Inhibition of intercellular adhesion molecule-1 with antisense deoxynucleotides prolongs renal isograft survival in the rat

BACKGROUND: Delayed graft function from ischemia-reperfusion injury has a negative impact on long-term renal graft survival. We tested the utility of antisense oligodeoxynucleotide (ODN) against intercellular adhesion molecule-1 (ICAM-1) in the pretransplant treatment of renal isografts in improving long-term graft survival. METHODS: Three groups of 16 inbred Lewis rats each underwent unilateral nephrectomy and were then transplanted with a kidney from a Lewis donor rat, which had received antisense ODN, reverse sense ODN, or saline vehicle six hours prior to nephrectomy. The kidneys were subjected to one hour of warm ischemia and 30 minutes of cold ischemia, which when untreated results in delayed graft function. The remaining native kidney was removed 10 days later. Serum creatinine and urinary protein excretion were measured in surviving rats at weeks 2, 4, 6, 8, 12, 16, and 20 after native nephrectomy. RESULTS: A Kaplan-Meier analysis revealed that by week 6 one half of the animals receiving reverse sense ODN and saline vehicle treatment had died, while all but 2 rats in the antisense ODN-treatment group survived to 20 weeks. Serum creatinine concentrations and urine protein excretion of surviving reverse sense and saline vehicle-treated rats were significantly higher than antisense treated rats at every time point. Histology at week 20 revealed marked interstitial fibrosis, focal glomerular sclerosis, vascular intimal and medial thickening and tubular atrophy in reverse sense and saline vehicle-treated kidneys, while antisense ODN-treated kidneys showed only modest changes. Immunohistochemistry showed macrophage and lymphocyte infiltration, as well as substantial up-regulation of MHC class II, in reverse sense and saline vehicle-treated kidneys compared to antisense ODN-treated kidneys. CONCLUSIONS: These results suggest that by ameliorating acute nonimmunological renal isograft injury, the long-term chronic nonimmunologic processes are improved as well. Furthermore, the data suggest that an antisense ODN strategy directed against ICAM-1 may have utility in human kidney transplantation.     

In vivo cyclin E expression as a marker for early cervical neoplasia

BACKGROUND: Heat shock has been associated with the acquisition of tolerance to a wide variety of stressful conditions, including ischemia. This is partly mediated by the production of various heat shock proteins (HSP), including HSP70. One novel approach to the reduction of ischemia-reperfusion injury after lung transplantation is the induction of HSP70 by heat pretreatment of the donor. The purpose of this study was to investigate the feasibility of this approach in an animal model of lung transplantation. METHODS: Animals were divided into six main groups, with groups I to III representing transplanted animals: In groups I and II, donor animals were anesthetized and then underwent heat stress 6 and 12 hours before organ harvest, respectively. Control animals underwent general anesthesia but no heat stress. After harvest, left lungs from groups I to III were preserved for 18 hours at 40 degrees C and then implanted into isogeneic recipients, which were killed 24 hours after reperfusion to assess graft function. Group IV and V animals underwent heat stress followed by a recovery period of 6 and 12 hours, respectively. Lungs were collected both at the time of harvest (right lungs) and after 18 hours of cold preservation (left lungs). Group VI served as nontransplanted controls. Groups IV to VI did not undergo lung transplantation. RESULTS: At the time of harvest but before implantation, HSP70 was significantly increased in heat-shocked nontransplanted donor lungs (groups IV and V) compared with group VI controls. After 18 hours of cold preservation, HSP70 levels were higher in group IV compared with group V and group VI controls. At 24 hours after reperfusion, mean arterial oxygenation was significantly higher in group I compared with group II and group III controls (290.25+/-24.5 vs 154.5+/-23.9 and 119.6+/-11.3 mm Hg, respectively; P < .001). Myeloperoxidase activity was improved in group I compared with group III controls (0.048+/-0.018 vs 0.137+/-0.036 deltaOD/mg/min, respectively; P < .05). The wet/dry weight ratio was also improved in group I compared with group III controls (6.2+/-0.3 vs. 7.8+/-0.4, respectively; P < .05). CONCLUSIONS: Heat pretreatment of the donor 6 hours before harvest results in increased synthesis of HSP70, which offers a dramatic protective effect against subsequent ischemia-reperfusion injury in the lung isograft.     

