, these experiments illustrated that bolused hemin and free hemoglobin could induce HO-1, but they were prohibitively toxic. In this study, we used a clinically accessible tool to selectively induce HO-1 expression in the lung and heart. Interestingly, brain tissue did not have evidence of HO-1 induction. Although we have not investigated the mechanism of HO-1 induction, it is probable that cellular regulation of HO-1 by HBOC requires direct interaction of the target cells with the hemoglobin molecules. So, an intact blood-brain barrier may preclude induction of HO-1 in the brain. But it is also possible that HBOC could access neurons after I/R.
J Am Coll Surg
Figure 5. Hemoglobin-based oxygen carrier (HBOC)-induced heme oxygenase (HO)-1 is predominantly in the cardiac myocyte. Heart samples of animals treated with HBOC (10% v/v) were taken after 12 hours of treatment and frozen in embedding medium. Sections were stained and examined with immunohistochemistry. Sections were stained with HO-1 (red), biz-benzimide (nuclear stain, blue), and wheat germ agglutinins (cell outline, green). The predominant expression of HO-1 occurs in the myocyte, as demonstrated by diffuse staining throughout each of the cells, which are cardiac myocytes.
The induction of HO-1 by HBOC could also be a consequence of some cellular injury from the HBOC itself. Three points argue against this. First, one would expect an induction of HSP72, a generalized, sensitive marker of stress response, if this was the case. We do not see any induction of HSP72, suggesting that the cells and tissues are under no increased stress. Next, microscopic analysis would also suggest tissue injury, which was not demonstrated during analysis. Finally, several earlier studies documented the safety of HBOCs in vivo and in vitro, examining both clinical outcomes and biochemical markers of injury.12,26,27 The major limitation of this study is the lack of I/R data. Although there are extensive data supporting the role of HO-1 induction in protection against I/R injury, these studies have used other techniques to induce HO-1. It is possible that the HO-1 may not be the mechanism for protection. But we recently showed, in a liver model of I/R, that HBOC pretreatment can attenuate liver injury. We further demonstrated that this protection is dependent on HO-1, and selective inhibition of HO-1 obliterates the protection offered by HBOC pretreatment. So this induction of HO-1 confers protection from I/R injury in the liver (Damle S and associates, presented at the American College of Surgeons Committee on Trauma meeting, Denver, CO, March 2007, under review).
Vol. 208, No. 4, April 2009
Damle et al
Blood Substitute Induces Heme Oxygenase-1
597
REFERENCES
Figure 6. Hemoglobin-based oxygen carrier (HBOC)-induced heme oxygenase (HO)-1 is predominantly in the alveolar macrophages. Lung samples of animals treated with HBOC (10% v/v) were taken after 12 hours of treatment, infused with embedding medium back to normal lung volumes, and frozen in embedding medium. Sections were initially stained with HO-1 (red), biz-benzimide (nuclear stain, blue), and wheat germ agglutinins (cell outline, green); they demonstrated particular cells concentrating HO-1 expression (images not shown). Subsequent sections were stained with HO-1 (red) and CD68 (macrophage specific marker, green). Shown here is a representative section of one alveolus. There is some expression of HO-1 in a variety of cell types, but the predominant cellular expression of HO-1 localizes to cells that also stain with CD68, suggesting that the predominant expression of HO-1 in the lung occurs in the alveolar macrophages (arrows).
In conclusion, this work confirms that an HBOC can induce the expression of HO-1 in the heart and lung. Ultimately, this may be used as a preconditioning tool to minimize organ dysfunction from I/R injury. In the heart, HBOC could potentially be given 24 hours before cardiopulmonary bypass. In either organ, HBOC may potentially be used to induce expression of HO-1 before transplantation. But further work delineating the precise time for maximal induction and demonstrating abrogation of reperfusion injury will need to be performed before clinical application. These findings lay the groundwork for future studies exploring the potential use of HBOC to ameliorate the deleterious effects of I/R on organ function in a variety of clinical scenarios.
