Subsequent Ischemia-reperfusion Injury Research Articles

Pharmacological Preconditioning of Rat Liver by Up-Regulation of Heme Oxygenase 1

Purpose We investigated whether pharmacologically induced up-regulation of heme oxygenase 1 by pyrrolidine dithiocarbamate (PDTC) conferred protection against subsequent ischemia-reperfusion injury (IRI) to the rat liver after temporary vascular occlusion of 70% of the organ. Methods Female Wistar rats (200 to 250 g body weight) anesthetized with pentobarbitone were cannulated in the carotid artery and jugular vein. After laparotomy, a rubber band was applied around the entire vascular supply to the median and left lateral lobes, enabling vascular occlusion of 70% of the liver. A laser Doppler miniprobe was placed on the left lateral lobe to monitor peripheral liver blood flow (PLBF). Immediately upon completion of the surgery, the rats were administered either PDTC (50 mg/kg intravenously; n = 8) or its solvent (isotonic NaCl; n = 8). After 60 minutes, regional ischemia was induced for 30 minutes. The animals were then monitored for 2 hours of reperfusion. Blood samples for alanine transferase (ALT) estimation (as a measure of parenchymal injury) were drawn immediately prior to ischemia and reperfusion, as well as 60 and 120 minutes after reperfusion; PLBF was calculated at these times. Results ALT increased in the course of the experiments but there was no difference between the groups. The reduction in PBLF due to ischemia-reperfusion was significantly lower in the PDTC group: about 16% versus 40%, after 2 hours of reperfusion. Conclusion Pretreatment with PDTC attenuated the disturbance of hepatic microcirculation, but not parenchymal injury, in the early phase of IRI.

Interstitial Lactic Acidosis in the Graft During Organ Harvest, Cold Storage, and Reperfusion of Human Liver Allografts Predicts Subsequent Ischemia Reperfusion Injury

The impact of the process of liver transplantation on glucose metabolism in the graft was studied using microdialysis. Microdialysis catheters were inserted into 15 human livers to monitor metabolic changes that took place during organ harvest, the process of backtable preparation of the graft, and following implantation in the recipient where it remained in situ for 48 hours. The cannula was perfused with isotonic solution and hourly samples of perfusate were collected and analyzed. Six livers showed serum biochemical evidence of ischemia/reperfusion (IR) injury with 24 hours aspartate transaminase (AST) levels >2000 IU/L (Group A) whereas the remaining patients showed little evidence of IR injury (Group B). In Group A, lactate levels in the donor microdialysate rose to >6 mM (P < 0.05), were significantly higher during backtable preparation of the liver (>15 mM; P < 0.03), and took longer to normalize in the recipient following implantation (18 vs. 8 hours, P < 0.03) than lactate levels of the livers of patients in Group B who did not develop ischemia reperfusion injury. No significant differences were observed in glucose, pyruvate, or glycerol concentrations between the two groups. Interstitial lactic acidosis in the donor allograft is associated with significant reperfusion injury on implantation.

Uncoupling Proteins 2 and 3 Function in Concert to Augment Tolerance to Cardiac Ischemia

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.

Pharmacological preconditioning with low-dose FK506 reduces subsequent ischemia-reperfusion injury in rabbit allograft knee joint