The role of xanthine dehydrogenase (xanthine oxidase) in ischemia-reperfusion injury in rat kidney

Xanthine dehydrogenase (XDH) and xanthine oxidase (XO) are enzymes involved in the metabolism of purines in various organisms. XO produces superoxide radicals, suggesting that is responsible for tissue ischemia-reperfusion injury. To test this notion further studies were performed on rat kidneys and the time course of changes in purine nucleotides, oxypurines and XDH and XO activity was determined. At 24 hours after reperfusion subsequent to 30-minute ischemia, serum creatinine increased to 0.83 +/- 0.74 mg/dl from 0.28 +/- 0.06 mg/dl (the level prior to ischemia, the control). Renal ATP and ADP contents were reduced after ischemia lasting for 30 minutes and restored 10 minutes after reperfusion following 30 minutes of ischemia. The renal AMP content increased after 30 minutes of ischemia and recovered within 10 minutes after reperfusion. The total adenine nucleotide (TAN) content was reduced gradually during ischemia-reperfusion in the rat kidney. Although the energy charge was reduced following 30 minutes of ischemia, it was restored to the control level 10 minutes following reperfusion. Hypoxanthine (HX) and xanthine (X), which had accumulated at 30 minutes after ischemia, were reduced to the control levels 10 minutes after reperfusion. There were no significant changes in the pre-ischemia values of total XDH and XO activities or XDH/XO ratio during the period nor at various time intervals (up to 24 hours) during reperfusion. It was shown that HX and X accumulate without significant conversion of XDH to XO during ischemia. Therefore the putative role of XO in ischemia-reperfusion injury seems to more complex than initially predicted.     

Metabolic factors have a major impact on kidney allograft survival

BACKGROUND: At the present time, late graft loss is the major cause of kidney failure after transplantation. However, the influence of metabolic factors on this process is ill-defined. METHODS: To identify the impact of lipid metabolism, glucose metabolism, and blood pressure and their prognostic value for graft survival, data for all recipients of a kidney allograft with a potential graft survival of >15 years and a minimum graft survival of 1 month were analyzed retrospectively. Recipients of kidney grafts functioning more than 15 years (n=32) were compared with those with a graft function of less than 10 years (n=152, controls) and evaluated in a multivariate analysis. RESULTS: Low levels of serum cholesterol, triglycerides, and glucose, before and after transplantation, were accompanied by a prolonged graft survival. Prognostic factors for early graft failure included serum triglycerides >300 mg/dl, cholesterol >250 mg/dl before transplantation, serum creatinine >4.0 mg/dl 1 month after transplantation, and donor age above 45 or less than 10 years. Additionally, systolic and, particularly, diastolic blood pressure was lower in the group with a prolonged graft function as compared with controls immediately before and after transplantation. In addition, the incidence of primary graft function was lower and the incidence of acute rejection episodes higher in controls. Cold and warm ischemic time, body mass index, recipient age, and gender did not differ significantly. CONCLUSIONS: Our data suggest that metabolic parameters play an important role in the process of late graft loss after kidney transplantation.     

Locally applied antisense oligonucleotide to proliferating cell nuclear antigen inhibits intimal thickening in experimental vein grafts

This study examines the effect of antisense oligonucleotide to proliferating cell nuclear antigen (PCNA) on the formation of vein graft intimal hyperplasia in vivo, using localized administration. Twenty-four New Zealand white rabbits had a right carotid interposition bypass graft using the external jugular vein and were sacrificed on the 28th postoperative day. To determine the effect of PCNA on the development of intimal hyperplasia, 6 animals had their grafts coated with a pluronic gel containing 18 base antisense oligonucleotide to PCNA (1 mg/ml), 6 received a pluronic gel containing an 18 base nonsense oligonucleotide (1 mg/ml), and 12 animals were controls (6 with and 6 without pluronic gel). These grafts were harvested for morphology and videomorphometry. There was no change in the intimal thickness between the control and gel-treated groups. (70 +/- 4 microm versus 72 +/- 4 microm; mean +/- s.e.m.; p = ns). The presence of nonsense oligonucleotide had no further effect. Antisense PCNA produced a 26% decrease in intimal thickness to 50 +/- 4 microm in the treated vein grafts (p < 0.03) without a change in medial thickness. This study shows that a local single application of antisense oligonucleotide to PCNA will reduce the intimal hyperplasia in experimental vein grafts over 28 days.     

