Journal of Surgical Research 109, 24 –30 (2003) doi:10.1006/jsre.2002.6562
Clinically Relevant Thermal Preconditioning Attenuates Ischemia-Reperfusion Injury P.H. McCormick, AFRCSI, G. Chen, M.B., S. Tlerney, FRCSI, C.J. Kelly, FRCSI, and D.J. Bouchier–Hayes, FRCSI 1 Department of Surgery, Royal College of Surgeons of Ireland, Beaumont Hospital, Dublin 9 Republic of Ireland Submitted for publication March 4, 2002
strated increased expression of HSP72 in the preconditioned group vs control and I/R alone. Conclusions. We conclude that clinically applicable thermal preconditioning can attenuate ischemiareperfusion induced lung injury, possibly through increased expression of HSP72. © 2003 Elsevier Science (USA) Key Words: heat shock protein 72; thermal preconditioning; clinically relevant; ischemia-reperfusion.
Introduction. Thermal preconditioning has previously been shown to attenuate ischemia-reperfusion induced injuries, possible due to increased expression of heat shock proteins (HSP). The model of thermal preconditioning used, however, was not clinically relevant as preconditioning was to 41°C, leading to cellular damage. Our aim was thus to establish a novel and clinically applicable method of preconditioning. Materials and Methods. Twenty-six male SpragueDawley rats were split into three groups (nine control, nine ischemia-reperfusion, and eight preconditioned followed by ischemia-reperfusion). To precondition the animals, they were anesthetized and, using a water bath, their core temperature was raised by 1°C for 15 min once a day for five successive days. I/R injury consisted of 30 min of aortic cross-clamping followed by 120 min of reperfusion; control animals had a laparotomy only. Indicators of lung injury were tissue myeloperoxidase, broncho-alveolar lavage protein concentration, and tissue edema. Tissue heat shock protein expression was detected by Western blot analysis. Results. Lower torso ischemia-reperfusion causes significant lung injury versus control, with raised levels of myeloperoxidase 4.53 iu/g to 7.88 iu/g (P < 0.05), raised B.A.L. protein concentration 419 g/ml to 684 g/ml (P < 0.05) and altered wet dry ratio 4.63 to 5.50. Clinically relevant thermal preconditioning attenuates all of these parameters back to control levels: myeloperoxidase 3.87 iu/g (P < 0.05 vs I/R), B.A.L. to 284 g/ml (P < 0.01 vs I/R) and wet dry ratio to 4.44 (P < 0.05 vs I/R). Western blot demonstrated increased expression of H.S.P. 72 in the preconditioned group versus control and I/R alone. Western blot demon-
INTRODUCTION
Preconditioning is a process where cells or tissues exposed to a sublethal stimulus are transiently protected from a subsequent normally lethal stress. Many forms of preconditioning have been investigated, in particular, ischemic, pharmacologic, and of most focus in our group, thermal. It is suggested that the beneficial effect of thermal preconditioning is mediated by heat shock proteins (HSP), in particular HSP 72 [1]. These HSPs act as molecular chaperones, preventing premature protein folding and denaturation and allowing normal protein folding, assembly, and interactions to proceed in adverse cellular environmental conditions [2, 3]. It has been demonstrated that thermotolerance attenuates ischemia-reperfusion induced endothelial–leukocyte interactions, possible through increased expression of HSP72 [4]. In vivo studies have shown that thermotolerance is protective against I/R injury in the heart [5], small intestine [6], skeletal muscle [7, 8], kidney [9], and the lungs [10] in a manner proportional to the expression of HSP72 [5]. The clinical significance of these studies is compromised as thermotolerance was achieved by increasing the core temperature to 41°C and the damage thus caused by the hyperthermia limits its role as a preconditioning tool. It has been shown recently that chronic mild hyperthemia protects cardiac myoblasts against subse-
1
To whom correspondence should be addressed at Department of Surgery, Beaumont Hospital, Dublin 9, Rep. of Ireland. Fax: 00-3531-8093335. E-mail:
[email protected].
0022-4804/03 $35.00 © 2003 Elsevier Science (USA) All rights reserved.
