| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Ann Thorac Surg 1999;67:1421-1427
© 1999 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, Washington University School of Medicine, Barnes Jewish Hospital, St. Louis, Missouri, USA
b Department of Surgery, Washington University School of Medicine, Barnes Jewish Hospital, St. Louis, Missouri, USA
Address reprint requests to Dr Patterson, Division of Cardiothoracic Surgery, Washington University School of Medicine, One Barnes-Jewish Hospital Plaza, 3108 Queeny Tower, St. Louis, MO 63110
Presented at the Thirty-fifth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 25–27, 1999.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and methodsResultsCommentsAcknowledgmentsReferences |
|---|
Methods. In preliminary studies to determine the optimal titer, animals were injected with various titers of adenovirus-HSP70 (saline, 5 x 109, 1 x 1010, and 2 x 1010 plaque forming units [pfu]) and sacrificed 5 days after injection. To determine the optimal exposure time, animals were sacrificed at different times (0, 6, 24, and 72 hours) after intravenous injection of adenovirus-HSP70. In a subsequent series of transplant experiments, donors were allocated to three groups according to transfection strategy. Group 1 (n = 8) donors received 5 x 109 pfu adenovirus-HSP70 intravenously, group 2 (n = 7) donors received 5 x 109 pfu adenovirus-ß-galactosidase (as a virus control), and group 3 (n = 7) donors received saline and served as a negative control. Twenty-four hours after treatment all grafts were harvested and stored for 18 hours before orthotopic left lung transplantation. Twenty-four hours after implantation animals were sacrificed for assessment. The expression of HSP70 was assessed by Western blot analysis.
Results. In preliminary studies, HSP70 was detectable even at low titers (5 x 109 pfu) of adenovirus-HSP70, and was detectable at low levels as early as 6 hours after intravenous administration. Heat shock protein 70 expression was maximal at 24 hours. In transplant experiments, Western blot analysis showed that overexpression of HSP70 occurred in the HSP70-transfected lungs. The mean arterial oxygenation 24 hours after reperfusion in group 1 was superior in comparison with other groups (p < 0.05). Wet to dry weight ratio (p < 0.05) and myeloperoxidase activity (p < 0.05) were also significantly less in group 1 grafts compared with the other groups.
Conclusions. This study demonstrates that in vivo, donor adenovirus-mediated gene transfer of HSP70 decreases subsequent ischemia-reperfusion injury in rat lung isografts.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentsAcknowledgmentsReferences |
|---|
We have recently shown that the induction of heat shock protein 70 (HSP70) by whole body heat stress to donor animals before lung harvest decreases reperfusion injury in preserved rat lung isografts [6]. However, it was unclear from our previous study which of the heat shock proteins increased by whole body hyperthermia is responsible for the observed protective effects as heat stress can also induce an increase in other cytoprotective proteins, such as catalase, superoxide dismutase, or other members of the HSP family. Marber and associates [7] have shown that overexpression of the rat inducible 70-kd heat stress protein in a transgenic mouse model increases the resistance of the heart to ischemic injury. They also reported that gene transfection with HSP70 using an adenoviral vector protects against myocardial ischemic injury [8]. To further dissect the role of HSP70 in lung graft ischemia-reperfusion injury, we transfected an adenoviral vector containing HSP70 to rat lung isografts before harvest and a subsequent severe ischemia-reperfusion injury.
|
Material and methods |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentsAcknowledgmentsReferences |
|---|
First-generation replication-deficient adenovirus serotype 5 carrying the Escherichia coli LacZ gene encoding for ß-galactosidase and driven by the constitutive cytomegalovirus promoter (Ad5.CMV.ß-gal) served as controls. The LacZ gene was chosen because ß-galactosidase activity is easily measured in situ with reliable and sensitive histochemical assays that use chromogenic substrates.
Adenoviral amplification was propagated in 293 cells for several passages to obtain high-titer stocks, as determined by the plaque assay (Drs R. Jude Samulski and Douglas McCarty, gene therapy center vector core facility, The University of North Carolina, Chapel Hill). Viral stocks of 2 x 1011 plaque-forming units per milliliter (pfu/mL) were prepared as previously described [10]. Immediately before use, these stocks were thawed and diluted in 1 mL of normal saline.