A comparison of the efficacy and tolerability of dorzolamide and acetazolamide as adjunctive therapy to timolol. Oral to Topical CAI Study Group

BACKGROUND: Recent observations provide evidence that complement is involved in the pathophysiology of ischemia/reperfusion injury. In this study, we assessed the impact of complement inhibition on hepatic microcirculation and graft function using a rat model of liver transplantation. METHODS: Arterialized orthotopic liver transplantation was performed in Lewis rats after cold preservation (University of Wisconsin solution, 4 degrees C, 24 h). Eight animals received the physiological complement regulator soluble complement receptor type 1 (sCR1) intravenously 1 min before reperfusion. Controls received Ringer's solution (n=8). Microvascular perfusion, leukocyte adhesion, and Kupffer cell phagocytic activity were studied 30-100 min after reperfusion by in vivo microscopy. RESULTS: Microvascular perfusion in hepatic sinusoids was improved in the sCR1 group (87+/-0.7% vs. 50+/-1%; P < 0.001). The number of adherent leukocytes was reduced in sinusoids (68.3+/-4.7 vs. 334.1+/-15.8 [adherent leukocytes per mm < or = liver surface]; P < 0.001) and in postsinusoidal venules after sCR1 treatment (306.6+/-21.8 vs. 931.6+/-55.9 [adherent leukocytes per mm < or = endothelial surface]; P < 0.001). Kupffer cell phagocytic activity was decreased in the sCR1 group compared to controls. Postischemic bile production reflecting hepatocellular function was increased by almost 200% (P = 0.004) after complement inhibition. Plasmatic liver enzyme activity was decreased significantly upon sCR1 treatment, indicating reduced parenchymal cell injury. CONCLUSIONS: Our results provide further evidence that the complement system plays a decisive role in hepatic ischemia/reperfusion injury. We conclude that complement inhibition by sCR1 represents an effective treatment to prevent reperfusion injury in liver transplantation.     

Localization of Six4/AREC3 in the developing mouse retina; implications in mammalian retinal development

BACKGROUND: We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125). METHODS: IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting. RESULTS: IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days). CONCLUSIONS: Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.     

Donor treatment with the lazeroid U74389G reduces ischemia-reperfusion injury in a rat lung transplant model

BACKGROUND: Antioxidant treatment with lazeroids has proven beneficial for the amelioration of reperfusion injury in experimental lung transplantation. This study compares the effect of donor versus recipient treatment on immediate postoperative graft function. METHODS: A model of acute double-lung transplantation in rats was used to assess graft function. Transplanted controls after 2 (group I) and 16 hours of ischemia (group II) were compared to a recipient (group III; 16-hour ischemia) and a donor treatment group (group IV; 16-hour ischemia) using the lazeroid U74389G (6 mg/kg). Serial assessment of alveolar-arterial oxygen difference, dynamic lung compliance, airway and pulmonary vascular resistance was obtained during a 2-hour reperfusion period. Final analysis included survival, weight gain, and histologic examination. RESULTS: Graft function was significantly better after 2 hours of ischemia than in any of the three 16-hour ischemia groups (II, III, IV). After 16 hours of ischemia, donor treatment provided superior graft function with respect to dynamic lung compliance, airway resistance, and alveolar-arterial oxygen difference when compared with groups II and III. The pulmonary vascular resistance was significantly higher in group III when compared with groups II and IV. Graft weight increase reflecting edema was highest in groups III (104%) and II (98%). CONCLUSIONS: After prolonged ischemia only donor treatment with the lazeroid U74389G was able to significantly reduce ischemia-reperfusion-related graft dysfunction.     

