Carbon monoxide (CO), an endogenous cytoprotective product of heme oxygenase type-1 regulates target thrombotic and inflammatory genes in ischemic stress. Regulation of the gene encoding early growth response 1 (Egr-1), a potent transcriptional activator of deleterious thrombotic and inflammatory cascades, may govern CO-mediated ischemic lung protection. The exact signaling mechanisms underlying CO-mediated cytoprotection are not well understood. In this study we tested the hypothesis that inhibition of mitogen-activated protein kinase-dependent Egr-1 expression may be pivotal in CO-mediated ischemic protection. In an in vivo isogeneic rat lung ischemic injury model, inhaled CO not only diminished fibrin accumulation and leukostasis and improved gas exchange and survival but also suppressed extracellular signalregulated kinase (ERK) activation, Egr-1 expression, and Erg DNAbinding activity in lung tissue. Additionally, CO-mediated inhibition of Egr-1 reduced expression of target genes, such as tissue factor, serpine-1, interleukin-1, and TNF-␣. However, CO failed to inhibit serpine-1 expression after unilateral lung ischemia in mice null for the Egr-1 gene. In RAW macrophages in vitro, hypoxiainduced Egr-1 mRNA expression was ERK-dependent, and COmediated suppression of ERK activation resulted in Egr-1 inhibition. Furthermore, CO suppression of ERK phosphorylation was reversed by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one but was insensitive to cAMP-dependent protein kinase A inhibition with H89 and NO synthase inhibition with L-nitroarginine methyl ester. This finding indicates that CO suppresses ERK in a cGMP-dependent but cAMP͞protein kinase A-and NO-independent manner. Together, these data identify a unifying molecular mechanism by which CO interrupts proinflammatory and prothrombotic mediators of ischemic injury.
Obliterative bronchiolitis (OB) develops insidiously in nearly half of all lung transplant recipients. Although typically preceded by a CD8+ T cell–rich lymphocytic bronchitis, it remains unresponsive to conventional immunosuppression. Using an airflow permissive model to study the role of gases flowing over the transplanted airway, it is shown that prolonged inhalation of sublethal doses of carbon monoxide (CO), but not nitric oxide (NO), obliterate the appearance of the obstructive airway lesion. Induction of the enzyme responsible for the synthesis of CO, heme oxygenase (Hmox) 1, increased carboxyhemoglobin levels and suppressed lymphocytic bronchitis and airway luminal occlusion after transplantation. In contrast, zinc protoporphyrin IX, a competitive inhibitor of Hmox, increased airway luminal occlusion. Compared with wild-type allografts, expression of inducible NO synthase (iNOS), which promotes the influx of cytoeffector leukocytes and airway graft rejection, was strikingly reduced by either enhanced expression of Hmox-1 or exogenous CO. Hmox-1/CO decreased nuclear factor (NF)-κB binding activity to the iNOS promoter region and iNOS expression. Inhibition of soluble guanylate cyclase did not interfere with the ability of CO to suppress OB, implicating a cyclic guanosine 3′,5′-monophosphate–independent mechanism through which CO suppresses NF-κB, iNOS transcription, and OB. Prolonged CO inhalation represents a new immunosuppresive strategy to prevent OB.
Background-The interaction between CD40 on antigen-presenting cells and CD40L on T cells is critical in allograft rejection. CD154 blockade suppresses allograft rejection, but the role of this pathway in allograft vasculopathy remains obscure. Methods and Results-A vascularized murine heterotopic cardiac transplant model was used to test whether perioperative CD154 blockade suppresses allograft vasculopathy or whether long-term CD154 blockade is required to suppress allograft vasculopathy. Perioperative CD154 blockade consisted of MR1 given on days Ϫ1, 1, and 3; long-term blockade consisted of MR1 given on days Ϫ1, 1, and 3 and continued twice weekly for 8 weeks. Allografts treated with perioperative or long-term CD154 blockade survived indefinitely. Perioperative and long-term treatment with control antibody (Ha4/8) resulted in uniform early rejection. Perioperative CD154 blockade transiently reduced early T-cell and macrophage infiltration in parallel with a transient reduction in endothelial adhesion receptor expression. Although perioperative CD154 blockade prevented allograft failure, it did not reduce allograft vasculopathy; mean neointimal cross-sectional area in perioperative MR1-treated and Ha4/8-treated recipients was 43Ϯ7% and 50Ϯ12%, respectively (PϭNS). In contrast, mean neointimal cross-sectional area in long-term, MR1-treated recipients was 19Ϯ3% (PϽ0.001 versus perioperative MR1). Long-term CD154 blockade also suppressed endothelial E-selectin, P-selectin, and intracellular adhesion molecule-1 expression and improved graft function 3.5-fold versus control (PϽ0.05). Conclusions-These data show that perioperative CD154 blockade mitigates acute rejection but long-term CD154 blockade may result in decreased allograft endothelial activation and is required to suppress allograft arteriopathy.