Objective To explore the effect of pharmacological preconditioning wtith low-dose FK506 reducing subsequent ischemia-reperfusion injury in rabbit knee joint and to explore the possible mechanism ofthat. Methods Twenty four animal model of allograft heterootopic knee joint were established and divided randonly into three groups： IR group （lschemia-reperfusion group）, FK506 group （FK506 preconditioning group） and ST group（ Sham control group）. Vessels were clamped for 1 hour, and then grafts received reperfusion. Tissues（for example muscles tissues） of pro-ischemia and reperfusion after 12 hours were obtained,and they were observed by microscope and electron microscope and were analysised with reverse transcription polymerase chain reaction（RT-PCR）, Western Blot, DNA ladder, flow cytometer. Moreover, the contents of MDA, NO and SOD in these tissues were examined by spectrophotometer. Results As a result, we observed disoranized tissues and swollen cells in IR groups but trenchant tissues and intact cells in FK506 groups by microscope, vacuolated and tumid mitochondrion in IR groups but non-vacuolated and intact that in FKS06groups. With FK506 preconidtioning, the contents MDA and NO were distinctly reduced but significant raisedin FK506 group. Compared with IR group, DNA ladder of FK506 group were weakly, and apoptotic ractes reduce from 20% to 10% and the expression of TNF, IL-1, Fas and FasL mRNA obviously down-regulated. At the same time, expression of HSP70 significantly up-regulated on protein and mRNA levels with RT-PCR andWestern blotting analysis in FK506 group. Conclusion Pharmacological preconditioning with low-dose FK506 （0. 3mg/kg） can induce tolerance of ischemia-reperfusion injury in rabbit allograft knee joint. Over-expression of HSP70 may be key factor of that. Key words: Allograft; Knee joint Ischemia-reperfusion injury; Heat shock protein 70; Taerolimus（FK506）

Preconditioned Hyperbaric Oxygenation Protects the Liver against Ischemia-Reperfusion Injury in Rats

Hyperbaric oxygen (HBO) therapy is an effective adjunct in treating ischemia-reperfusion (I/R) injury of brain, small intestine, testis, and crushing extremities. This study was designed to test the hypotheses that preconditioning the rats with HBO could protect the liver against subsequent I/R injury. Daily treatment with one-dose HBO (90 min, 2.5 ATA) was brought about for male Sprague Dawley rats for 1 to 3 days before an I/R injury of liver. Hepatic expression of heat-shock protein 70 (Hsp70), total concentration of glutathione (GSH), activity of catalase, superoxide dismutase (SOD), and serum AST and ALT were estimated before and after HBO, as well as after I/R injury. The results showed that activity of hepatic catalase was decreased by one dose, but not three doses, of HBO as compared with baseline data. However, hepatic Hsp70 expression fluctuated insignificantly. AST and ALT increase less in rats preconditioned with one-dose HBO as compared with those without HBO or with three-dose HBO. Our results showed preconditioning by one-dose HBO protects rat liver against subsequent ischemia-reperfusion injury.

Role of Poly-(ADP-Ribose) Polymerase in Transplant Acute Tubular Necrosis and Its Relationship With Delayed Renal Function

The enzyme poly(ADP-ribose) polymerase (PARP-1) participates in the repair of DNA damaged by genotoxic agents such as oxygen-derived free radicals. If the allograft suffers pretransplant cold ischemia and subsequent ischemia–reperfusion injury (IR), overactivation of PARP-1 can be induced, which may lead to an increase in acute tubular necrosis (ATN) and a delay in total recovery of renal function (RRF) of the transplanted organ. We studied the nuclear expression of PARP-1 in tubular cells by immunohistochemistry with the monoclonal antibody PAR01 in 104 kidney transplant biopsies from allografts with ATN. In 50% of biopsies with ATN, >50% of tubular nuclei were PARP-1 +; only 9.6% of biopsies were negative. The increase in the immunohistochemical expression of PARP-1 showed a statistically significant relationship with the duration of cold ischemia, with serum creatinine levels, and with the time required to achieve effective diuresis ( P < .0001, Spearman test). Cold ischemia of >24 hours and serum creatinine levels >1.7 mg/dL showed a statistically significant relationship with the highest PARP-1 expression levels (2.83 ± 0.4 vs 1.36 ± 0.8, P < .0001, Mann–Whitney U test). We conclude that PARP-1 plays an important role in ATN and RRF and is related to the extent and severity of ATN and to the renal allograft function.

