Subsequent Ischemia-reperfusion Injury Research Articles

The augmentative effect of repeated heat shock preconditioning on the production of heat shock protein 72 and on ischemic tolerance in rat liver tissue

Objective : Heat shock pretreatment induces heat shock protein (HSP)72 strongly in rat livers and provides the tolerance against subsequent ischemia-reperfusion injury. In this study, the effects of repeated heat shock pretreatment on the production of HSP72 in rat livers and on subsequent ischemic tolerance were investigated. Methods : Rats pretreated with repeated heat shock were compared with those that received a single heat shock pretreatment. The production of HSP72 was analysed using Western-blotting and densitometer. At 48h after heat shock pretreatment, all rats were subjected to warm liver ischemia for 30 or 45min and then reperfused. Survival rate of the animals and liver functions during reperfusion were analysed. Results : The production of HSP72 increased in the repeated heat shock group more than in the single heat shock group. Although there were no significant differences in animal survival or in liver functions after a 30-min ischemia between the single heat shock group and the repeated heat shock group, animal survival and liver functions after a 45-min ischemia were significantly better in the repeated heat shock group. Conclusion : In rats, repetition of heat shock pretreatment augmented the production of HSP72 in liver tissue and protected the liver from ischemia-reperfusion injury.

Gene transfer of heat shock protein 70 protects lung grafts from ischemia-reperfusion injury

Background. We recently demonstrated that heat stress induction of heat shock protein 70 (HSP70) in donor animals before harvest decreases posttransplant ischemia-reperfusion injury in preserved rat lung isografts. The purpose of this study was to investigate the feasibility of HSP70 gene transfection into rat lung isografts using an adenoviral vector, and to study the effects of gene expression on subsequent ischemia-reperfusion injury. Methods. In preliminary studies to determine the optimal titer, animals were injected with various titers of adenovirus-HSP70 (saline, 5 × 10 9, 1 × 10 10, and 2 × 10 10 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 × 10 9 pfu adenovirus-HSP70 intravenously, group 2 (n = 7) donors received 5 × 10 9 pfu adenovirus-β-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 × 10 9 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.

The role of coronary flow and adenosine in postischemic recovery of septic rat hearts.

Sepsis depresses myocardial function but prevents subsequent ischemia-reperfusion injury. Elevated coronary flow (CF) and endogenous adenosine may be important factors in the complete recovery of postischemic myocardial function observed in septic rat hearts. The purpose of this study was to determine the effects of manipulating CF and of antagonizing adenosine receptors on the postischemic recovery of left ventricular developed pressure (LVDP) in septic and control rat hearts. The relationship between CF and LVDP in septic rat hearts before ischemia was depressed compared with control. However, this relationship was unaltered by ischemia in septic hearts, whereas in control hearts it was severely depressed. Preventing the elevation of CF during reperfusion did not significantly affect the recovery of LVDP in septic rat hearts. Adenosine antagonism by 8-phenyltheophylline (0.1 and 1 nM) prevented the elevated CF during reperfusion, and the higher dose significantly depressed postischemic function. We conclude that elevated CF did not contribute to the recovery of postischemic LVDP in septic rat hearts but that endogenous adenosine may provide protection from ischemia.

CYTOPROTECTION INDUCED BY GENETIC MODIFICATION OF HUMAN ENDOTHELIAL CELLS(EC) DURING COLD PRESERVATION WITH AN ADENOVIRUS VECTOR ENCODING THE ANTI-APOPTOTIC Bcl-2 GENE.