Animals
Inbred male F344 rats (Harlan Sprague-Dawley Inc, Indianapolis, IN), weighing 250 to 290 g, were used in all experiments. All animal procedures were approved by the Animal Studies Committee at Washington University. Animals received humane care in compliance with "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Experimental groups
Preliminary experiments: in vivo transfection of normal lungs
The first group of experiments aimed to determine (1) the optimal titer of adenovirus vector; and (2) the optimal exposure time for in vivo transfection of normal lungs.
Rats were anesthetized by subcutaneous injection of ketamine chloride (25 mg/kg) and atropine sulfate (0.25 mg/kg). After endotracheal intubation with a 14-gauge catheter, animals were mechanically ventilated using a small animal Harvard ventilator (tidal volume, 3 mL; respiratory rate, 60 breaths/min) (Harvard Apparatus, Holliston, MA) with 0.5% halothane and 99.5% oxygen. A small incision was made in the left lower neck, and the left external jugular vein was mobilized. The adenovirus construct was injected intravenously for a period of 5 minutes. The incision was then closed, and the animals allowed to recover from anesthesia. (1) To determine optimal titer, animals were divided into four groups (n = 2) according to the titers of administered adenovirus construct encoding HSP70 (saline, 5 x 109, 1 x 1010, and 2 x 1010 pfu). All animals were sacrificed 5 days after injection. (2) To determine optimal exposure time, animals were divided into eight groups (n = 2) according to time of sacrifice after intravenous injection of adenovirus encoding HSP70. The expression of HSP70 in both experimental groups was assessed by Western blot analysis.
Transplant experiments: in vivo graft transfection
F344 rats were divided into three groups. Group 1 (n = 8) donor animals were injected with 5 x 109 pfu of adenovirus encoding HSP70. Group 2 (n = 7) donors served as adenoviral controls and received 5 x 109 pfu of adenovirus encoding ß-galactosidase. Group 3 (n = 7) donors served as negative controls and received normal saline without adenovirus. Twenty-four hours after injection, donor lungs were harvested and preserved for 18 hours at 4°C, then implanted into isogeneic recipients. Recipient rats were sacrificed and assessed 24 hours after transplant.
Rat lung transplantation
Rat left lungs were harvested and transplanted into isogeneic recipients as previously described [11, 12]. In brief, donors were anesthetized, intubated, and heparinized, then underwent a median sternolaparotomy. The abdominal aorta, inferior vena cava, and the left atrial appendage were incised, and the lungs were flushed through the main pulmonary artery with 20 mL of cold low-potassium dextran-1% glucose (LPDG) solution after the heart-lung block was excised with the lungs inflated at end-tidal volume. Three cuffs made from 14-gauge grooved polyethylene tubing were placed in the donor left pulmonary artery, vein, and bronchus, respectively. Donor left lungs were then preserved in LPDG solution at 4°C for 18 hours until implantation. Donor right lungs were snap-frozen in liquid nitrogen for subsequent analysis. The grafts were implanted with the use of a cuff technique for all anastomoses.
Assessment
For all groups, recipient animals were reanesthetized 24 hours after reperfusion using the donor technique described above, then mechanically ventilated with 100% oxygen. Median laparotomy-sternotomy was performed, and the contralateral right hilum was clamped. Animals were then ventilated for 5 minutes using a tidal volume of 1.5 mL, respiratory rate of 100 breaths/min, and positive end-expiratory pressure of 1.0 cm H2O to assess the function of the left lung isograft by arterial blood gas analysis using blood samples obtained from the abdominal aorta. After sacrifice, each left lung graft was divided into two specimens: the upper half was snap-frozen in liquid nitrogen for myeloperoxidase (MPO) activity measurements, and the lower half was weighed, dried at 70°C for 48 hours, then weighed again for calculation of the wet to dry weight ratio.
Lungs from animals in preliminary, nontransplant experiments were snap-frozen in liquid nitrogen. These frozen samples were subsequently analyzed for HSP70 production by Western blot analysis.