Effects of NMDA-R1 antisense oligodeoxynucleotide administration: behavioral and radioligand binding studies

The effects of an antisense phosphodiester oligodeoxynucleotide (ODN) directed to the NR1 subunit of the NMDA receptor mRNA and of its corresponding sense ODN were investigated in mice. Treatment with the antisense ODN significantly increased the time mice spent in the open arms of an elevated maze while the total number of arm entries was unaltered. Furthermore, seizure latencies after the administration of an ED100 dose of NMDA (150 mg/kg) were significantly higher in antisense treated animals compared to vehicle controls. At the same time, treatment with NR1 antisense ODN significantly reduced the Bmax of [3H]CGS-19755 binding (2101 fmol/mg protein) compared to both vehicle (2787 fmol/mg protein) and sense (2832 +/- 39 fmol/mg protein) controls without any significant change in KD (33 nM). A corresponding reduction of [3H]CGP-39653 binding was also observed after treatment with NR1 antisense compared to both sense and vehicle controls. In contrast, neither antisense nor sense ODNs altered the proportion of high affinity glycine sites or the potency of glycine at either high or low affinity glycine binding sites to inhibit [3H]CGP-39653 binding. These results show that in vivo treatment with NR1 antisense ODNs to the NMDA receptor complex reduces antagonist binding at NMDA receptors and has pharmacological effects similar to those observed with some NMDA receptor antagonists. These results also suggest that treatment with antisense ODNs may provide another means to investigate allosteric modulation of receptor subtypes in vivo.     

ICAM-1 upregulation in distant tissues after hepatic ischemia/reperfusion: a clue to the mechanism of multiple organ failure

BACKGROUND/PURPOSE: Endothelial cell adhesion molecules (ECAMs) are felt to play an important role in ischemia/reperfusion (I/R) injury by causing adhesion of leukocytes to endothelial cells. It is possible that ECAMs play a role in multiple organ system failure. ICAM-1 is one of the adhesion molecules that has been shown to be upregulated in response to cytokines. This upregulation leads to leukocyte endothelial cell interaction (adhesion) and to neutrophil infiltration of the affected tissue. The purpose of our study was to measure ICAM-1 expression in the liver and other organs after hepatic ischemia/reperfusion (I/R). METHODS: A laparotomy was performed on 14 Sprague-Dawley rats; 45 minutes of occlusive ischemia to the left lateral lobe was followed by 5 hours of reperfusion. The rat was injected with I125-labeled ICAM-1 MAb and I131-labeled nonbinding MAb (to control for nonspecific accumulation of ICAM-1 MAb). Entire organs were harvested and accumulated activity was measured in each organ. ICAM-1 levels were expressed as percent injected dose per gram of tissue. Control animals underwent sham laparotomy. RESULTS: ICAM-1 was upregulated in the ischemic lobe of the liver, nonischemic lobe of the liver, heart, kidney, intestine, and pancreas. Up-regulation in the lung was not significant. Both the lung and liver had high constitutive levels of ICAM-1. CONCLUSIONS: These data show that (1) significant hepatic upregulation of ICAM-1 after hepatic ischemia/reperfusion and (2) significant ICAM-1 upregulation in other tissues (heart, kidney, and intestine) after hepatic ischemia/reperfusion. The ICAM-1 upregulation in distant organs is likely mediated by cytokines such as tumor necrosis factor (TNF). These data show that leukocyte endothelial cell interactions in distant organs may be mediated by hepatic ischemia/reperfusion. This is a possible explanation for how failure of one organ can lead to failure of others in multiple organ system failure.     

Effects of acute ethanol intoxication on experimental brain injury in the rat: neurobehavioral and phosphorus-31 nuclear magnetic resonance spectroscopy studies