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quent H 2O 2 exposure with increased HSP 70 expression [11]. Furthermore, an in vivo study has demonstrated an improvement in endothelial function with repeated moderate hyperthermia [12]. Ischemia-reperfusion (I/R) injuries are an inevitable consequence of aortic surgery and transplantation, leading to both a local injury and systemic inflammatory reaction [13]. Resulting pulmonary microvascular permeability, plus renal and cardiac injuries, lead to severe and often fatal complications [14, 15]. A central component in the pathogenesis of I/R injury is the upregulation of surface adhesion molecules on the vascular endothelium [9] and subsequent interaction between these molecules and activated neutrophils [16, 17]. This facilitates transendothelial migration of neutrophils, leading to the subsequent release of reactive oxygen species and cytokines causing interstitial tissue injury [18]. The aim of this study was thus to evaluate the efficiency of clinically relevant thermal preconditioning in upregulating the expression of HSP 72 and in subsequently preventing I/R induced lung injury in an animal model of lower torso ischemia reperfusion. MATERIALS AND METHODS
Animals and Thermal Preconditioning All animal procedures were carried out in an approved animal research facility, under license from the Department of Health, Republic of Ireland. Twenty-six Sprague-Dawley rats (250 –300 g) were maintained on a diet of purified chow pellet and water. The rats were randomized into three groups: a control group subjected to laparotomy and sham I/R (nine rats), an I/R group that had infrarenal aortic cross clamping for 30 min followed by 120 min of reperfusion (nine rats), and a group that received thermal preconditioning before undergoing I/R (eight rats). Animals were sacrificed and studied at the end of the 120 min of reperfusion and after 150 min of anesthesia in the control group. Thermal preconditioning was induced by raising the core body temperature to 38.5–39°C by partial immersion in a water bath (Grant Instruments, Sheprath, UK), under anesthesia with inhalational halothane (May and Baker, Dagenham, UK). The animals’ temperatures were gradually increased and were continuously monitored by rectal thermometer. This increased temperature was maintained for 15 min and repeated on five consecutive days. I/R took place 18 h after the fifth thermal preconditioning.
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ing it at 90°C in a gravity convection oven for 72 h to attain its dry weight.
Myeloperoxidase Assay Myeloperoxidase (MPO) is a heme-containing enzyme which is found within the azurophil granules of neutrophils, and so can be used as an indirect measure of tissue neutrophil infiltration. The right ventricle was cannulated with a 25-gauge needle and the right pulmonary hilum was clamped, after which the pulmonary vasculature of the left lung was “washed out” with 50 ml of normal saline to clear the lung of intravascular neutrophils. After weighing, the left lower lobe was homogenized in 10 ml of 0.5% hexadecyl trimethyl ammonium bromide (HTAB)(Sigma Chemical Co., St. Louis, MO) in potassium phosphate buffer at pH 6. The homogenates were freeze– thawed twice and centrifuged at 2000 rpm for 10 min. The resultant supernatant was assayed spectrophotometrically for MPO activity by incubating 10 l of the supernatant with 290 l of a developing solution. This solution contained 5 ml of a 0.017% suspension of O-dioniside, and 5 ml of 0.006% hydrogen peroxide with 40 ml of phosphate buffered saline. The absorbance was measured at 450 nm after 10 min incubation and MPO activity calculated per gram of tissue.
Western Immunoblotting for HSP72 Expression Tissue samples from the lung and diaphragm were removed, homogenized in phosphate-buffered saline (1 ml), and centrifuged at 4100g for 30 min at 4°C. The supernatants were collected and protein concentration was quantified using a Coomasie protein assay reagent (Rockford, IL). The final protein concentration of the samples was diluted to 50 g/10 l. The protein was denatured at 100°C for 10 min and aliquots containing equal amounts of proteins were then suspended in sodium dodecyl sulfate (SDS)-glycerol loading buffer (pH 6.8, 62.5 mmol/L Tris, 2% SDS, 10% glycerol, 5% mercaptoethanol, 0.01% bromophenol blue) and proteins were seperated by SDS polyacrylamide gel electrophoresis (PAGE) (ExcelGel, Pharmacia, Sweden). Proteins were then transferred to a nitrocellulose membrane (Sigma Chemical Co., St. Louis, MO) and labeled with a primary monoclonal antibody, mouse anti-rat immunoglobulin specific for the HSP 72 (StressGen, Victoria, British Columbia, Canada). After the secondary monoclonal antibody (goat anti-mouse) was added, the protein was visualized using bromochloroindolyl phosphaye/nitro blue tetrazolium (BCIP/NBT, Sigma Chemical Co.)
Statistical Analysis All data presented as mean (⫹/⫺ SEM). Statistical analysis was performed using analysis of variance (ANOVA) with Scheffe post hoc test. All statistical analysis was carried out on SPSS.