Author Contributions Study conception and design: Damle, Moore, Babu, Fullerton Acquisition of data: Damle, Babu Analysis and interpretation of data: Moore, Meng, Fullerton, Banerjee Drafting of manuscript: Damle, Moore, Babu, Meng, Fullerton, Banerjee Critical revision: Damle, Moore, Babu, Meng, Fullerton, Banerjee
1. Flameng W, Herjgers P, Szecsi J, et al. Determinants of early and late results of combined valve operations and coronary artery bypass grafting. Ann Thor Surg 1996;61:621–628. 2. King R, Binns OAR, Rodriguez F, et al. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation. Ann Thor Surg 2000;69:1681–1685. 3. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic application. Physiol Rev 2006;86:583–650. 4. Clark JE, Roberta F, Sarathchandra P, et al. Heme oxygenase1-derived bilirubin ameliorates postischemic myocardial dysfunction. Am J Physiol: Heart Circ Physiol 2000;278:H643– H651. 5. Das S, Fraga C, Das D. Cardioprotective effect of resveratrol via HO-1 expression involves p38 map kinase and PI-3-kinase signaling, but does not involve NFkB. Free Rad Res 2006;40: 1066–1075. 6. Hangaishi M, Ishizaka N, Aizawa T, et al. Induction of heme oxygenase-1 can act protectively against cardiac ischemia/ reperfusion in vivo. Biochem Biophys Res Comm 2000;279: 582–588. 7. Liu X, Pachori A, Ward C, et al. Heme oxygenase-1 inhibits postmyocardial infarct remodeling and restores ventricular function. FASEB J 2006;20:207–216. 8. Yet S-F, Tian R, Layne MD, et al. Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice. Circ Res 2001;89:168–173. 9. Fredenburgh L, Baron R, Carvajal I, et al. Absence of heme oxygenase-1 expression in the lung parenchyma exacerbates endotoxin-induced acute lung injury and decreases surfactant protein-B levels. Cell Mol Biol 2005;51:513–520. 10. Otterbein LE, Kolls JK, Mantell LL, et al. Exogenous administration of heme oxygease-1 by gene transfer provides protection against hyperoxia-induced lung injury. J Clin Invest 1999;103: 1047–1054. 11. Moore EE, Johnson JL, Cheng AM, et al. Insights from studies of blood substitutes in trauma. Shock 2005;24:197– 205. 12. Johnson JL, Moore EE, Gonzales RJ, et al. Alteration of the postinjury hyperinflammatory response by means of resuscitation with a red cell substitute. J Trauma 2003;54:133– 140. 13. Cheng AM, Moore EE, Johnson JL, et al. Polymerized hemoglobin induces heme oxygenase-1 protein expression and inhibits intercellular adhesion molecule-1 protein expression in human lung microvascular endothelial cells. J Am Coll Surg 2005; 201:579–584. 14. Tenhunen R, Marver HS, Schmid R. Microsomal heme oxygenase. Characterization of the enzyme. J Biol Chem 1969;244: 6388–6394. 15. Maines MD, Trakshel GM, Kutty RK. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J Biol Chem 1986;261:411–419. 16. Chabannes D, Hill M, Merieau E, et al. A role for heme oxygenase-1 in the immunosuppressive effect of adult rat and human mesenchymal stem cells. Blood 2007;110:3691– 3694.
598
Damle et al
Blood Substitute Induces Heme Oxygenase-1
17. Rushword S, MacEwan D. HO-1 underlies resistancec of AML cells to TNF-induced apoptosis. Blood 2008;111:379– 801. 18. Shokawa T, Yoshizumi M, Yamamoto H, et al. Induction of heme oxygenase-1 inhibits monocyte chemoattractant protein-1 mRNA expression in U937 cells. J Pharmacol Sci 2006;100:162–166. 19. Geuken E, Buis CI, Visser DS, et al. Expression of heme oxygenase-1 in human livers before transplantation correlates with graft injury and function after transplantation. Am J Transplant 2005;5:1875–1885. 20. Rensing H, Bauer I, Datene V, et al. Differential expression pattern of heme oxygenase-1/heat shock protein 32 and nitric oxide synthase-II and their impact on liver injury in a rat model of hemorrhage and resuscitation. Crit Care Med 1999;27:2766–2775. 21. Tang YL, Qian K, Zhang C, et al. A vigilant, hypoxia-regulated heme oxygenase-1 gene vector in the heart limits cardiac injury after ischemia-reperfusion in vivo. J Cardiovasc Pharmacol Ther 2005;10:251–263.