Using the lateral fluid-percussion model of experimental brain injury in the rat, the authors investigated the effect of acute ethanol (EtOH) intoxication on cardiovascular changes, neurological motor deficits, brain bioenergetics, and mortality associated with traumatic brain injury. Two hours after gastric administration of EtOH (low dose in 20 animals, 1.5 g/kg; high dose in 28, 3.0 g/kg) or saline (equal volume), animals were subjected to a fluid-percussion brain injury centered over the left parietal cortex. These injuries were of either moderate (X = 2.2 atm; 10 animals/treatment) or high severity (X = 3.0 atm; 18 animals/saline, 10 animals/low-dose EtOH, and 18 animals/high-dose EtOH). Neurological motor function was evaluated daily over a 1-week period, while a subset of eight animals receiving high-dose EtOH and subjected to brain injury of high severity were monitored for 4 hours using phosphorus-31 nuclear magnetic resonance spectroscopy to determine intracellular pH, free magnesium, and brain cytosolic phosphorylation potential. A significant (p < 0.05) and prolonged (up to 1 hour) hypotension was observed in animals pretreated with either low- or high-dose EtOH. Neither low-dose (blood-EtOH concentration = 110 +/- 40 mg/dl) nor high-dose (blood-EtOH = 340 +/- 70 mg/dl) EtOH had any effect on survival or neurological motor function after moderate brain injury. Following severe brain injury, animals pretreated with high-dose (blood-EtOH concentration = 352 +/- 65 mg/dl) EtOH showed a significantly increased mortality and markedly worsened neurological deficits at 24 hours postinjury. Following injury, free magnesium and cytosolic phosphorylation potential declined in both groups by approximately 50% to 60%, with no significant differences between groups with respect to these variables. In contrast, brain intracellular pH in the EtOH-treated animals was consistently higher than in the control group after injury. These data suggest that prior exposure to EtOH, particularly at high concentrations, may have detrimental effects on neurobehavioral function and survival in the acute period (up to 24 hours) after severe brain injury, and may be associated with posttraumatic cerebral alkalosis.     

Long-term outcome of kidney-pancreas transplant recipients with good graft function at one year

To assess the long-term outcome of kidney/pancreas transplantation, patients were identified who had good graft function at one year posttransplant and a minimum of 3 years' follow-up. Fifty recipients from 1987-92 met these criteria. Records were reviewed for graft survival, graft function, readmissions, and medical complications. Psychosocial adjustment and quality of life were assessed using the SCL-90-R and SIP surveys, respectively. Patient, kidney, and pancreas survivals were 94%, 86%, and 85% at five years (Kaplan-Meier), with a mean follow-up of 4.3 years. The 3 deaths were due to 2 sudden arrests at home (presumed to be cardiac events) and 1 episode of sepsis. Other graft losses were due to rejection, except for one case of sepsis. The remaining patients are normoglycemic (glucose 92 +/- 23 mg/dl) and have a creatinine of 1.8 +/- 0.6 mg/dl. Mortality after the first year was 0.9%/year. Estimated kidney and pancreas half-lives were 15 +/- 2 and 23 +/- 7 years, respectively. Hospitalization, acute rejection, graft pancreatitis, dehydration, and severe infections all decreased dramatically after the first year. While CMV was the most common infection in the first year, foot infections predominated thereafter. Retinal hemorrhage was infrequent. Sudden death (presumably cardiac) was the chief cause of mortality, while peripheral vascular disease resulted in several amputations. Fractures were common, suggesting the need for increased attention to bone demineralization. Psychosocial and quality of life evaluations were within normal limits. In conclusion, most complications specifically related to transplantation occur in the first year, but underlying disease renders these patients susceptible to a variety of cardiovascular, bone, and other disorders.     

Protection from renal ischemia-reperfusion injury by the 2-methylaminochroman U83836E

BACKGROUND: In a prior study the 21-aminosteroid (lazaroid) U74389F provided in vivo protection from oxidative stress when used as a preventive therapy in ischemia-reperfusion injury in the kidney. As the cell membrane is the principal site for lipoperoxidation, in the current study the very lipophilic 2-methylaminochroman U83836E, a recently developed lazaroid, was administered to rats at 3 mg/kg before renal ischemia-reperfusion. In addition to the biochemical parameters, the renal function and the histological appearance were carefully evaluated. METHODS: Glutathione, adenine nucleotides and lipid peroxidation products were determined in kidneys reperfused for 2 and 24 hours after 90 minutes of ischemia. Renal function was assessed by plasma creatinine, and renal injury by histological examination. RESULTS: Reperfusion-induced glutathione oxidation, expressed as an oxidized-to-total glutathione ratio, was significantly attenuated both after 2 and 24 hours of reperfusion by treatment with U83836E. Adenosine triphosphate (ATP) was still significantly depleted after 24 hours in the control group, while at the same time treated animals had already recovered to baseline values. Lipid peroxidation products were significantly lower in lazaroid-groups both after 2 and 24 hours of reperfusion. Renal function after 24 hours of reperfusion was notably better in the treated rats. Histological examination confirmed the protective action of the drug. After 24 hours the control group showed large areas of parenchymal hemorrhage and necrosis with dilated tubules and blood vessel thrombosis, while treated animals showed small necrotic areas with a background of mild interstitial inflammatory cells. CONCLUSIONS: Our results suggest that there is a protective effect of U83836E in ischemia-reperfusion injury, in that tissue damage due to oxidative stress is reduced, thus ameliorating renal function impairment.     