Preconditioning with 1,25-Dihydroxyvitamin D3 Protects against Subsequent Ischemia-Reperfusion Injury in the Rat Kidney

Background/Aim: Induction of heat shock protein 70 (HSP70) is important in the tolerance of subsequent ischemia-reperfusion (I/R) injury. The aim of this study was to evaluate the effect of HSP70 induction by 1,25-dihydroxyvitamin D₃ (VD3) on subsequent I/R injury in rats. Methods: HSP70 was induced in Sprague-Dawley rats by VD3 treatment for 7 days, and the effect of VD3 pretreatment on subsequent I/R injury was evaluated in terms of renal function, tubular necrosis score, tumor necrosis factor alpha mRNA expression, mitogen-activated protein kinase expression, and proliferating cell nuclear antigen expression. Results: VD3 treatment increased HSP70 expression which was localized to renal tubular cells in the outer medulla. Pretreatment with VD3 before I/R injury resulted in (1) decreased blood urea nitrogen and serum creatinine levels; (2) decreased tubular cell necrosis; (3) increased tubular cell proliferation as determined by proliferating cell nuclear antigen expression; (4) decreased tumor necrosis factor alpha mRNA expression, and (5) increased extracellular signal regulated protein kinase and decreased c-Jun N-terminal kinase expression. Conclusion: Our study demonstrates that VD3 is a nontoxic inducer of HSP70 and exerts a protective effect against subsequent I/R injury.

Correlation of morphological findings with functional reserve in the aging donor: role of the poly (ADP-ribose) polymerase

IntroductionThe enzyme poly (ADP-ribose) polymerase (PARP-1) participates in the first events of DNA repair in higher organisms. Under conditions of tissue ischemia, this action can lead to significant decreases in NAD(+), massive adenosine triphosphate (ATP) depletion, and cell death. In renal grafts with pretransplantation cold ischemia and subsequent ischemia-reperfusion injury, overactivation of PARP-1 may lead to a higher index of acute tubular necrosis, a delay in total recovery of the function of the transplanted organ, and an early progression to chronic graft nephropathy. The present study examined whether increased tubular expression of PARP-1 in kidneys from aged donors contributed to recipient renal function. Material and methodWe studied the nuclear expression of PARP-1 using immunohistochemistry with monoclonal antibody PAR01 in 75 kidney biopsy specimens from 40 aged donors. ResultsImmunohistochemical expression of PARP-1 showed a statistically significant relationship with donor age (r = .408, P = .006, Spearman test), with time required to achieve effective diuresis (r = .386, P = .01, Spearman test) and with creatinine levels in the first 3 months. We also highlighted a greater intensity of PARP-1 expression in suboptimal donor kidneys that failed to reduce the serum creatinine levels to <1.7 mg/dL (creatinine <1.7 PARP: 1.29 ± 1.49 vs creatinine >1.7 PARP: 2.29 ± 1.33, P = .047, Mann-Whitney U test). ConclusionWe conclude that the determination of PARP-1 in biopsy specimens from aged donors may be a useful predictive factor for renal graft function.

Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney.

Improving the ability of the kidney to tolerate ischemic injury has important implications. We investigated the effect of recombinant human erythropoietin (rHuEPO) treatment on subsequent ischemia/reperfusion (I/R) injury and evaluated the role of heat shock protein (HSP) 70 in rHuEPO-induced renal protection. rHuEPO (3000 U/kg) was administered 24 h before I/R injury, and rats were killed at 24, 48, and 72 h after I/R injury. Pretreatment of rHuEPO resulted in the following: i) decreased serum creatinine level; ii) decreased tubular cell apoptosis and necrosis, measured by DNA fragmentation analysis and TUNEL staining and histomorphological criteria; iii) decreased tubular cell proliferation as determined by proliferating cell nuclear antigen expression; iv) increased bcl-2 protein and decreased caspase 3 activity; and v) decreased JNK expression. rHuEPO treatment increased HSP70 expression in a dose-dependent manner in normal rat kidneys, and inhibition of HSP70 expression by quercetin eliminated the renoprotective effect of rHuEPO in ischemic kidneys. Our study demonstrates that rHuEPO has a protective effect on subsequent I/R injury and that this effect is associated with induction of HSP70. Our study provides a new avenue for therapy to prevent renal damage after I/R injury.

Clinically applicable thermal preconditioning attenuates leukocyte-endothelial interactions.