J Am Coll Surg
22. Yamashita K, Ollinger R, McDaid J, et al. Heme oxygenase-1 is essential for and promotes tolerance to transplanted organs. FASEB J 2006;20:776–778. 23. Roach J, Moore EE, Patrick D, et al. Heme-oxygenase-1 induction in macrophages by a hemoglobin-based oxygen carrier reduces endotoxin stimulated cytokine secretion. Shock; in press. 24. Moore EE. Blood substitutes: the future is now. J Am Coll Surg 2003;196:1–17. 25. Kubulus D, Rensing H, Paxian M, et al. Influence of hemebased solutions on stress protein expression and organ failure after hemorrhagic shock. Crit Care Med 2005;33:629–637. 26. Cothren CC, Moore EE, Long JS, et al. Large volume polymerized haemoglobin solution in a Jehovah’s Witness following abruptio placentae. Transfusion Med 2004;14:241–246. 27. Gould SA, Moore EE, Hoyt DB, et al. The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergent surgery. J Am Coll Surg 1998;187: 113–120.
MOVING? MOVED? Please go to www.efacs.org Provide your new address, telephone number, fax number, or email changes.
mary graft dysfunction and failure.2 Developing strategies to attenuate the adverse effects of reperfusion injury may have direct clinical benefit. Heme oxygenase-1 (HO-1) induction has emerged as potential therapy to minimize the effects of ischemia and reperfusion.3 HO-1, also known as heat shock protein (HSP)32, is a member of the heme oxygenase family of proteins, which function primarily to metabolize heme to iron, biliverdin, and carbon monoxide. Experimentally, HO-1 induction has been shown to be protective in a variety of models of I/R injury in the heart4-8 and lung.9,10 But these promising investigations have used clinically inaccessible or unsafe methods, such as heat shock, arsenic, endotoxin, and hemin, to induce HO-1. The current generation of hemoglobin-based oxygen carriers (HBOCs) are solutions of tetrameric hemoglobin chemically polymerized to prevent endothelial extravasation and vasoconstriction, dissolved in an isotonic solution.11 Clinical investigation has documented the safety of
Ischemia and reperfusion (I/R) injury remains a major contributor to morbidity and mortality in diverse clinical scenarios. After cardiac bypass, ischemic time correlates with postoperative morbidity and mortality.1 In the setting of lung transplantation, prolonged I/R time can lead to priDisclosure Information: Nothing to disclose. The project described was supported by award numbers T32GM008315 and P50GM049222 from the National Institutes of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of General Medical Sciences or the National Institutes of Health. Abstract presented at the American College of Surgeons 93rd Annual Clinical Congress, Surgical Forum, New Orleans, LA, October 2007. Received November 26, 2008; Revised January 7, 2009; Accepted January 9, 2009. From the Departments of Surgery, University of Colorado at Denver and Health Sciences Center (Damle, Moore, Babu, Meng, Fullerton, Banerjee) and Denver Health Medical Center (Moore), Denver, CO. Correspondence address: Ernest E Moore, MD, Denver Health Medical Center, 777 Bannock St, Denver, CO 80204.
© 2009 by the American College of Surgeons Published by Elsevier Inc.
592
ISSN 1072-7515/09/$36.00 doi:10.1016/j.jamcollsurg.2009.01.015
Vol. 208, No. 4, April 2009
Abbreviations and Acronyms
HBOC HO-1 HSP I/R
⫽ ⫽ ⫽ ⫽
hemoglobin-based oxygen carrier heme oxygenase-1 heat shock protein ischemia and reperfusion
Damle et al
Blood Substitute Induces Heme Oxygenase-1
593
blood volume, normal saline (10% calculated blood volume), over 30 minutes, or received no fluid (sham). All animals were sacrificed 12 hours after infusion for tissue collection. Tissue protein isolation
these solutions in clinical trials, and in a Food and Drug Administration phase II trial, we observed a marked attenuation of circulating interleukin 6 (IL-6) and interleukin 8 (IL-8) in severely injured patients resuscitated with an HBOC.12 We have subsequently shown that HBOC induces the expression of HO-1 in isolated cells in vitro.13 We have also recently demonstrated that HBOC attenuates liver dysfunction after I/R injury (under review). This prompted us to explore the potential use of HBOC in other organs. In this study, we determined the organ- and cellspecific induction of HO-1 after administration of an HBOC.