Chronic airway rejection is characterized by prolonged inflammation, epithelial damage, and eventual luminal obliterative bronchiolitis (OB). In cardiac allografts, the inducible nitric oxide synthase (iNOS) promotes acute rejection but paradoxically reduces neointimal formation, the hallmark of chronic rejection. The specific roles of NOS isoforms in modulating lymphocyte traffic and airway rejection are not known. Using a double lumen mouse tracheal transplant model, tracheal grafts from B10.A (allo) or C57BL/6J (iso) mice were transplanted into cyclosporine-treated wild-type (WT) iNOS−/− or endothelial NOS (eNOS)−/− recipients. OB was observed in WT tracheal allografts at 3 weeks (53 ± 2% luminal occlusion vs. 17 ± 1% for isografts, P < 0.05) with sites of obstructive lesion formation coinciding with areas of CD3+ CD8+ T cell–rich lymphocytic bronchitis. In contrast, allografts in iNOS−/− recipients exhibited reductions in local expression of proinflammatory chemokines and cytokines, graft T cell recruitment and apoptosis, and luminal obliteration (29 ± 2%, P < 0.05 vs. WT allografts). Recipient eNOS deficiency, however, suppressed neither chemokine expression, lymphocyte infiltration, nor airway occlusion (54 ± 2%). These data demonstrate that iNOS exacerbates luminal obliteration of airway allografts in contrast with the known suppression by iNOS of cardiac allograft vasculopathy. Because iNOS−/− airways transplanted into WT allograft hosts are not protected from rejection, these data suggest that iNOS expressed by graft-infiltrating leukocytes exerts the dominant influence on airway rejection.
Abstract-Expression of the zinc finger transcription factor early growth response gene-1 (Egr-1) is triggered rapidly after mechanical vascular injury or after a precipitous drop in ambient oxygen, whereupon it induces the expression of diverse gene families to elicit a pathological response. Initially characterized as an early response transcriptional activator, the role of Egr-1 in more chronic forms of vascular injury remains to be defined. Studies were designed to examine whether Egr-1 induction may serve as a causal link between early preservation injury and delayed vascular consequences, such as coronary allograft vasculopathy (CAV). The preservation and transplantation of heterotopic murine cardiac allografts strongly induce Egr-1 expression, leading to increased expression of its downstream target genes, such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and platelet-derived growth factor A chain. Expression of these Egr-1-inducible gene targets is virtually obliterated in homozygous Egr-1-null donor allografts, which also exhibit attenuated parenchymal rejection and reduced CAV as long as 60 days. Congruous data are observed by treating donor hearts with a phosphorothioate antisense oligodeoxyribonucleotide directed against Egr-1 before organ harvest, which blocks subsequent expression of Egr-1 mRNA and protein and suppresses the late development of CAV. These data indicate that Egr-1 induction represents a central effector mechanism in the development of chronic rejection characterized by CAV. Blocking the expression of this proximal transcription factor solely at the time of organ harvest elicits beneficial delayed consequences for the cardiac allograft.
Little is known about interactions between endogenous anti-inflammatory paradigms and microvascular thrombosis in lung ischemia/reperfusion (I/R) injury. Interleukin (IL)-10 suppresses macrophage activation and down-regulates proinflammatory cytokine production, but there are no available data to suggest a link between IL-10, thrombosis, and fibrinolysis in the setting of I/R. We hypothesized that hypoxia/ischemia triggers IL-10 production, to dampen proinflammatory cytokine and adhesion receptor cascades and to restore vascular patency by fibrinolytic potentiation. Studies were performed in a mouse lung I/R model. IL-10 mRNA levels in lung were increased 43-fold over base line by 1 h of ischemia/2 h of reperfusion, with a corresponding increase in plasma IL-10. Expression was prominently localized in bronchial epithelial cells and mononuclear phagocytes. To study the link between IL-10 and fibrinolysis in vivo, the induction of plasminogen activator inhibitor-1 (PAI-1) was evaluated. Northern analysis demonstrated exaggerated pulmonary PAI-1 expression in IL-10 (؊/؊) mice after I/R, with a corresponding increase in plasma PAI/tissue-type plasminogen activator activity. In vivo, IL-10 (؊/؊) mice showed poor postischemic lung function and survival after I/R compared with IL-10 (؉/؉) mice. Despite a decrease in infiltration of mononuclear phagocytes in I/R lungs of IL-10 (؊/؊) mice, an increased intravascular pulmonary fibrin deposition was observed by immunohistochemistry and Western blotting, along with increased IL-1 expression. Recombinant IL-10 given to IL-10 (؊/؊) mice normalized the PAI/tissue-type plasminogen activator ratio, reduced pulmonary vascular fibrin deposition, and rescued mice from lung injury. Since recombinant hirudin (direct thrombin inhibitor) also sufficed to rescue IL-10 (؊/؊) mice, these data suggest a preeminent role for microvascular thrombosis in I/R lung injury. Ischemiadriven IL-10 expression confers postischemic pulmonary protection by augmenting endogenous fibrinolytic mechanisms. Ischemia/reperfusion (I/R)1 lung injury plays a significant role in clinical situations such as lung transplantation (1-3). Lung failure associated with I/R is characterized by increased microvascular permeability, pulmonary vascular resistance with subsequent edema formation and impairment of gas exchange, and microembolism. The lungs are particularly susceptible to ischemia/reperfusion injury, presumably due to the rich vascularity of the lungs and the relatively large surface area over which blood-borne components can interact with endothelium. The proximate mechanisms of ischemic lung injury are diverse and include leukocyte activation and recruitment (1), complement activation (4), abnormalities in pulmonary vascular tone, and increased procoagulant activity, resulting in microcirculatory failure, cellular dysfunction, edema, and cell death. The local production of proinflammatory cytokines, such as IL-1␣ and tumor necrosis factor-␣, is considerably increased in I/R injury (5, 6), which can also feed...