We have previously demonstrated that clinically applicable thermal preconditioning induces heat shock protein 72 (HSP72) and protects against a subsequent ischemia-reperfusion (I/R) injury in an animal model. A core component of I/R injuries is the interaction between activated leukocytes and endothelial cells. We hypothesized that the effects of clinically applicable thermal preconditioning are mediated through attenuation of this leukocyte-endothelial (L-E) interaction. Twenty-one male Sprague Dawley rats were divided into control, I/R, and preconditioning plus I/R groups. Preconditioning was done under general anesthesia and the animals' temperature raised by 1 degrees C for 15 minutes in a water bath. This was repeated once a day for 5 successive days. I/R injury was caused by occlusion of the superior mesenteric artery for 10 minutes followed by 1 hour of reperfusion. L-E interactions were analyzed using intravital microscopy of a mesenteric vessel in vivo. L-E interactions were determined using leukocyte velocity (which decreases as cells interact), and number of adherent and migrated leukocytes. HSP72 was assessed by Western blot. Ischemia-reperfusion caused a decrease in leukocyte rolling velocity at all timepoints (p < 0.05 versus controls). Preconditioning attenuated the effects of I/R, and leukocyte rolling velocity was significantly improved versus I/R (p < 0.05) to levels similar to those in controls. Similarly, the number of adherent and migrating leukocytes increased significantly (p < 0.05) after I/R versus control at all time points, and preconditioning attenuated these to control levels, (p < 0.05 versus I/R) at both the 30- and 60-minute postischemia time points. Upregulation of HSP72 was demonstrated on Western blot. These results demonstrate that the benefit of clinically applicable thermal preconditioning is at least partially because of an immunomodulatory role in attenuating leukocyte-endothelial interactions associated with an increased expression of HSP 72.

Preconditioned somatothermal stimulation on median nerve territory increases myocardial heat shock protein 70 and protects rat hearts against ischemia-reperfusion injury

Objective: This study was designed to test the hypotheses that local somatothermal stimulation on the left median nerve territory increases myocardial heat shock protein 70 and that preconditioning of rats with local somatothermal stimulation protects the hearts against subsequent ischemia-reperfusion injury. Methods: Local somatothermal stimulation was brought about by means of application of a heating rod over and above the left median nerve territory (1.5 cm proximal to the palm crease) in male Sprague-Dawley rats. After rats were treated with local somatothermal stimulation, the gene expression of heat shock protein 70 in regional muscle, heart, and liver was assessed by means of Western blotting and reverse transcription—polymerase chain reaction. In addition, durations of arrhythmia, mortality rates, and mitochondrial functions were compared between groups preconditioned with or without local somatothermal stimulation followed by subsequent myocardial ischemia-reperfusion injury. Results: The results showed that the gene expression of heat shock protein 70 was upregulated in the muscle beneath the area of local somatothermal stimulation, as well as in the heart, although not in the liver. When animals were preconditioned with local somatothermal stimulation on the left median nerve territory followed by subsequent ischemia-reperfusion injury of the heart, there were significant decreases of creatine kinase level from the heart, duration of arrhythmia, mortality rate, and improved mitochondrial respiratory function compared with that seen in those without local somatothermal stimulation preconditioning. Conclusion: We conclude that local heat stress preconditioning on the left median nerve territory has a potential cardioprotective effect against subsequent ischemia-reperfusion injury.J Thorac Cardiovasc Surg 2003;125:678-85

Pharmacological hepatic preconditioning: involvement of 70-kDa heat shock proteins (HSP72 and HSP73) in ischaemic tolerance after intravenous administration of doxorubicin.