METHODS Materials
HBOC (PolyHeme, pyridoxyilated, polymerized human hemoglobin) was donated as a generous gift by Northfield Laboratories, Inc. Saline was obtained from Sigma-Aldrich Corp. Antibodies against HO-1 and heat shock protein 72 (HSP72) were obtained from Assay Designs, Inc. All fluorescent and isotype control antibodies were obtained from Jackson ImmunoResearch Laboratories unless otherwise stated.
Animals were sacrificed by aortic exsanguination. The vascular space was then flushed with cold saline until the aortic effluent became clear. Organs (specifically, brain, heart, and lung) were immediately excised and samples were flash frozen for protein analysis. Frozen organs were homogenized in commercial homogenization buffer (1:10 w/v; Pierce Biotechnology, Inc). Samples were spun twice at 14,000 g for 10 minutes at 4°C. Supernatants were removed, aliquoted, and snap frozen. Protein concentrations were determined before freezing using the Bradford Colorimetric Assay kit (Pierce Chemical). Immunoblot
Equal protein amounts of tissue homogenates were loaded into 8% to 16% acrylamide gels (Pierce), fractioned with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc). Membranes were blocked with 5% nonfat milk solution overnight and immunoprobed for HO-1 and HSP72. Bound antibodies were detected with an enhanced chemoluminescence detection system (Pierce). Densitometry was performed using Kodak 1D software. All plots were normalized to protein load and results were expressed as a percent of control.
Animals
Animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee at Denver Health Medical Center, Denver, CO. Adult male Sprague-Dawley rats (Harlan Laboratories) weighing 320 to 350 g were housed under barrier-sustained conditions and allowed free access to chow and water before use. All animals were maintained in accordance with the recommendations of the NIH Guide for the Care and Use of Laboratory Animals. Experimental design
HBOC or saline was administered to animals through their femoral vein (5 animals per group). For time course studies, animals were sacrificed immediately, 12 hours, and 24 hours after infusion to determine HO-1 and HSP72 induction. HSP72 was measured as a marker of nonspecific stress induction. For dose-response studies, animals were first infused with either HBOC at 5% or 10% of calculated
Immunohistochemistry
Representative samples of organs were taken and frozen in embedding medium for immunohistochemistry. Tissue blocks were cut and sections were allowed to air dry at room temperature for 24 hours. Slides were fixed and permeabilized with 70:30 acetone:methanol solution at ⫺20°C. Nonspecific antigen sites were blocked with 10% donkey serum (Jackson Laboratories) in 1% BSA/PBS for 1 hour at room temperature. Slides were then probed with rabbit polyclonal anti-HO-1 antibody (AssayDesign) and mouse polyclonal antirat CD68 antibody (Santa Cruz), a specific marker of macrophages, in 1% BSA/PBS for 1 hour. Negative control slides were incubated with isotype controls. After three PBS washes, the slides were incubated for 1 hour at room temperature with donkey antirabbit Cy3-conjugated antibody, donkey antimouse Alexa488conjugated antibody (Molecular Probes), and bisbenzimide nuclear stain (Sigma-Aldrich).
594
Damle et al
Blood Substitute Induces Heme Oxygenase-1
J Am Coll Surg
Images were acquired with a Zeiss Axiovert fitted with a Cooke CCD SensiCam using Chroma Sedat cubes with single excitation and emission filters and a multiple bandpass dichroic and narrow motorized Sutter filter control. The 3 channel images were analyzed with Intelligent Imaging Innovations Slidebook 4.1 software (Intelligent Imaging Innovations, Inc). Statistical analysis
Data were analyzed by ANOVA for statistical significance followed by Tukey’s multiple comparison adjustment test. All ANOVA models were adjusted for the effect of daily variability and different gels. Statistical significance was accepted as p ⬍ 0.05. For the purpose of cleaner graphic presentation of the data, after confirmation of statistical significance, experimental data were normalized by dividing individual values by the average of the control values for that specific experiment or gel. The values represented in the graphs are the average of these fold changes over the average control values.