Myeloperoxidase activity
Quantitative MPO activity was determined as previously described [13]. In brief, frozen lung samples were homogenized in 1 mL of 0.5% hexadecyltrimethyl-ammonium bromide, 5 mmol/L EDTA, and 50 mmol/L potassium phosphate buffer (pH 6.2) with a Broeck tissue grinder (Kontes Glass Co, Vineland, NJ). Hexadecyltrimethyl-ammonium bromide is a detergent that releases MPO from the primary granules of neutrophils. The homogenate was centrifuged at 10,000g for 15 minutes at 4°C. The supernatant was subsequently assayed for total soluble protein by the method of Pierce Laboratories (Rockford, IL) [14]. Myeloperoxidase activity was measured spectrophotometrically as follows: 10 µL of five-fold supernatant was combined with 0.6 mL Hanks BSA (0.255 bovine serum albumin added to Hanks solution), 0.5 mL of 100 mmol/L potassium phosphate buffer (pH 6.2), 0.1 mL 0.05% H2O2, and 0.1 mL of 1.25 mg/mL o-dianisidine. Color development was stopped by the addition of 0.1 mL of 1% NaN3 after 5 minutes and after 20 minutes at room temperature. Optical density was measured at 460 nm with a spectrophotometer (Model PMQ II, Carl Zeiss, Jena, West Germany). Color development was linear from 5 to 20 minutes. One unit of enzyme activity was defined as the amount of 1.0 optical density units per minute per milligram of tissue protein at room temperature.
Western blot analysis of HSP70 and HSP90
The presence of HSP70 or HSP90 was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting as previously described [15–17]. In brief, frozen specimens were homogenized in cold buffer A (consisting of 10 mmol/L Tris/HCL, pH 7.5; 250 mmol/L sucrose; and 0.1 mmol/L phenylmethylsulfonyl fluoride) using a ratio of 2 mL buffer A per gram of tissue. The homogenate was centrifuged at 30,000g for 10 minutes at 4°C. The supernatant was removed and centrifuged at 100,000g for 45 minutes at 4°C. The high-speed supernatant was rapidly frozen and stored at -80°C until analysis. Protein content of the supernatants was measured by the bicinchonic acid protein reagent assay system (Pierce). Thereafter, 20 µg of total protein was loaded in each lane. Proteins were separated by SDS-PAGE on 0.75-mm thick, 12.5% acrylamide gels. The proteins on the gel were immediately transferred to nitrocellulose membranes (Hybond C, Amersham, Bucks, UK) by Western blotting. The other identical gel was stained with Coomassie brilliant blue to visualize the protein bands. The nitrocellulose membrane was washed in phosphate-buffered saline, 0.1% Tween-20 with 5% dried skimmed milk powder to block nonspecific binding sites. The membrane was then incubated at room temperature for 1 hour with a primary monoclonal antibody, mouse anti-human immunoglobulin G (IgG), specific against the inducible form of HSP70 or HSP90 anti-mouse monoclonal IgG1 (Stressgen Biotechnologies Corp, Sydney, BC, Canada) at 1:1000 dilution. After repeated washings, the membrane was incubated with anti-mouse peroxidase-labeled secondary antibody (at 1:1000 dilution at room temperature for 1 hour), then with enhanced chemiluminescence detection reagent (Amersham International Ltd, Little Chalfont, Bucks, UK). The nitrocellulose paper was then exposed to Fujifilm Medical X-Ray film (Fuji Photo Film Co, Tokyo, Japan). Purified recombinant human HSP70 or HSP90 protein (bovine) served as a positive control (Stressgen Biotechnologies Corp). The degree of HSP70 expression was semiquantitatively evaluated with computed densitometry using NIH Image software (National Institutes of Health, Bethesda, MD).
Statistical analysis
Values are reported as mean ± standard error of the mean (SEM). One-way analysis of variance with pairwise comparison by the Bonferroni method was used to compare overall differences among groups. Differences were considered significant if the p value was less than 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentsAcknowledgmentsReferences |
|---|
|
|
|
|
Wet to dry weight ratio
Mean wet/dry weight ratios of transplanted lungs in group 1 were significantly less than those observed in controls (group 1 vs groups 2 and 3, 6.024 ± 0.9 versus 7.791 ± 1.48 and 7.574 ± 1.32, p = 0.0133, p = 0.0329). There were no significant differences between groups 2 and 3 (Fig 5).
|
OD · mg-1 · min-1; p = 0.0404, p = 0.0091, respectively). There were no significant differences between groups 2 and 3 (Fig 6).
|
|
Comments |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentsAcknowledgmentsReferences |
|---|
Several authors have recently demonstrated the effectiveness of gene transfection with HSP70, done either in vivo or in vitro in the setting of ischemia-reperfusion injury [8, 18]. On the basis of these encouraging results, we transfected the HSP70 adenoviral construct into rat lungs to study its effects on subsequent lung graft ischemia-reperfusion injury.