Abstract-Transiently increased expression of leukocyte adhesion receptors after lung preservation contributes to early graft demise by recruiting leukocytes, activating complement, and causing microcirculatory stasis. We hypothesized that inhibiting intercellular adhesion molecule-1 (ICAM-1) expression even briefly may significantly improve lung graft function and that the preservation period might provide a unique window to deliver a therapeutic pulse of antisense oligonucleotide ICAM-1 to inhibit ICAM-1 expression after transplantation. Interleukin-1␤-treated rat pulmonary endothelial cells given a 20-mer phosphorothioate oligonucleotide comprising an antisense span targeted to the 3Ј-untranslated region of rat ICAM-1 demonstrated an oligonucleotide dose-dependent reduction in ICAM-1 expression. Using a cationic liposomal carrier, this same antisense oligonucleotide (but not the sense control) instilled into the pulmonary vasculature at the time of preservation reduced subsequent graft ICAM-1 expression and graft leukostasis and markedly improved oxygenation, pulmonary blood flow, and graft survival. These experiments demonstrate that the preservation period presents a window during which to target an anti-ICAM-1 expression strategy to inhibit early adhesion receptor expression and improve functional outcome after lung transplantation. (Circ Res. 2000;86:166-174.)Key Words: intercellular adhesion molecule-1 Ⅲ lung transplantation Ⅲ isograft Ⅲ leukocyte adhesion receptor C linical lung transplantation at its best is a harrowing experience, because lung grafts can fail catastrophically shortly after reperfusion for reasons that are often not understood. 1,2 Clinical lung preservation strategies are directed toward maintaining proper electrolyte and osmotic homeostasis, but surprisingly little is done to protect the vast pulmonary vascular network on which the lung depends for both integrity and function. Because the early expression of the leukocyte adhesion receptor P-selectin can result in rapid sequestration of neutrophils (polymorphonuclear leukocytes [PMNs]) after lung transplantation, 3 promoting microcirculatory stasis and local tissue destruction, we hypothesized that a strategy that protects the lungs from early leukocyte recruitment could confer clinical benefit. Because P-selectin places neutrophils into a favorable steric relationship for intercellular adhesion molecule-1 (ICAM-1) binding to ␤ 2 -integrins, we hypothesized that early inhibition of inducible ICAM-1 expression might serve as a useful target for therapeutic intervention in lung transplantation to prevent acute graft injury. Toward this end, we have focused on the unique opportunity provided by the lung harvest procedure, during which it is possible to deliver agents (such as antisense oligodeoxynucleotides) ex vivo directly into the vasculature, which can block adhesion receptor expression during the first few critical hours after reperfusion. The current studies were designed to (1) elucidate the nature and functional relevance of endo...
Nitroglycerin (NTG) given to donor lungs improves lung preservation for transplantation, but the mechanism(s) underlying this therapeutic benefit remain incompletely understood. Furthermore, it is not known whether the therapeutic window of opportunity for NTG administration is temporally-restricted. Because endothelin-1 (ET-1), a potent vasoconstrictor, and nitric oxide (NO) are reciprocally regulated in vitro, we hypothesized that early administration of the NO donor NTG may suppress ET-1 and thereby improve lung preservation. Using an isogeneic rat left lung transplantation model, four groups were studied (n = 12 transplant/group): (1) NTG given during flush/ preservation (Early NTG); (2) NTG given in the ex vivo flush (Late NTG); (3) No NTG; and (4) a nonselective ET-receptor antagonist (PD156252) given during flush/preservation. Early NTG decreased vascular tone in lung grafts measured ex vivo as well as in vivo following lung transplantation, and resulted in improved survival (100%) and gas exchange (pO2 209 +/- 19 mm Hg) compared with Late (17%, 62 +/- 16 mm Hg) or No NTG (25%, 59 +/- 9 mm Hg) (P < 0.05 for Early NTG versus all other groups for both survival and pO2). PD156252 was associated with an intermediate level of survival (50%) and function (104 +/- 23 mm Hg). Transplanted lung graft ET-1 mRNA, measured by Northern blotting and in situ hybridization, and protein, measured by Western blotting and immunohistochemistry, were suppressed only with Early NTG (P < 0.05 versus all other groups). Post-transplantation benefits of NTG are restricted to lung grafts which received NTG during the early harvest and immersion periods, and are coincident with suppression of graft ET-1 expression. When viewed in the context of improved graft survival and function with ET-1 receptor blockade, these data suggest that early administration of NTG to donor lungs improves primary graft function, in part, by suppressing graft ET-1 expression.
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