Pharmacological preconditioning may induce a stress response which protects liver against ischaemia-reperfusion injury (IRI). The aim of this study was to determine, in an animal model, whether intravenous administration of doxorubicin induces heat shock proteins (HSPs) in liver tissue and facilitates liver tolerance to subsequent warm IRI. Male Wistar rats were used. Production of HSPs was determined in liver tissue sequentially after the injection of doxorubicin 1 mg/kg body-weight. Acquisition of tolerance for 30 min warm ischaemia and reperfusion of the liver was determined in animals pretreated (48 h beforehand) with doxorubicin, and in controls. Biochemical liver function and liver adenine nucleotide concentration 40 min after reperfusion and survival rate at 7 days after the ischaemic insult were recorded. Expression of HSP72 and HSP73 in the liver was confirmed 48 h after doxorubicin administration. Biochemical parameters and survival rates were significantly better in pretreated animals than in controls. These results indicate that doxorubicin has the potential to provide the liver with tolerance against IRI. A simultaneous increase of both HSP72 and HSP73 in liver tissue may explain the acquisition of tolerance following the administration of doxorubicin.

Hepatic ischemic preconditioning in mice is associated with activation of NF-κB, p38 kinase, and cell cycle entry

A brief period of hepatic ischemia protects the liver against subsequent ischemia-reperfusion (IR) injury, but the mechanism of such preconditioning is poorly understood. We examined whether preconditioning activated nuclear factor kappa B (NF-κB), the stress-activated protein kinases (SAPK), c-Jun N-terminal kinase-1 (JNK-1) and p38, and entry into the cell cycle. We used a murine model of partial hepatic ischemia. Preconditioning was performed by clamping the vasculature for 2 to 20 minutes, and allowing reperfusion for 10 minutes before 90-minute ischemia or IR. As assessed by serum alanine aminotransferase (ALT) levels and liver histology, preconditioning periods of 5 and 10 minutes were highly protective against IR injury, whereas 2-, 15-, and 20-minute intervals were ineffective. Preconditioning was associated with entry of hepatocytes into the cell cycle within 2 hours of subsequent IR, as indicated by proliferating cell nuclear antigen (PCNA) nuclear staining, induction of cyclin D1 and numerous mitotic figures; in the absence of preconditioning, such changes were not seen until 24 hours. Preconditioning increased nuclear binding of NF-κB within 30 minutes of the subsequent ischemic interval, paralleled by degradation of inhibitory (binding) protein for NF-κB (IκBα). Ischemic preconditioning also activated p38 kinase and JNK-1, which are known to converge on cyclin D1 regulation. The protective effect of the preconditioning regimen was more closely associated with p38 kinase than JNK-1 activation. In conclusion, the hepatoprotective effects of ischemic preconditioning are associated with activation of NF-κB and SAPKs that are associated with entry of hepatocytes into the cell cycle, a critical biological effect that favors survival of the liver against ischemic and IR injury. (H EPATOLOGY 2002;36:94-102.)

Preconditioning Somatothermal Stimulation on Right Seventh Intercostal Nerve Territory Increases Hepatic Heat Shock Protein 70 and Protects the Liver from Ischemia–Reperfusion Injury in Rats

Hyperthermic preconditioning attenuates the heat-induced cellular response to a subsequent severe heat challenge. However, it is impractical to perform whole-body hyperthermia in humans. This study was designed to test the hypotheses that hepatic heat shock protein 70 (Hsp70) could be induced by local somatothermal stimulation (LSTS) on right seventh intercostal nerve territory and that preconditioning the rats with LSTS protects the liver from subsequent ischemia–reperfusion injury. LSTS was brought about by application of a heating rod above right seventh intercostal nerve territory in male Sprague–Dawley rats. Hepatic gene expression of Hsp70 was assessed by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Finally, serum ALT and AST and the lipid peroxidation product malondialdehyde (MDA) were evaluated in ischemic–reperfused rats preconditioned by application of LSTS on right seventh intercostal nerve territory. The results showed that hepatic gene expression of Hsp70 was upregulated in rats treated with LSTS. When animals were preconditioned with LSTS, followed by subsequent ischemia–reperfusion injury of the liver, there were significant decreases in liver enzymes (ALT/AST) and MDA formation in rats pretreated with one dose of LSTS (LSTS-1 group) as compared with those not treated with LSTS (control group) or treated with three doses of LSTS (LSTS-3 group). We conclude that mild local heat stress (one dose) on right seventh intercostal nerve territory upregulates hepatic gene expression of Hsp70 and protects the liver from subsequent ischemia–reperfusion injury. This might provide an easily applicable method for those patients facing ischemia–reperfusion challenge of the liver, as in liver resection and liver transplantation.