The lung is a particularly attractive organ for gene therapy because of its accessibility by way of the airways or the vasculature. Both the airway epithelium and the vasculature endothelium represent potential targets for gene therapy [19]. In a recent study of endobronchial gene delivery, expression of a reporter gene was observed in epithelial cells lining conducting airways and distal lung segments but not in vascular endothelium, suggesting that this may not be the optimal route for gene delivery to pulmonary vascular cells [20]. In contrast, adenovirus vectors administered directly into the lumen of large vessels are capable of transferring genes to the endothelium of both arteries and veins [21]. We have previously reported the use of a liposomal vector to transfect lung grafts before harvest (in vivo donor transfection by intravenous injection) [22]. However, viruses are considered more efficient vectors compared with liposomal vectors, partly because they have evolved excellent mechanisms for cellular attachment, penetration, and avoidance of intracellular lysosomal degradation. Adenovirus vectors are especially efficient for in vivo delivery of exogenous DNA into a wide variety of tissue types, and do not require the target cells to undergo replication to express the exogenous gene of interest.
In the preliminary experiments, our results demonstrate that gene expression, albeit low, was present as early as 6 hours after intravenous injection and increased significantly after 24 hours. Long exposure times may not be suitable in the setting of clinical organ transplantation. We therefore used a relatively short exposure time of 24 hours in these experiments. In addition, a variety of titers of adenovirus-HSP70 were used to investigate the optimal transfection titer in this setting. The lowest titer group, at 5 x 109 pfu, showed no significant difference compared with the high-titer groups. Thus, to avoid possible cytotoxicity, we selected a titer of 5 x 109 pfu for our subsequent experiments. In a contemporaneous series of preliminary experiments (data not shown), donor animals received 5 x 109 pfu of adenovirus encoding ß-galactosidase. Subsequent to harvest, transgene expression was noted in endothelial cells of large pulmonary vessels and not in bronchial epithelium. These observations are consistent with those reported by Lemarchand and colleagues [23] in the sheep model. They demonstrated that the administration of adenovirus vectors into the pulmonary artery without vascular occlusion led to gene transfer to the endothelium of the pulmonary arteries but not to the endothelium of the bronchial circulation or airway epithelium.
Many experiments using gene transfection have demonstrated the direct role of overexpression of HSP70 in protection from various types of injury [8, 18]. In the present study, we demonstrate that rat lung grafts transfected with HSP70 (group 1) gene showed overexpression of HSP70 at the time of harvest compared with adenoviral control (group 2) or saline control (group 3) groups. The present study also demonstrated that graft function 24 hours after reperfusion was markedly improved in HSP70-transfected lungs (group 1) compared with the other two groups.
Several authors have reported that high level of overexpression of HSP70 induced by gene transfection is more effective in protecting organs from ischemia-reperfusion injury compared with self-protective mechanisms mobilized by heat stress containing the normal high level expression of HSP70 [18, 24, 25]. On the other hand, several investigators have observed a significant inflammatory response when adenoviral vectors were used [26, 27]. In addition, preliminary data in cotton rats and rhesus monkeys indicate that there is a dose-dependent inflammatory response in the airway after intratracheal delivery of adenovirus vector. In our preliminary study using a titer of 5 x 109 pfu, there was only a minimal host inflammatory response to adenoviral administration on pathologic sections (data not shown). If high titers of adenovirus constructs could be administered to the donors without inciting an immune response, then higher expression of HSP70 might be present in the grafts at the time of harvest. Furthermore, optimization of the in vivo adenovirus delivery system (for example, by higher dosages or repeated administration) may offer significant increases in exogenous gene expression and duration of expression of the transferred gene. However, studies of gene transfection with HSP70 are just beginning, especially in the field of lung transplantation. Further research will be needed to elucidate the exact role of HSP70 and to assess the feasibility of in vivo gene transfection in the setting of lung transplantation.