RESULTS HBOC induces a dose-dependent increase in HO-1 expression in the heart and lung but not in the brain
In the heart, there were 1.5- and 3-fold increases in HO-1 levels over baseline after 5% and 10% v/v administration of HBOC, respectively (Fig. 1). By contrast, saline administered at 10% v/v did not significantly increase levels of HO-1 over baseline. In addition, HSP72 levels did not change in any group over baseline levels, suggesting the HO-1 induction is not from a generalized stress response. Similar results are noted in the lung, with 2- and 3.7-fold increases in tissue HO-1 levels over baseline after 5% and 10% v/v administration, respectively (Fig. 2). Saline administration at 10% v/v did not result in a significant increase in HO-1. HSP72 levels were also unchanged in all groups, compared with baseline. In contrast, in the brain there was minimal HO-1 at baseline and no significant induction after 5% or 10% v/v administration of HBOC (data not shown). HSP72 levels also remained unchanged. The time-dependent induction of HO-1 by HBOC was organ specific
Heart HO-1 induction was measured at 3 time intervals during the initial 24 hours after induction (Fig. 3). There was minimal HO-1 expression at the time of infusion. Thereafter, HO-1 increased 2.5- and 3.5-fold over these baseline levels at 12 and 24 hours, respectively, after HBOC administration. In contrast, HO-1 expression in
Figure 1. Hemoglobin-based oxygen carrier (HBOC) induces a dosedependent increase in heme oxygenase (HO)-1 in the heart. Animals were injected with normal saline (10% v/v) or HBOC (5% and 10% v/v) and then sacrificed 12 hours after injection. After flushing the organs with cold saline, organs were homogenized and then analyzed by Western blot for tissue levels of HO-1. HBOC induced a dose-dependent increase in tissue levels of HO-1: 1.5- and 3-fold increases in HO-1 expression over that in sham animals after infusion of 5% and 10% HBOC, respectively. HSP72 (heat shock protein 72, generalized marker for stress) levels did not change in any group.
the lung was maximal 12 hours after HBOC infusion, with a 3-fold increase, and was approaching baseline at 24 hours (Fig. 4). Brain tissue did not have evidence of HO-1 induction at the 3 time points examined (data not shown). HSP72 levels did not change significantly in any organ at any of the times examined. Cellular localization of HO-1 was tissue specific and predominated in the cardiac myocyte and alveolar macrophages, respectively
In the heart, immunohistochemistry was used to localize tissue expression of HO-1. HO-1 expression occurred in most cells of the heart, suggesting that the predominant cell type to express the HO-1 is the cardiac myocyte (Fig. 5). In the lung, cellular localization of HO-1 appeared to be concentrated in a subset of cells. Counterstaining sections with HO-1 and CD68, a macrophage-specific cellular stain, indicated that the predominant cell expressing HO-1 in the lung was the alveolar macrophage (Fig. 6).
DISCUSSION Tissue I/R injury remains a major contributor to organ dysfunction in a variety of clinical settings. HO-1 activity has been shown to provide protection against I/R injury in an array of tissues, but clinically safe methods to induce HO-1 have been lacking.3 Our ongoing work with an
Vol. 208, No. 4, April 2009
Figure 2. Hemoglobin-based oxygen carrier (HBOC) induces a dosedependent increase in heme oxygenase (HO)-1 in the lung. Animals were injected with normal saline (10% v/v) or HBOC (5% and 10% v/v) and then sacrificed 12 hours after injection. After flushing the organs with cold saline, organs were homogenized and analyzed by Western blot for tissue levels of HO-1. Saline alone did not increase tissue levels of HO-1 over levels in sham animals. There were 2- and 4-fold increases in HO-1 expression over that in sham animals after infusion of 5% and 10% HBOC, respectively. Heat shock protein (HSP) 72 levels did not change in any group, suggesting the HO-1 induction was specific and not from a generalized stress response.