Sample Collection
Broncho-Alveolar Lavage After sternotomy, the left bronchus was clamped. Bronchoalveolar lavage (BAL) of the right lung was performed with 2 ml of saline containing 0.07 M EDTA. The lavage fluid was centrifuged at 1500g for 20 min at 4°C, and the supernatant frozen at ⫺80°C. BAL fluid was subsequently used for measurement of protein concentration by the spectrophotometric dye method of Lowry.
Wet/dry Lung Weight Ratio Lung wet:dry (W:D) weight ratios were used as a measure of pulmonary edema. The W:D weight ratio of the right lower lobe was calculated by weighing the freshly harvested organ, and then heat-
RESULTS Myeloperoxidase
Neutrophil infiltration as indicated by MPO activity significantly increased in the I/R group [7.88 (⫹/⫺1.17) U/g vs controls 4.53 (⫹/⫺0.8) U/g (P ⬍ 0.05, CI 6.6 to 8.2)] (Fig. 2). Pretreatment with clinically relevant temperature increases attenuate this with an activity per gram of tissue of 3.88 (⫹/⫺0.45) U/g (P ⬍ 0.05 vs I/R, CI 7.5 to 0.51, P ⬍ 0.889 vs control, CI ⫺2.8 to 4.1).
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FIG. 1. Level of myeloperoxidase in units per gram of tissue in lungs of control, ischemia-reperfused and heat shocked and ischemiareperfused animals. Myeloperoxidase is a measure of neutrophil infiltration.
BAL
Pulmonary microvascular permeability as assessed by BAL protein concentration was significantly elevated in the I/R group [684 (⫹/⫺73) g/ml vs controls 419 (⫹/⫺75) g/ml (P ⬍ 0.05, CI 23 to 505)] (Fig. 1). This was significantly attenuated by thermal preconditioning to a protein concentration of 284 (⫹/⫺38.4)
g/ml (P ⫽ 0.001 vs I/R, CI 151 to 648, P ⫽ 0.378 vs control, CI⫺384 to 113). Wet/Dry Ratio
Pretreatment significantly attenuated the development of lung edema caused by I/R. Wet/dry ratio was 4.44 (⫹/⫺0.3) in the thermal preconditioned group vs
FIG. 2. Protein concentration of broncho-alveolar lavage fluid from control, ischemia-reperfused, and heat shocked and ischemiareperfused animals in g/ml. Protein concentration is a measure of pulmonary microvascular permeability.
MCCORMICK ET AL.: CLINICALLY APPLICABLE THERMAL PRECONDITIONING
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FIG. 3. Wet/dry ratio of lung tissue from control, ischemia-reperfused and heat shocked and ischemia-reperfused animals. Tissue edema is evaluated by wet/dry ratio.
5.5 (⫹/⫺0.12) in the I/R group (P ⬍ 0.05, CI 0.115 to 2). The ratio in the control group [4.63 (⫹/⫺0.28)] was similar to the heated group (P ⫽ 0.873, CI ⫺1.13 to 0.759) (Fig. 3). HSP 72 Western Immunoblotting
In this experiment, repeated low magnitude (1°) temperature increases resulted in an increased expres-
sion of HSP 72 in the lung and to a lesser extent the diaphragm of treated animals against control and I/R animals (Fig. 4). DISCUSSION
Our hypothesis was that a clinically relevant model of thermal preconditioning could upregulate the ex-
FIG. 4. Western blot for HSP 72 in diaphragm and lung of control, ischemia-reperfused animals and heat shocked and ischemiareperfused animals.