Erythropoietin-receptor expression and function during the initiation of murine yolk sac erythropoiesis

Although erythropoietin is necessary for definitive (fetal liver and bone marrow) erythropoiesis, the role of erythropoietin signaling in primitive (yolk sac) hematopoiesis has not been well defined. In situ hybridization studies have revealed that erythropoietin-receptor (EPOR) mRNA accumulation begins in mesoderm cell masses of the developing yolk sac of the neural plate stage embryo (E7.5) before the development of morphologically recognizable erythroblasts. EPOR mRNA is also present in yolk sac blood islands at early somite stages (E8.5). These findings suggest that EPOR functions during early stages of yolk sac erythropoiesis. We have used a serum-free murine yolk sac explant system (Palis et al., Blood 86:156, 1995) to investigate the initial differentiation of primitive erythroblasts from extraembryonic mesoderm cells. Exogenous erythropoietin increased both erythroblast numbers and betaH1-globin accumulation in yolk sac explants, suggesting that primary yolk sac erythroblasts are directly responsive to erythropoietin. An antisense oligodeoxynucleotide (ODN) experimental approach was used to examine the functional role of erythropoietin signaling during the initiation of yolk sac hematopoiesis in yolk sac explants. Antisense EPOR ODN produced a >50% reduction (p < 0.005) in the number of differentiating primitive erythroblasts, >95% reduction in betaH1-globin accumulation (p < 0.001), and a >50% reduction (p < 0.01) in the number of CFU-E and BFU-E compared with missense EPOR ODN-treated and untreated control explants. Antisense EPOR ODN also blocked the increase in primitive erythroblast number induced by exogenous erythropoietin. We conclude that erythropoietin/EPOR signaling is functionally active during the initial proliferation and differentiation of primary yolk sac erythroblasts. These results also suggest that other growth factor signaling cascades are active during the onset of mammalian erythropoiesis.     

A comparison of in vivo gene delivery methods for antisense therapy in ligament healing

To determine the most efficient in vivo delivery method of oligonucleotides for antisense therapy in ligament healing, fluorescence-labelled phosphorothioate oligodeoxynuleotides (ODN) were introduced into 12 rabbit ligament scars 2 weeks after injury using haemagglutinating virus of Japan (Sendai virus: HVJ)-conjugated liposomes. We compared the efficiency of cellular uptake of fluorescence as a percentage of all cells in each scar using three delivery procedures: (1) direct free-hand injection into the ligament scar using a conventional syringe; (2) systematic direct sca injection using a repeating 10 microliters dispenser and a square mesh grid system; and (3) injection into the feeding (femoral) artery. Results showed that there was a significant difference in fluorescence uptake by scar cells on day 1 after injection between the three delivery methods: (1) direct free-hand, 9.7 +/- 7.6% (average +/- s.d.); (2) systematic direct, 58.4 +/- 15.9%; and (3) intra-arterial, 0.2 +/- 0.1%. Systematic direct injection was most efficient and it resulted in 25.9 +/- 13.0% of scar cells being labeled at 7 days after transfection. We then introduced antisense ODN for the rabbit proteoglycan, decorin, into ligament scars with this delivery method and confirmed a significant inhibition of decorin mRNA expression in antisense-treated scar tissues in vivo both at 2 days (42.3 +/- 14.7% of sense control +/- s.d.; P < 0.0025) and 3 weeks (60.5 +/- 28.2% of sense control +/- s.d.; P < 0.024) after treatment, compared with sense ODN-treated scars. Decorin was significantly suppressed also at protein level in antisense-treated scars at 4 weeks (66.6 +/- 35.7% of sense control +/- s.d.; P < 0.045) after treatment. These results demonstrate that in vivo transfection efficiency in ligament scars is 'delivery system dependent' and that introduction of antisense ODN for the small proteoglycan, decorin, with this delivery method can lead to significant suppression of its expression over 3 weeks both at mRNA and protein levels. Thus, an effective model for the potential manipulation of scar composition and quality in ligament healing has been established.     