HBOC prompted us to examine the use of this solution to selectively induce HO-1 expression in tissue beds. The major findings of this investigation are: circulating HBOC can profoundly induce HO-1 levels in the heart and lung but has no induction of HO-1 in the brain; and the predominant cellular expression of HO-1 depends on the organ examined: the cardiac myocyte in the heart or the pulmonary alveolar macrophage in the lung. Heme oxygenase was originally described in the 1960s to be the enzyme responsible for metabolizing heme to biliverdin, carbon monoxide, and free iron.14 The first two of these products are believed to be the active cytoprotective agents, but their mechanisms remain to be elucidated. Once the HO-1 isoform was discovered and found to be highly inducible,15 subsequent studies showed HO-1 to have potent antiinflammatory properties. The concept that HO-1 induction can be immunomodulatory and protective has been established in a number of experimental models,4,6,7,10,16-18 including models of I/R.8,19-22 Although there are extensive data documenting the protective effects of HO-1 and its byproducts, there have been no safe clinical therapies to increase this protein’s expression in tissue. Methods such as hemin, adenoviral transfection, ischemic preconditioning, and protoporphyrins are useful tools in animal models but are not clinically applicable. Given our previous experience with HBOC, we hypothesized that HBOC would induce in vivo expression of
Damle et al
Blood Substitute Induces Heme Oxygenase-1
595
Figure 3. Hemoglobin-based oxygen carrier (HBOC) infusion stimulates progressive induction of heme oxygenase (HO)-1 in the myocardium over 24 hours. Animals infused with 10% HBOC were sacrificed at varying time intervals and homogenized samples of myocardium were analyzed for tissue expression of HO-1 by Western blot. There were 2.5- and 3.5-fold increases in tissue levels of HO-1 over baseline at 12 and 24 hours, respectively. Heat shock protein (HSP) 72 levels did not change significantly over baseline levels.
HO-1. We began our investigations in isolated cell lines. We found that HO-1 can be induced by HBOC in isolated pulmonary endothelial cells.13 In addition, we noted that there was a significant reduction in the expression of intercellular adhesion molecule (ICAM)-1 after stimulation with lipopolysaccharide.13 More recently, we showed that this HBOC induces HO-1 in isolated peritoneal macrophages, which results in attenuated cytokine production in response to lipopolysaccharide.23 This study extends our previous work by demonstrated that this HBOC can be exploited to induce HO-1 in vivo. Hemoglobin-based oxygen carriers are currently under investigation for trauma resuscitation in Food and Drug Administration-approved phase III clinical trials.24 The current generation of HBOC, consisting of polymerized tetrameric hemoglobin, eliminates the vasoconstrictive problems encountered with earlier-generation HBOCs. This is accomplished by polymerizing several hemoglobin tetramers with glutaraldehyde and thoroughly removing all native a2b2 tetramers such that circulating hemoglobin does not enter the subendothelial spaces.11,24 The differences in hemoglobin configuration between the earliergeneration HBOCs and the HBOC used in our work may
596
Damle et al
Blood Substitute Induces Heme Oxygenase-1
Figure 4. Hemoglobin-based oxygen carrier (HBOC) infusion demonstrated a maximal induction of heme oxygenase (HO)-1 12 hours after infusion in the lung. Animals infused with 10% HBOC were sacrificed at varying time intervals, and homogenized samples of myocardium were analyzed for tissue expression of HO-1 by Western blot. Maximal expression of HO-1 occurred at 12 hours after infusion, with a 3-fold increase in levels of HO-1 over baseline levels. By 24 hours, levels of HO-1 were returning to baseline levels but remained elevated compared with baseline. Heat shock protein (HSP) 72 levels did not change in any group.
explain conflicting data from previous similar studies. Kubulus and colleagues25 reported that although the diaspirin-cross-linked hemoglobin (modified tetramer) induced HO-1 modestly in rodent models of hemorrhagic shock, it also increased the release of the hepatic enzymes alanine transaminase and aspartate transaminase. These authors also found that although hemin bolused at high doses was the best inducer of HO-1 in this model, it was associated with high mortality. On the other end, the smallest dose of hemin preparation that was not hepatotoxic induced HO-1 minimally compared with the smallest doses of tetrameric hemoglobin. In sum, these experiments illustrated that bolused hemin and free hemoglobin could induce HO-1, but they were prohibitively toxic. In this study, we used a clinically accessible tool to selectively induce HO-1 expression in the lung and heart. Interestingly, brain tissue did not have evidence of HO-1 induction. Although we have not investigated the mechanism of HO-1 induction, it is probable that cellular regulation of HO-1 by HBOC requires direct interaction of the target cells with the hemoglobin molecules. So, an intact blood-brain barrier may preclude induction of HO-1 in the brain. But it is also possible that HBOC could access neurons after I/R.
J Am Coll Surg
Figure 5. Hemoglobin-based oxygen carrier (HBOC)-induced heme oxygenase (HO)-1 is predominantly in the cardiac myocyte. Heart samples of animals treated with HBOC (10% v/v) were taken after 12 hours of treatment and frozen in embedding medium. Sections were stained and examined with immunohistochemistry. Sections were stained with HO-1 (red), biz-benzimide (nuclear stain, blue), and wheat germ agglutinins (cell outline, green). The predominant expression of HO-1 occurs in the myocyte, as demonstrated by diffuse staining throughout each of the cells, which are cardiac myocytes.