168 The vascular EC of donor organs have been shown to be primarily injured during the cold preservation and reperfusion. The integrity of the vascular endothelium in transplantation plays a critical role for the survival of the graft. EC are central to the development of immune inflammatory processes such as graft rejection and EC death has long been associated with vascular disease such as atherosclerosis. Despite the better understanding of the pathogenesis of EC damage during preservation and the subsequent ischemia-reperfusion injury, effective treatment remains elusive. In this study, we examined the possibility of genetic modification of EC to increase their resistance during the preservation time with an adenovirus vector encoding the anti-apoptotic human Bcl-2 gene. METHODS: An adenovirus vector (ΔE1) containing an expression cassette with the human Bcl-2 gene under the control of a cytomegalovirus promoter (AdCMVhBcl-2) was developed in our laboratory. An adenovirus vectors encoding an irrelevant gene (Luciferase, AdCMVLuc) and a reported gene (E coliβ-galactosidase, AdCMVLacZ) were used as a control and for transfection efficiency, respectively. Human EC (HUVECs) were maintained in culture in standard conditions, experiments were initiated when a confluent monolayer was established. For gene transfer, adenovirus vectors at different concentrations were added in sufficient volume to cover the cells, at 37°C, 48 hrs before the experiments. After gene transfer, culture medium was removed and UW solution was added and the plates were stored at 4°C for 24 and 48 hrs. Apoptosis of EC was determined by Annexia V FITC assay by FACS. Transfection rate was determined by β-galactosidase (Lac-Z) expression by FACS in AdCMVLacZ EC, expression of Bcl-2 was determined by Western Blot. The experiments were performed with 2 independent cell lines.RESULTS: The transfection efficiency of the adenovirus vector on EC at different concentrations was: 8, 22 and 99% at 10, 100 and 500 pfu, respectively. Cold preservation induced EC damage and apoptosis. The percentage of apoptotic cells after 48 hours of cold preservation was: 17% in control (non-transfected) EC, 15% in AdCMVLuc (500 pfu), 8% in AdCMVhBcl-2(100 pfu), and 2% in AdCMVhBcl-2 (500 pfu). CONCLUSIONS: In this study we showed that genetically modified human EC with the anti-apoptotic hBcl-2 gene induced a highly significant cytoprotective effect during the cold preservation time. The possibility of cytoprotection of EC by gene therapy may potentially prevent the upregulation of proinflammatory genes after transplantation, increase the survival of xenografts and the avoidance of transplant arteriosclerosis.

CYTOPROTECTION INDUCED BY GENETIC MODIFICATION OF HUMAN ENDOTHELIAL CELLS(EC) DURING COLD PRESERVATION WITH AN ADENOVIRUS VECTOR ENCODING THE ANTI-APOPTOTIC Bcl-2 GENE.

168 The vascular EC of donor organs have been shown to be primarily injured during the cold preservation and reperfusion. The integrity of the vascular endothelium in transplantation plays a critical role for the survival of the graft. EC are central to the development of immune inflammatory processes such as graft rejection and EC death has long been associated with vascular disease such as atherosclerosis. Despite the better understanding of the pathogenesis of EC damage during preservation and the subsequent ischemia-reperfusion injury, effective treatment remains elusive. In this study, we examined the possibility of genetic modification of EC to increase their resistance during the preservation time with an adenovirus vector encoding the anti-apoptotic human Bcl-2 gene. METHODS: An adenovirus vector (ΔE1) containing an expression cassette with the human Bcl-2 gene under the control of a cytomegalovirus promoter (AdCMVhBcl-2) was developed in our laboratory. An adenovirus vectors encoding an irrelevant gene (Luciferase, AdCMVLuc) and a reporter gene (E coli β-galactosidase, AdCMVLacZ) were used as a control and for transfection efficiency, respectively. Human EC (HUVECs) were maintained in culture in standard conditions, experiments were initiated when a confluent monolayer was established. For gene transfer, adenovirus vectors at different concentrations were added in sufficient volume to cover the cells, at 37 °C, 48 hrs before the experiments. After gene transfer, culture medium was removed and UW solution was added and the plates were stored at 4 °C for 24 and 48 hrs. Apoptosis of EC was determined by Annexin V FITC assay by FACS. Transfection rate was determined byβ-galactosidase (Lac-Z) expression by FACS in AdCMVLacZ EC, expression of Bcl-2 was determined by Western Blot. The experiments were performed with 2 independent cell lines. RESULTS: The transfection efficiency of the adenovirus vector on EC at different concentrations was: 8, 22 and 99% at 10, 100 and 500 pfu, respectively. Cold preservation induced EC damage and apoptosis. The percentage of apoptotic cells after 48 hours of cold preservation was: 17% in control (non-transfected) EC, 15% in AdCMVLuc (500 pfu), 8% in AdCMVhBcl-2 (100 pfu), and 2% in AdCMVhBcl-2 (500 pfu).CONCLUSIONS: In this study we showed that genetically modified human EC with the anti-apoptoic hBcl-2 gene induced a highly significant cytoprotective effect during the cold preservation time. The possibility of cytoprotection of EC by gene therapy may potentially prevent the upregulation of proinflammatory genes after transplantation, increase the survival of xenografts and the avoidance of transplant arteriosclerosis.