Hyperbaric oxygenation pretreatment induces catalase and reduces infarct size in ischemic rat myocardium.

Ischemia-reperfusion injury is a major complication occurring in heart stroke, cardiopulmonary bypass surgeries, and heart transplantation. Reactive oxygen species generated during the reperfusion phase overwhelm the scavenging capacities of antioxidant enzymes, and result in oxidative damage to the myocardium. We examined whether hyperbaric oxygenation (HBO) pretreatment induces antioxidant enzymes and protects the heart from subsequent ischemia-reperfusion injury. Rats were intermittently exposed to 100% O2 at 3 ATA (where ATA is absolute atmosphere) for 1 h daily and then sacrificed after 24 h of recovery in room air. Isolated hearts were subjected to 40 min of ischemia and 90 min of reperfusion. HBO pretreatment was found to condition the heart and enhance enzymatic activity and gene expression of catalase, thereby significantly reducing infarct size after reperfusion. A catalase inhibitor, 3-amino-1,2,4-triazole, completely abolished the infarct-limiting effect of HBO pretreatment, which suggests that HBO-induced tolerance against ischemia-reperfusion injury is due to catalase induction. Our results imply that HBO preconditioning may be developed as a new preventive measure for reperfusion injury in the heart.

Adenovirus-mediated gene transfer of human interleukin 10 ameliorates reperfusion injury of rat lung isografts.

The objective of this study was to examine the feasibility of human interleukin 10 gene transfer into rat lung isografts and to investigate the effect of gene transfer on subsequent ischemia-reperfusion injury. Male F344 rats were divided into 4 groups and underwent left lung isotransplantation. Twenty-four hours before harvest, 5 x 10E9 pfu (group I, n = 6) or 1 x 10E10 pfu (group II, n = 7) of AdRSVhIL-10 was intravenously administered to donor rats. In group I-C (n = 6) and group II-C (n = 6), serving as controls, 5 x 10E9 pfu and 1 x 10E10 pfu of AdCMVLacZ were administered, respectively. Grafts were preserved for 18 hours at 4 degrees C before implantation and assessed 24 hours after reperfusion. Transgene expression of human interleukin 10 was assessed by both reverse transcriptase-polymerase chain reaction and immunohistochemistry. Graft inducible nitric oxide synthase, tumor necrosis factor alpha, intercellular adhesion molecule-1, growth-regulated gene product/cytokine-induced neutrophil chemoattractant-1, and monocyte chemotactic protein-1 mRNA expression were assessed by reverse transcriptase-polymerase chain reaction. Isograft gas exchange, exhaled nitric oxide, and myeloperoxidase activity were also analyzed. Dose-dependent transgene expression was detected by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Arterial PO (2) in groups I (164.72 +/- 85.3 mm Hg) and II (153.19 +/- 113 mm Hg) was significantly higher than in groups I-C (82.37 +/- 19.1 mm Hg) and II-C (77.95 +/- 33.4 mm Hg) (P =.022 and P =.031, respectively). Arterial PCO (2) in group I (33.40 +/- 6.80 mm Hg) was significantly lower than in group I-C (51.23 +/- 11.9 mm Hg) (P =.0096). Myeloperoxidase activity in group II (0.083 +/- 0.031 DeltaOD. min(-1). mg(-1)) was significantly lower than in group II-C (0.117 +/- 0.028 DeltaOD. min(-1). mg(-1)) (P =.044). The inducible nitric oxide synthase mRNA expression in group II (0.627 +/- 0.28) was significantly lower than in group II-C (1.125 +/- 0.63) (P =. 039). Adenovirus-mediated human interleukin 10 gene transfer in vivo into lung isografts ameliorates subsequent ischemia-reperfusion injury. This results in improved graft gas exchange, reduced neutrophil sequestration, and down-regulation of graft inducible nitric oxide synthase mRNA expression.

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.