The induction of HO-1 by HBOC could also be a consequence of some cellular injury from the HBOC itself. Three points argue against this. First, one would expect an induction of HSP72, a generalized, sensitive marker of stress response, if this was the case. We do not see any induction of HSP72, suggesting that the cells and tissues are under no increased stress. Next, microscopic analysis would also suggest tissue injury, which was not demonstrated during analysis. Finally, several earlier studies documented the safety of HBOCs in vivo and in vitro, examining both clinical outcomes and biochemical markers of injury.12,26,27 The major limitation of this study is the lack of I/R data. Although there are extensive data supporting the role of HO-1 induction in protection against I/R injury, these studies have used other techniques to induce HO-1. It is possible that the HO-1 may not be the mechanism for protection. But we recently showed, in a liver model of I/R, that HBOC pretreatment can attenuate liver injury. We further demonstrated that this protection is dependent on HO-1, and selective inhibition of HO-1 obliterates the protection offered by HBOC pretreatment. So this induction of HO-1 confers protection from I/R injury in the liver (Damle S and associates, presented at the American College of Surgeons Committee on Trauma meeting, Denver, CO, March 2007, under review).
Vol. 208, No. 4, April 2009
Damle et al
Blood Substitute Induces Heme Oxygenase-1
597
REFERENCES
Figure 6. Hemoglobin-based oxygen carrier (HBOC)-induced heme oxygenase (HO)-1 is predominantly in the alveolar macrophages. Lung samples of animals treated with HBOC (10% v/v) were taken after 12 hours of treatment, infused with embedding medium back to normal lung volumes, and frozen in embedding medium. Sections were initially stained with HO-1 (red), biz-benzimide (nuclear stain, blue), and wheat germ agglutinins (cell outline, green); they demonstrated particular cells concentrating HO-1 expression (images not shown). Subsequent sections were stained with HO-1 (red) and CD68 (macrophage specific marker, green). Shown here is a representative section of one alveolus. There is some expression of HO-1 in a variety of cell types, but the predominant cellular expression of HO-1 localizes to cells that also stain with CD68, suggesting that the predominant expression of HO-1 in the lung occurs in the alveolar macrophages (arrows).
In conclusion, this work confirms that an HBOC can induce the expression of HO-1 in the heart and lung. Ultimately, this may be used as a preconditioning tool to minimize organ dysfunction from I/R injury. In the heart, HBOC could potentially be given 24 hours before cardiopulmonary bypass. In either organ, HBOC may potentially be used to induce expression of HO-1 before transplantation. But further work delineating the precise time for maximal induction and demonstrating abrogation of reperfusion injury will need to be performed before clinical application. These findings lay the groundwork for future studies exploring the potential use of HBOC to ameliorate the deleterious effects of I/R on organ function in a variety of clinical scenarios.
Author Contributions Study conception and design: Damle, Moore, Babu, Fullerton Acquisition of data: Damle, Babu Analysis and interpretation of data: Moore, Meng, Fullerton, Banerjee Drafting of manuscript: Damle, Moore, Babu, Meng, Fullerton, Banerjee Critical revision: Damle, Moore, Babu, Meng, Fullerton, Banerjee
1. Flameng W, Herjgers P, Szecsi J, et al. Determinants of early and late results of combined valve operations and coronary artery bypass grafting. Ann Thor Surg 1996;61:621–628. 2. King R, Binns OAR, Rodriguez F, et al. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation. Ann Thor Surg 2000;69:1681–1685. 3. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic application. Physiol Rev 2006;86:583–650. 4. Clark JE, Roberta F, Sarathchandra P, et al. Heme oxygenase1-derived bilirubin ameliorates postischemic myocardial dysfunction. Am J Physiol: Heart Circ Physiol 2000;278:H643– H651. 