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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 15, 2001 as doi:10.1096/fj.01-0490fje. |
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College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA; and
* Harvard Medical School, Boston, Massachusetts 02115, USA
2Correspondence: Columbia University, College of Physicians and Surgeons, PH 10 Stem, 630 W. 168th St., New York, NY 10032, USA. E-mail: djp5{at}columbia.edu
SPECIFIC AIMS
Experiments tested the hypothesis that lung transplantation triggers early growth response-1 (Egr-1) expression and nuclear DNA binding activity corresponding to Egr-1, leading to increased expression of downstream target genes such as interleukin 1ß (IL-1ß), tissue factor, and plasminogen activator inhibitor 1 (PAI-1) and accompanying vascular stress. Furthermore, experiments tested the hypothesis that the devastating functional consequences of Egr-1 up-regulation can be prevented using a phosphorothioate antisense oligodeoxyribonucleotide (ODN) directed against the Egr-1 translation initiation site in order to block expression of Egr-1 and its gene targets.
PRINCIPAL FINDINGS
1. Expression of Egr-1 in an isogeneic orthotopic rat lung transplant model
Although hypothermic preservation did not increase Egr-1 expression measurably, there was a rapid and striking increase in Egr-1 mRNA levels after reperfusion, which peaked at 30 min of reperfusion and subsequently tapered off. A rapid increase in Egr-1 protein was observed along nearly the same time course with a minor rightward temporal shift, as could be expected based on the time required to translate nascent mRNA. Peak Egr-1 protein expression was observed at 1 h after reperfusion. Immunohistochemical studies showed that in addition to vascular smooth muscle staining for Egr-1, Egr-1 immunoreactivity and immunostaining for mononuclear phagocytes colocalize within the transplanted lungs.
2. Effects of antisense Egr-1 oligonucleotides on lung graft Egr-1 expression
Egr-1 mRNA levels after transplantation/reperfusion were markedly elevated in the group of lungs preserved with vehicle alone (Fig. 1
A). A similar degree of Egr-1 mRNA induction was noted in the group of lungs preserved with scrambled-sequence ODNs (3'-TCGTGCCGCTGCCAT-5'). However, when lungs were preserved with antisense Egr-1 ODNs (3'-TACCGTCGCCGGTTC-5') but otherwise subjected to identical transplantation/reperfusion procedures, Egr-1 mRNA levels were halved. Concordant with these observations, analyses of the expression of Egr-1 protein showed that only antisense ODNs, but not scrambled-sequence ODNs, significantly blocked increased levels of Egr-1 protein in the control group after transplantation/reperfusion (Fig. 1B
). Electrophoretic gel mobility shift assays demonstrated that although there was no apparent gel shift band in nuclear extracts of untreated lung tissue, a dense band was observed in nuclear extracts of both vehicle-preserved and scrambled-sequence ODN-preserved lungs (Fig. 1C
). In sharp contrast, preservation of lungs in the presence of antisense Egr-1 virtually abrogated the gel retardation band associated with Egr-1/DNA binding.
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3. Effects of antisense Egr-1 oligonucleotides on graft function and leukocyte accumulation
In transplanted lungs that had been preserved with Lipofectin and vehicle without ODNs, pulmonary vascular resistance (PVR) was significantly elevated and arterial oxygenation deteriorated markedly despite inhalation of pure oxygen. With the addition of antisense Egr-1 ODNs, lung preservation was significantly improved as manifest by reduced PVR and improved arterial oxygenation. Scrambled-sequence ODNs exhibited no effect, either positive or negative, on lung preservation relative to vehicle alone (Fig. 2
A, B). Myeloperoxidase assay demonstrated that only antisense Egr-1 ODNs, but not scrambled-sequence ODNs or vehicle, significantly reduced elevated levels of myeloperoxidase activity after transplantation (Fig. 2C
).