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pression of HSP 72 and could attenuate the pulmonary damage caused by a lower torso ischemia-reperfusion injury. These results demonstrate that lower torso ischemia-reperfusion leads to increased pulmonary microvascular permeability, as demonstrated by significant increases in broncho-alveolar lavage protein concentrations and altered wet/dry ratios, and causes increased transendothelial migration of neutrophils, indicated by significantly raised levels of tissue myeloperoxidase. Uniquely, this study demonstrates that thermal preconditioning, in a manner which is clinically applicable, causes a significant increase in the expression of HSP 72 and attenuates all of these parameters to control levels demonstrating protection against an ischemia-reperfusion injury. A significant element of distant organ dysfunction secondary to lower torso ischemia-reperfusion is due to the interaction between activated neutrophils and the microvascular endothelium [16, 19]. Neutrophil activation has been identified as a key mediator of injury postaortic cross-clamping in intestine [17], skeletal muscle [7, 8], hepatic tissue [20], cardiac tissue [21], and in particular, in the lungs [18, 22]. This activation secondary to ischemia-reperfusion takes the form of a neutropenia, both locally and systemically [18], and an upregulation of the CD11b–CD18 adhesion glycoprotein complex expressed on the neutrophils [20, 23]. This glycoprotein is essential for transendothelial migration, as this complex binds with the intracellular adhesion molecule (ICAM) ligand on the endothelial cell. The expression of this ligand is upregulated by I/R injury, possibly secondary to the production of free radicals and/or cytokines such as TNF and/or IL1 [15, 24]. The production of these molecules is enhanced in tissue subjected to I/R and they have been implicated in endothelial dysfunction and adhesion molecule upregulation [12, 25, 26]. The combined effect of I/R on neutrophils and the endothelium is an increased interstitial neutrophil invasion and subsequent release of proteolytic enzymes leading to tissue damage [18]. Previous work by our group and others has demonstrated that exposure of cells and tissues to a sublethal stimulus protects them against a subsequent, normally lethal assault. This “preconditioning” assault has many forms, thermotolerance [9], ischemia [27], hypothermia [28], and pharmacological intervention [22]. In most situations, the level of subsequent protection is associated with and proportional to the level of expression of heat shock protein [29]. These proteins, first discovered in 1962, are highly conserved proteins, constitutively expressed and upregulated by stress [2, 30]. Evidence has demonstrated that a variety of environmental and pathological stresses induce HSPs. The degree of induction depends on the level of and duration of the exposure to the stress. It is now understood that a cellular stress leads to an activation of heat
shock transcription factors (HSF), which bind to a region on the DNA called the heat shock elements (HSE). These HSEs are upstream of the HSP gene, and once the HSFs bind to the HSEs, transcription is then initiated, followed by translation. Once sufficient HSPs have been manufactured to supply the cells’ demands, the excess binds to the cytosolic HSFs and inactivates them once more [31]. These proteins act as “molecular chaperones,” playing a central role in the binding of denatured proteins and allowing normal protein construction in adverse cellular conditions [3, 32]. There are several groups of HSPs but the 70 kDa family and in particular, HSP72, have been strongly implicated in this preconditioning role. The expression of HSP72 has been proportionately associated with attenuation of I/R induced injury to the lungs [9], kidney [9], and endothelium [4, 29]. The manner of the protective effect of HSP72 is unclear. Post I/R injury it has been shown to reduce endothelial ICAM upregulation [15] and to reduce the expression of leukotriene B4 [6], a molecule which is known to induce leukocyte chemotaxis, adherence, and degranulation [33]. Thermotolerance also augments the cellular antioxidant defense system by increasing the synthesis of superoxide dismutase leading to destruction of damaging free radicals [34, 35]. Data also suggest that HSP72 has significant anti-apoptotic properties in relation to caspase 3, [36, 37] and that it reduces both levels of TNF␣ and TNF␣ cytotoxicity [38 – 40]. Recent findings suggest that a potential mechanism may be the ability of HSPs to inhibit proinflammatory responses in the lungs, as prior HSP induction has been demonstrated to inhibit inducible nitric oxide synthase gene expression [41, 42]. While the exact mechanism of protection is not clearly understood, elegant experiments using anti-HSP antibodies and transgenic mice with increased levels of HSP 72 have demonstrated that the protection afforded by thermal preconditioning is due to the HSPs and not to another benefit of the heating process [43, 44]. This study and others demonstrate that thermal preconditioning leads to increased expression of HSP72 and subsequently protects against I/R induced pulmonary injury. The increased expression of HSPs is a defense mechanism in response to a potential cellular injury. In attempting to harness HSP as a preconditioning tool, therefore, the difficulty lies in inducing upregulation and subsequent protection without causing cellular injury, as preconditioning optimally requires a safe, noninjurious, side effect-free method of pretreatment [45]. Previous studies have demonstrated that the temperature regimen required to induce HSPs can be manipulated using pharmacological agents [46] and the mechanism of HSP upregulation can itself be short circuited [47]. Cellular studies have demonstrated that manipulation of heating patterns
MCCORMICK ET AL.: CLINICALLY APPLICABLE THERMAL PRECONDITIONING
may allow lower temperature elevations to induce HSPs [48]. These studies led us to the hypothesis that a clinically applicable method of thermal preconditioning could be developed. This study demonstrates for the first time, that HSP72 and subsequent protection against an I/R induced pulmonary injury can be induced in a manner which is clinically relevant using a low magnitude repeated heat shock. This proves that thermal preconditioning shows significant potential as a therapeutic intervention, and hence we are now involved in further studies evaluating the role of similar regimens in inducing HSPs in humans.
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