Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress.

Activation of the zinc-finger transcription factor early growth response (Egr)-1, initially linked to developmental processes, is shown here to function as a master switch activated by ischemia to trigger expression of pivotal regulators of inflammation, coagulation and vascular hyperpermeability. Chemokine, adhesion receptor, procoagulant and permeability-related genes are coordinately upregulated by rapid ischemia-mediated activation of Egr-1. Deletion of the gene encoding Egr-1 strikingly diminished expression of these mediators of vascular injury in a murine model of lung ischemia/reperfusion, and enhanced animal survival and organ function. Rapid activation of Egr-1 in response to oxygen deprivation primes the vasculature for dysfunction manifest during reperfusion. These studies define a central and unifying role for Egr-1 activation in the pathogenesis of ischemic tissue damage.

Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis.

Carbon monoxide (CO) can arrest cellular respiration, but paradoxically, it is synthesized endogenously by heme oxygenase type 1 (Ho-1) in response to ischemic stress. Ho-1-deficient (Hmox1-/-) mice exhibited lethal ischemic lung injury, but were rescued from death by inhaled CO. CO drove ischemic protection by activating soluble guanylate cyclase and thereby suppressed hypoxic induction of the gene encoding plasminogen activator inhibitor-1 (PAI-1) in mononuclear phagocytes, which reduced accrual of microvascular fibrin. CO-mediated ischemic protection observed in wild-type mice was lost in mice null for the gene encoding PAI-1 (Serpine1). These data establish a fundamental link between CO and prevention of ischemic injury based on the ability of CO to derepress the fibrinolytic axis. These data also point to a potential therapeutic use for inhaled CO.

Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage.

Agents that restore vascular patency in stroke also increase the risk of intracerebral hemorrhage (ICH). As Factor IXa is a key intermediary in the intrinsic pathway of coagulation, targeted inhibition of Factor IXa-dependent coagulation might inhibit microvascular thrombosis in stroke without impairing extrinsic hemostatic mechanisms that limit ICH. A competitive inhibitor of native Factor IXa for assembly into the intrinsic Factor X activation complex, Factor IXai, was prepared by covalent modification of the Factor IXa active site. In a modified cephalin clotting time assay, in vivo administration of Factor IXai caused a dose-dependent increase in time to clot formation (3.6-fold increase at the 300 micrograms/kg dose compared with vehicle-treated control animals, P < 0.05). Mice given Factor IXai and subjected to middle cerebral artery occlusion and reperfusion demonstrated reduced microvascular fibrin accumulation by immunoblotting and immunostaining, reduced 111In-labeled platelet deposition (42% decrease, P < 0.05), increased cerebral perfusion (2.6-fold increase in ipsilateral blood flow by laser doppler, P < 0.05), and smaller cerebral infarcts than vehicle-treated controls (70% reduction, P < 0.05) based on triphenyl tetrazolium chloride staining of serial cerebral sections. At therapeutically effective doses, Factor IXai was not associated with increased ICH, as opposed to tissue plasminogen activator (tPA) or heparin, both of which significantly increased ICH. Factor IXai was cerebroprotective even when given after the onset of stroke, indicating that microvascular thrombosis continues to evolve (and may be inhibited) even after primary occlusion of a major cerebrovascular tributary.

Critical role for IL-6 in hypertrophy and fibrosis in chronic cardiac allograft rejection.

Chronic cardiac allograft rejection is the major barrier to long term graft survival. There is currently no effective treatment for chronic rejection except re-transplantation. Though neointimal development, fibrosis, and progressive deterioration of graft function are hallmarks of chronic rejection, the immunologic mechanisms driving this process are poorly understood. These experiments tested a functional role for IL-6 in chronic rejection by utilizing serial echocardiography to assess the progression of chronic rejection in vascularized mouse cardiac allografts. Cardiac allografts in mice transiently depleted of CD4+ cells that develop chronic rejection were compared with those receiving anti-CD40L therapy that do not develop chronic rejection. Echocardiography revealed the development of hypertrophy in grafts undergoing chronic rejection. Histologic analysis confirmed hypertrophy that coincided with graft fibrosis and elevated intragraft expression of IL-6. To elucidate the role of IL-6 in chronic rejection, cardiac allograft recipients depleted of CD4+ cells were treated with neutralizing anti-IL-6 mAb. IL-6 neutralization ameliorated cardiomyocyte hypertrophy, graft fibrosis, and prevented deterioration of graft contractility associated with chronic rejection. These observations reveal a new paradigm in which IL-6 drives development of pathologic hypertrophy and fibrosis in chronic cardiac allograft rejection and suggest that IL-6 could be a therapeutic target to prevent this disease.