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4. Egr-1 regulation of cytokine and coagulation-related factors
To investigate the contribution of proinflammatory cytokines to post-transplantation lung injury, IL-1ß mRNA expression was examined. IL-1ß expression was strongly up-regulated after lung transplantation; this up-regulation was suppressed significantly by administration of antisense Egr-1 ODN but not by scrambled- sequence ODN (Fig. 2D
). Fibrin immunoblots show that graft lungs preserved in the presence of vehicle or scrambled-sequence ODN exhibit a marked increase in fibrin deposition compared with nontransplanted control lung tissue (Fig. 2E
). Antisense Egr-1 treatment potently suppressed accumulation of fibrin in lung grafts. To explore likely mechanisms responsible for these observations, tissue factor and PAI-1 expression were examined. Rats subjected to lung transplantation showed elevated levels of steady-state tissue factor mRNA and PAI-1 mRNA (Fig. 2F, G
). Only antisense Egr-1 ODNs, but not scrambled-sequence Egr-1 ODNs, suppressed both tissue factor and PAI-1 mRNA levels. These findings are concordant with the hypothesis that induction of Egr-1 represents an upstream event that triggers increased transcription of proinflammatory, procoagulant, and antifibrinolytic genes, resulting in graft leukosequestration and thrombosis (Fig. 3
).
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5. Effects of antisense Egr-1 oligonucleotides on recipient survival
Although survival was extremely poor in recipients transplanted with vehicle-preserved (19%) or scrambled sequence Egr-1 ODN-preserved (25%) donor lungs, survival of recipients of antisense Egr-1 ODN-preserved donor lungs more than doubled (56%) (Fig. 2H
). These data indicate that Egr-1 blockade at the time of lung preservation confers remarkable functional and survival advantages to lung graft recipients.
CONCLUSIONS
Rapid induction and activation of Egr-1 after lung transplantation means that molecular events programming the vascular response are well under way in the earliest minutes after the severed organ is reconnected. The significance of Egr-1 activation in this setting cannot be understated, because the pleiotropic consequences of Egr-1 activation can be disastrous for the newly reattached organ. The studies presented here demonstrate for the first time that graft inflammation and thrombosis can be triggered by Egr-1 induction and that these pathological events can be diminished by a simple strategy of Egr-1 suppression. These data add to the growing body of evidence implicating a pathogenic role for microvascular accrual of fibrin, which impedes return of blood flow. Egr-1 induction, which triggers several downstream mechanisms leading to accumulation of fibrin, may actually be the prime actuator of thrombosis in the ischemic lungs. Not only can tissue factor induction by Egr-1 lead to tissue factor-dependent activation of the procoagulant pathway, but the fibrin ultimately formed by activation of the final common pathway of coagulation is likely to be relatively impervious to degradation consequent to Egr-1-mediated up-regulation of PAI-1. The simultaneous overexpression of procoagulant and fibrinolytic suppressor genes leads to explosive amplification of fibrin deposition in the transplanted vasculature. In the studies shown here, Egr-1 suppression reduces IL-1ß induction as well as graft leukostasis. As Egr-1 induction promotes activation of coagulation, inflammation, and leukocyte accumulation, the hallmarks of reperfusion injury in transplanted lungs, targeted inhibition of Egr-1 expression using an antisense Egr-1 ODN delivered at the time of lung harvest can have profound effects on primary graft function.
Several strategies may be able to inhibit the pathological consequences of Egr-1 induction. For the current experiments, an antisense approach was used. The start codon region of Egr-1 mRNA targeted by the antisense ODN was chosen because this was a particularly effective inhibitory target in vascular smooth muscle cells. Because of potential concerns regarding specificity of the phosphorothioate ODN for the chosen target, scrambled phosphorothioate ODNs of identical size and net charge as their parent counterparts, yet which do not form heteroduplexes with target mRNA, were used for control conditions. In the current studies, only an antisense Egr-1 ODN (but not scrambled sequence Egr-1 ODN) reduced both Egr-1 protein and mRNA levels. These data point to a direct inhibitory effect of this ODN on the targeted sequence, Egr-1. These data are the first to delineate the deleterious biological actions of Egr-1 in the setting of vascular injury induced by lung transplantation and to suggest a simple and effective therapy to improve lung preservation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0490fje; to cite this article, use FASEB J. (October 15, 2001) 10.1096/fj.01-0490fje 
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