Expression of endothelin-1 and effects of an endothelin receptor antagonist, TAK-044, at reperfusion after cold preservation in a canine lung transplantation model

BACKGROUND: Rapid increase of pulmonary vascular resistance (PVR) early after reperfusion remains a major issue in clinical lung transplantation. A potent vasoconstrictor peptide, endothelin- plays an important role in various pulmonary pathophysiologic conditions and might induce increased PVR. We investigated the expression and influence of endothelin-1, and the effects of an ETA and ETB nonselective endothelin receptor antagonist, TAK-044, at reperfusion after cold preservation in a canine lung transplantation model. METHODS: Left single lung allotransplantation procedures were performed in three groups of animals. In group I (n=5) lungs were preserved for 12 hours; in group II (n=5) lungs were preserved for 18 hours; and in group III (n=6) lungs were also preserved for 18 hours, and TAK-044 (5 mg/kg) was administered just before reperfusion. All donor lungs were flushed and preserved with low-potassium dextran glucose solution at 4 degrees C. RESULTS: Six hours after reperfusion, arterial oxygen tension (mm Hg, inspired oxygen fraction=1.0) was 512.9+/-34.7 in group I, 152.4+/-46.7 in group II, and 509.6+/-29.0 in group III; PVR index (dyne x sec x cm(-5) x m2) was 1130+/-142 in group I, 1820+/-142 in group II, and 1287+/-191 in group III. Plasma endothelin-1 level was elevated significantly, and endothelin-1-like immunoreactivity was found in a variety of pulmonary vascular tissue and was seen less with immunohistochemical evaluation in group II in bronchial tissue. Conclusions: These results suggest that endothelin-1 is expressed as a result of ischemia-reperfusion injury and may worsen early graft function. TAK-044 is beneficial in protecting the graft from high pulmonary vascular resistance and pulmonary edema during the early posttransplantation stage.     

Heat shock improves recovery and provides protection against global ischemia after hypothermic storage

BACKGROUND: Improved methods of donor heart preparation before preservation could allow for prolonged storage and permit remote procurement of these organs. Previous studies have shown that overexpression of heat-shock protein 72 provides protection against ischemic cardiac damage. We sought to determine whether rats subjected to heat stress with only 6-hour recovery could acquire protection to a subsequent heart storage for 12 hours at 4 degrees C. METHODS: Three groups of animals (n = 10 each) were studied: control, sham-treated, and heat-shocked rats (whole-body hyperthermia 42 degrees C for 15 minutes). After 12-hour cold ischemia hearts were reperfused on a Langendorff column. To confirm any differences in functional recovery, hearts were then subjected to an additional 15-minute period of warm global ischemia after which function and lactate dehydrogenase enzyme leakage were measured. RESULTS: Heat-shocked animals showed marked improvements compared with controls in left ventricular developed pressure (63+/-4 mm Hg versus 44+/-4 mm Hg, p<0.05) heart rate x developed pressure (13,883+/-1,174 beats per minute x mm Hg versus 8,492+/-1,564 beats per minute x mm Hg, p<0.05), rate of ventricular pressure increase (1,912+/-112 mm Hg/second versus 1,215+/-162 mm Hg/second, p<0.005), rate of ventricular pressure decrease (1,258+/-89 mm Hg/second versus 774+/-106 mm Hg/second, p<0.005). Diastolic compliance and lactate dehydrogenase release were improved in heatshocked animals compared with controls and sham-treated animals. Differences between heat-shocked animals and control or sham-treated animals were further increased after the additional 15-minute period of warm ischemia. Western blot experiments confirmed increased heat-shock protein 72 levels in heat-shocked animals (>threefold) compared with sham-treated animals and controls. CONCLUSIONS: Heat shock 6 hours before heart removal resulted in marked expression of heat-shock protein 72 and protected isolated rat hearts by increased functional recovery and decreased cellular necrosis after 12-hour cold ischemia in a protocol mimicking that of heart preservation for transplantation. Protection was further confirmed after an additional 15-minute period of warm ischemia.