Ischemic preconditioning of the liver in rats: Implications of heat shock protein induction to increase tolerance of ischemia-reperfusion injury

It has been reported that ischemic preconditioning of the heart or brain has a possible relevance to heat shock protein (HSP). It is still unknown, however, whether HSP induced by means of ischemic preconditioning of the liver is a direct factor in the acquisition of tolerance to succeeding ischemia-reperfusion injury. In the present study we used ischemic preconditioning of the liver to verify the effects of induced HSP72 in the liver on the subsequent longer warm ischemia and reperfusion. Rats preconditioned with short-term (15-minute) ischemia were compared with rats preconditioned by heat exposure or with control rats. After a 48-hour recovery from the sublethal stress for preconditioning, all rats were exposed to longer (30-minute) warm ischemia and reperfusion. Forty-eight hours after ischemic preconditioning, HSP72 was clearly induced in the liver, as well as in the liver preconditioned with heat shock, but not in the kidney or heart. This ischemic preconditioning also attenuated the liver damage in the subsequent ischemiareperfusion injury, improving the restoration of hepatic function during reperfusion and resulting in higher postischemic rat survival. According to the proposed model of tolerance acquisition for ischemia-reperfusion injury by stress preconditioning, these observations support the speculation that the induced HSP72 plays some beneficial role in this protection mechanism.

Molecular adaptation of cellular defences following preconditioning of the heart by repeated ischaemia

The molecular mechanism of preconditioning the heart by repeated ischaemia was investigated by examining the expression of stress related and antioxidative genes and proteins. Isolated buffer-perfused rat hearts were made ischaemic for 5 min followed by 10 min of reperfusion (1 x PC). Another group of experiments was performed by repeating the ischaemia/reperfusion episode four times (4 x PC). Both 1 x PC and 4 x PC groups were reperfused for 60 min. Control experiments were performed by perfusing the hearts with buffer using the same time frame, but without ischaemia/reperfusion. The induction of the expression of two oncogenes (c-fos and c-myc), three heat shock protein (HSP) genes (HSP 27, HSP 70, and HSP 89 mRNAs) and two antioxidative enzyme genes (Mn-superoxide dismutase (SOD) and catalase mRNAs) by northern hybridisation, as well as the activities of four major antioxidative enzymes (SOD, catalase, glutathione peroxidase, and glutathione reductase) were examined in control and 1 x PC and 4 x PC hearts. In addition, 2-D gel electrophoresis of the proteins in all groups was performed to examine the induction of any new protein. Myocardial preservation was studied by examining left ventricular functions. Northern hybridisation detected enhanced level of c-fos 2.2 kb mRNA and c-myc 2.4 kb mRNA in 4 x PC hearts after 60 min of reperfusion, induction being stronger for c-fos. 1 x PC hearts showed minimal expression of c-fos mRNA, but not c-myc mRNA. 4 x PC hearts also showed the induction of HSP 27, HSP 70, and HSP 89 mRNAs as well as catalase and Mn-SOD mRNAs, whereas 1 x PC hearts showed some induction of HSP 70 mRNA and catalase mRNA only. 2-D gel electrophoresis revealed the expression of 15-20 new proteins in 4 x PC hearts only. The activities of three major antioxidative enzymes, Mn-SOD, peroxisomal catalase, and glutathione peroxidase, but not Cu/Zn-SOD, cytosolic catalase, and glutathione reductase, were enhanced after 60 min of reperfusion in 4 x PC hearts only compared to both 1 x PC and baseline values. 4 x PC hearts, but not 1 x PC hearts, showed reduction of subsequent myocardial ischaemic and reperfusion injury. Repeated ischaemia and reperfusion (4 x PC), and not 1 x PC, caused the induction of several stress related and antioxidant genes including HSP 27, 70, and 89 genes, Mn-SOD and catalase genes, and proto-oncogenes (c-fos and c-myc), as well as the expression of several stress inducible proteins and stimulation of three antioxidative enzymes. Mn-SOD, peroxisomal catalase, and glutathione peroxidase, simultaneously protecting the heart from subsequent ischaemia-reperfusion injury. The expression of stress inducible and antioxidant genes and stimulation of antioxidant enzyme activities may reflect the heart's response enabling it to survive against ischaemic stress by eliminating the oxidative assault.