In summary, in vivo adenovirus-mediated gene transfection with HSP70 to the donor animals has protective effects against ischemia-reperfusion injury after prolonged preservation of transplanted lung isografts.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentsAcknowledgmentsReferences |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentsAcknowledgmentsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  E. M. Harrison, E. Sharpe, C. O. Bellamy, S. J. McNally, L. Devey, O. J. Garden, J. A. Ross, and S. J. Wigmore Heat shock protein 90-binding agents protect renal cells from oxidative stress and reduce kidney ischemia-reperfusion injury Am J Physiol Renal Physiol, August 1, 2008; 295(2): F397 - F405. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Godzich, M. Hodnett, J. A. Frank, G. Su, M. Pespeni, A. Angel, M. B. Howard, M. A. Matthay, and J. F. Pittet Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia-reperfusion injury in rats FASEB J, July 1, 2006; 20(9): 1519 - 1521. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ishiyama, S. Dharmarajan, M. Hayama, H. Moriya, K. Grapperhaus, and G. A. Patterson Inhibition of nuclear factor {kappa}B by I{kappa}B superrepressor gene transfer ameliorates ischemia-reperfusion injury after experimental lung transplantation J. Thorac. Cardiovasc. Surg., July 1, 2005; 130(1): 194 - 201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. Kiang, P. D. Bowman, B. W. Wu, N. Hampton, A. G. Kiang, B. Zhao, Y.-T. Juang, J. L. Atkins, and G. C. Tsokos Geldanamycin treatment inhibits hemorrhage-induced increases in KLF6 and iNOS expression in unresuscitated mouse organs: role of inducible HSP70 J Appl Physiol, August 1, 2004; 97(2): 564 - 569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hayashi, Y. Sawa, N. Fukuyama, H. Nakazawa, and H. Matsuda Preoperative Glutamine Administration Induces Heat-Shock Protein 70 Expression and Attenuates Cardiopulmonary Bypass-Induced Inflammatory Response by Regulating Nitric Oxide Synthase Activity Circulation, November 12, 2002; 106(20): 2601 - 2607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. de Perrot and S. Keshavjee Lung preservation Ann. Thorac. Surg., August 1, 2002; 74(2): 629 - 631. [Full Text] [PDF]
|
|
|
|
|
|
N. Daddi, T. Suda, F. D'Ovidio, S. A. Kanaan, T. Tagawa, K. Grapperhaus, B. D. Kozower, J. H. Ritter, N. S Yew, T. Mohanakumar, et al. Recipient intramuscular cotransfection of naked plasmid transforming growth factor {beta}1 and interleukin 10 ameliorates lung graft ischemia-reperfusion injury J. Thorac. Cardiovasc. Surg., August 1, 2002; 124(2): 259 - 269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Tagawa, T. Suda, N. Daddi, B. D. Kozower, S. A. Kanaan, T. Mohanakumar, and G. A. Patterson Low-dose endobronchial gene transfer to ameliorate lung graft ischemia-reperfusion injury J. Thorac. Cardiovasc. Surg., April 1, 2002; 123(4): 795 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Stoica, M. Goddard, and S. R. Large The endothelium in clinical cardiac transplantation Ann. Thorac. Surg., March 1, 2002; 73(3): 1002 - 1008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. D'Ovidio, N. Daddi, T. Suda, K. Grapperhaus, and A. G. Patterson Efficient naked plasmid cotransfection of lung grafts by extended lung/plasmid exposure time Ann. Thorac. Surg., June 1, 2001; 71(6): 1817 - 1823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Itano, W. Zhang, J. H. Ritter, T. J. McCarthy, T. Mohanakumar, and G. A. Patterson Adenovirus-mediated gene transfer of human interleukin 10 ameliorates reperfusion injury of rat lung isografts J. Thorac. Cardiovasc. Surg., November 1, 2000; 120(5): 947 - 956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Suda, B. N. Mora, F. D’Ovidio, J. A. Cooper, M. Hiratsuka, W. Zhang, T. Mohanakumar, and G. A. Patterson IN VIVO ADENOVIRUS-MEDIATED ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE TRANSFER AMELIORATES LUNG ALLOGRAFT ISCHEMIA-REPERFUSION INJURY J. Thorac. Cardiovasc. Surg., February 1, 2000; 119(2): 297 - 304. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