5. Das S, Fraga C, Das D. Cardioprotective effect of resveratrol via HO-1 expression involves p38 map kinase and PI-3-kinase signaling, but does not involve NFkB. Free Rad Res 2006;40: 1066–1075. 6. Hangaishi M, Ishizaka N, Aizawa T, et al. Induction of heme oxygenase-1 can act protectively against cardiac ischemia/ reperfusion in vivo. Biochem Biophys Res Comm 2000;279: 582–588. 7. Liu X, Pachori A, Ward C, et al. Heme oxygenase-1 inhibits postmyocardial infarct remodeling and restores ventricular function. FASEB J 2006;20:207–216. 8. Yet S-F, Tian R, Layne MD, et al. Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice. Circ Res 2001;89:168–173. 9. Fredenburgh L, Baron R, Carvajal I, et al. Absence of heme oxygenase-1 expression in the lung parenchyma exacerbates endotoxin-induced acute lung injury and decreases surfactant protein-B levels. Cell Mol Biol 2005;51:513–520. 10. Otterbein LE, Kolls JK, Mantell LL, et al. Exogenous administration of heme oxygease-1 by gene transfer provides protection against hyperoxia-induced lung injury. J Clin Invest 1999;103: 1047–1054. 11. Moore EE, Johnson JL, Cheng AM, et al. Insights from studies of blood substitutes in trauma. Shock 2005;24:197– 205. 12. Johnson JL, Moore EE, Gonzales RJ, et al. Alteration of the postinjury hyperinflammatory response by means of resuscitation with a red cell substitute. J Trauma 2003;54:133– 140. 13. Cheng AM, Moore EE, Johnson JL, et al. Polymerized hemoglobin induces heme oxygenase-1 protein expression and inhibits intercellular adhesion molecule-1 protein expression in human lung microvascular endothelial cells. J Am Coll Surg 2005; 201:579–584. 14. Tenhunen R, Marver HS, Schmid R. Microsomal heme oxygenase. Characterization of the enzyme. J Biol Chem 1969;244: 6388–6394. 15. Maines MD, Trakshel GM, Kutty RK. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J Biol Chem 1986;261:411–419. 16. Chabannes D, Hill M, Merieau E, et al. A role for heme oxygenase-1 in the immunosuppressive effect of adult rat and human mesenchymal stem cells. Blood 2007;110:3691– 3694.
598
Damle et al
Blood Substitute Induces Heme Oxygenase-1
17. Rushword S, MacEwan D. HO-1 underlies resistancec of AML cells to TNF-induced apoptosis. Blood 2008;111:379– 801. 18. Shokawa T, Yoshizumi M, Yamamoto H, et al. Induction of heme oxygenase-1 inhibits monocyte chemoattractant protein-1 mRNA expression in U937 cells. J Pharmacol Sci 2006;100:162–166. 19. Geuken E, Buis CI, Visser DS, et al. Expression of heme oxygenase-1 in human livers before transplantation correlates with graft injury and function after transplantation. Am J Transplant 2005;5:1875–1885. 20. Rensing H, Bauer I, Datene V, et al. Differential expression pattern of heme oxygenase-1/heat shock protein 32 and nitric oxide synthase-II and their impact on liver injury in a rat model of hemorrhage and resuscitation. Crit Care Med 1999;27:2766–2775. 21. Tang YL, Qian K, Zhang C, et al. A vigilant, hypoxia-regulated heme oxygenase-1 gene vector in the heart limits cardiac injury after ischemia-reperfusion in vivo. J Cardiovasc Pharmacol Ther 2005;10:251–263.
J Am Coll Surg
22. Yamashita K, Ollinger R, McDaid J, et al. Heme oxygenase-1 is essential for and promotes tolerance to transplanted organs. FASEB J 2006;20:776–778. 23. Roach J, Moore EE, Patrick D, et al. Heme-oxygenase-1 induction in macrophages by a hemoglobin-based oxygen carrier reduces endotoxin stimulated cytokine secretion. Shock; in press. 24. Moore EE. Blood substitutes: the future is now. J Am Coll Surg 2003;196:1–17. 25. Kubulus D, Rensing H, Paxian M, et al. Influence of hemebased solutions on stress protein expression and organ failure after hemorrhagic shock. Crit Care Med 2005;33:629–637. 26. Cothren CC, Moore EE, Long JS, et al. Large volume polymerized haemoglobin solution in a Jehovah’s Witness following abruptio placentae. Transfusion Med 2004;14:241–246. 27. Gould SA, Moore EE, Hoyt DB, et al. The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergent surgery. J Am Coll Surg 1998;187: 113–120.
MOVING? MOVED? Please go to www.efacs.org Provide your new address, telephone number, fax number, or email changes.