Neuronal protection in stroke by an sLex-glycosylated complement inhibitory protein.

Glycoprotein adhesion receptors such as selectins contribute to tissue injury in stroke. Ischemic neurons strongly expressed C1q, which may target them for complement-mediated attack or C1qRp-mediated clearance. A hybrid molecule was used to simultaneously inhibit both complement activation and selectin-mediated adhesion. The extracellular domain of soluble complement receptor-1 (sCR1) was sialyl Lewis x glycosylated (sCR1sLex) to inhibit complement activation and endothelial-platelet-leukocyte interactions. sCR1 and sCR1sLex colocalized to ischemic cerebral microvessels and C1q-expressing neurons, inhibited neutrophil and platelet accumulation, and reduced cerebral infarct volumes. Additional benefit was conferred by sialyl Lewis x glycosylation of the unmodified parent sCR1 molecule.

The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta.

Inducible nitric oxide (NO) produced by macrophages is cytotoxic to invading organisms and has an important role in host defense. Recent studies have demonstrated inducible NO production within the heart, and that cytokine-induced NO mediates alterations in cardiac contractility, but the cytotoxic potential of nitric oxide with respect to the heart has not been defined. To evaluate the role of inducible nitric oxide synthase (iNOS) on cardiac myocyte cytotoxicity, we exposed adult rat cardiac myocytes to either cytokines alone or to activated J774 macrophages in coculture. Increased expression of both iNOS message and protein was seen in J774 macrophages treated with IFN gamma and LPS and cardiac myocytes treated with TNF-alpha, IL-1 beta, and IFN gamma. Increased NO synthesis was confirmed in both the coculture and isolated myocyte preparations by increased nitrite production. Increased NO synthesis was associated with a parallel increase in myocyte death as measured by CPK release into the culture medium as well as by loss of membrane integrity, visualized by trypan blue staining. Addition of the competitive NO synthase inhibitor L-NMMA to the culture medium prevented both the increased nitrite production and the cytotoxicity observed after cytokine treatment in both the isolated myocyte and the coculture experiments. Because transforming growth-factor beta modulates iNOS expression in other cell types, we evaluated its effects on cardiac myocyte iNOS expression and NO-mediated myocyte cytotoxicity. TGF-beta reduced expression of cardiac myocyte iNOS message and protein, reduced nitrite production, and reduced NO-mediated cytotoxicity in parallel. Taken together, these experiments show the cytotoxic potential of endogenous NO production within the heart, and suggest a role for TGF-beta or NO synthase antagonists to mute these lethal effects. These findings may help explain the cardiac response to sepsis or allograft rejection, as well as the progression of dilated cardiomyopathies of diverse etiologies.

Cerebral protection in homozygous null ICAM-1 mice after middle cerebral artery occlusion. Role of neutrophil adhesion in the pathogenesis of stroke.

Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.

Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation.

Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis.

Cardiac preservation is enhanced in a heterotopic rat transplant model by supplementing the nitric oxide pathway.

Nitric oxide (NO) is a novel biologic messenger with diverse effects but its role in organ transplantation remains poorly understood. Using a porphyrinic microsensor, the first direct measurements of coronary vascular and endocardial NO production were made. NO was measured directly in the effluent of preserved, heterotopically transplanted rat hearts stimulated with L-arginine and bradykinin; NO concentrations fell from 2.1 +/- 0.4 microM for freshly explanted hearts to 0.7 +/- 0.2 and 0.2 +/- 0.08 microM for hearts preserved for 19 and 38 h, respectively. NO levels were increased by SOD, suggesting a role for superoxide-mediated destruction of NO. Consistent with these data, addition of the NO donor nitroglycerin (NTG) to a balanced salt preservation solution enhanced graft survival in a time- and dose-dependent manner, with 92% of hearts supplemented with NTG surviving 12 h of preservation versus only 17% in its absence. NTG similarly enhanced preservation of hearts stored in University of Wisconsin solution, the clinical standard for preservation. Other stimulators of the NO pathway, including nitroprusside, L-arginine, or 8-bromoguanosine 3',5' monophosphate, also enhanced graft survival, whereas the competitive NO synthase antagonist NG-monomethyl-L-arginine was associated with poor preservation. Likely mechanisms whereby supplementation of the NO pathway enhanced preservation included increased blood flow to the reperfused graft and decreased graft leukostasis. NO was also measured in endothelial cells subjected to hypoxia/reoxygenation and detected based on its ability to inhibit thrombin-mediated platelet aggregation and serotonin release. NO became undetectable in endothelial cells exposed to hypoxia followed by reoxygenation and was restored to normoxic levels on addition of SOD. These studies suggest that the NO pathway fails during preservation/transplantation because of formation of oxygen free radicals during reperfusion, which quench available NO. Augmentation of NO/cGMP-dependent mechanisms enhances vascular function after ischemia and reperfusion and provides a new strategy for transplantation of vascular organs.

Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.

Oxygen deprivation, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. To investigate the effects of low ambient oxygen tension on the fibrinolytic system, mice were placed in a hypoxic environment with pO2 < 40 Torr. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) antigen, detected by ELISA, increased in a time-dependent fashion after hypoxic exposure (increased as early as 4 h, P < 0.05 vs. normoxic controls), and were accompanied by an increase in plasma PAI-1 activity by 4 h (P < 0.05 vs. normoxic controls). Northern analysis of hypoxic murine lung demonstrated an increase in PAI-1 mRNA compared with normoxic controls; in contrast, transcripts for both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) decreased under hypoxic conditions. Immunocolocalization studies identified macrophages as the predominant source of increased PAI-1 within hypoxic lung. Using a transformed murine macrophage line, striking induction of PAI-1 transcripts occurred under hypoxic conditions, due to both increased de novo transcription as well as increased mRNA stability. Consistent with an important role of the fibrinolytic system in hypoxia-induced fibrin accumulation, PAI-1 +/+ mice exposed to hypoxia exhibited increased pulmonary fibrin deposition based upon a fibrin immunoblot, intravascular fibrin identified by immunostaining, and increased accumulation of 125I-fibrinogen/fibrin in hypoxic tissue. In contrast, mice deficient for the PAI-1 gene (PAI-1 -/-) similarly exposed to hypoxic conditions did not display increased fibrin accumulation compared with normoxic PAI-1 +/+ controls. Furthermore, homozygous null uPA (uPA -/-) and tPA (tPA -/-) mice subjected to oxygen deprivation showed increased fibrin deposition compared with wild-type controls. These studies identify enhanced expression of PAI-1 as an important mechanism suppressing fibrinolysis under conditions of low oxygen tension, a response which may be further amplified by decreased expression of plasminogen activators. Taken together, these data provide insight into an important potential role of macrophages and the fibrinolytic system in ischemia-induced thrombosis.

Reduced microvascular thrombosis and improved outcome in acute murine stroke by inhibiting GP IIb/IIIa receptor-mediated platelet aggregation.

Treatment options in acute stroke are limited by a dearth of safe and effective regimens for recanalization of an occluded cerebrovascular tributary, as well as by the fact that patients present only after the occlusive event is established. We hypothesized that even if the site of major arterial occlusion is recanalized after stroke, microvascular thrombosis continues to occur at distal sites, reducing postischemic flow and contributing to ongoing neuronal death. To test this hypothesis, and to show that microvascular thrombosis occurs as an ongoing, dynamic process after the onset of stroke, we tested the effects of a potent antiplatelet agent given both before and after the onset of middle cerebral arterial (MCA) occlusion in a murine model of stroke. After 45 min of MCA occlusion and 23 h of reperfusion, fibrin accumulates in the ipsilateral cerebral hemisphere, based upon immunoblotting, and localizes to microvascular lumena, based upon immunostaining. In concordance with these data, there is a nearly threefold increase in the ipsilateral accumulation of 111In-labeled platelets in mice subjected to stroke compared with mice not subjected to stroke. When a novel inhibitor of the glycoprotein IIb/IIIa receptor (SDZ GPI 562) was administered immediately before MCA occlusion, platelet accumulation was reduced 48%, and fibrin accumulation was reduced by 47% by immunoblot densitometry. GPI 562 exhibited a dose-dependent reduction of cerebral infarct volumes measured by triphenyltetrazolium chloride staining, as well as improvement in postischemic cerebral blood flow, measured by laser doppler. GPI 562 caused a dose-dependent increase in tail vein bleeding time, but intracerebral hemorrhage (ICH) was not significantly increased at therapeutic doses; however, there was an increase in ICH at the highest doses tested. When given immediately after withdrawal of the MCA occluding suture, GPI 562 was shown to reduce cerebral infarct volumes by 70%. These data support the hypothesis that in ischemic regions of brain, microvascular thrombi continue to accumulate even after recanalization of the MCA, contributing to postischemic hypoperfusion and ongoing neuronal damage.

Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation.

The period of hypoxia is an important priming event for the vascular dysfunction that accompanies reperfusion, with endothelial cells (ECs) and neutrophils (PMNs) playing a central role. We hypothesized that EC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic period during organ preservation permits brisk PMN recruitment into postischemic tissue, a process further amplified in an oxidant-rich milieu. Exposure of human umbilical vein ECs to a hypoxic environment (pO2 approximately 20 torr) stimulated release of von Willebrand factor (vWF), stored in EC WP bodies, as well as increased expression of the WP body-derived PMN adhesion molecule P-selectin at the EC surface. Increased binding of 111In-labeled PMNs to hypoxic EC monolayers (compared with normoxic controls) was blocked with a blocking antibody to P-selectin, but was not affected by a nonblocking control antibody. Although increased P-selectin expression and vWF release were also noted during reoxygenation, hypoxia alone (even in the presence of antioxidants) was sufficient to increase WP body exocytosis. To determine the relevance of these observations to hypothermic cardiac preservation, during which the pO2 within the cardiac vasculature declines to similarly low levels, experiments were performed in a rodent (rat and mouse) cardiac preservation/transplantation model. Immunodepletion of recipient PMNs or administration of a blocking anti-P-selectin antibody before transplantation resulted in reduced graft neutrophil infiltration and improved graft survival, compared with identically preserved hearts transplanted into control recipients. To establish the important role of endothelial P-selectin expression on the donor vasculature, murine cardiac transplants were performed using homozygous P-selectin deficient and wild-type control donor hearts flushed free of blood/platelets before preservation/transplantation. P-selectin-null hearts transplanted into wild-type recipients demonstrated a marked (13-fold) reduction in graft neutrophil infiltration and increased graft survival compared with wild-type hearts transplanted into wild-type recipients. To determine whether coronary endothelial WP exocytosis may occur during cardiac preservation in humans, the release of vWF into the coronary sinus (CS) was measured in 32 patients during open heart surgery. CS samples obtained at the start and conclusion of the ischemic period demonstrated an increase in CS vWF antigen (by ELISA) consisting of predominantly high molecular weight multimers (by immunoelectrophoresis). These data suggest that EC WP exocytosis occurs during hypothermic cardiac preservation, priming the vasculature to recruit PMNs rapidly during reperfusion.

Antisense intercellular adhesion molecule-1 (ICAM-1) oligodeoxyribonucleotide delivered during organ preservation inhibits posttransplant ICAM-1 expression and reduces primary lung isograft failure.

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-1beta-treated rat pulmonary endothelial cells given a 20-mer phosphorothioate oligonucleotide comprising an antisense span targeted to the 3'-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.

cAMP pulse during preservation inhibits the late development of cardiac isograft and allograft vasculopathy.

The causes of transplant-associated coronary artery disease remain obscure, and there is no known treatment. Preservation injury of murine heterotopic vascularized cardiac isografts caused a small, albeit significant, increase in neointimal formation; preservation injury of allografts markedly increased both the incidence and severity of transplant-associated coronary artery disease. As cAMP is an important vascular homeostatic mediator the levels of which decline during organ preservation, buttressing cAMP levels solely during initial preservation both improved acute allograft function and reduced the severity of transplant-associated coronary artery disease in grafts examined 2 months later. Inhibiting the cAMP-dependent protein kinase abrogated these beneficial effects. cAMP treatment was associated with an early reduction in leukocyte infiltration and a reciprocal decrease in superoxide and increase in NO levels. These data indicate that alloantigen-independent injury to the graft, which occurs at the time of cardiac preservation, can set in motion pathological vascular events that are manifest months later. Furthermore, a cAMP pulse during cardiac preservation reduces the incidence and severity of transplant-associated coronary artery disease.

QT dispersion as a marker of risk in patients awaiting heart transplantation.

OBJECTIVES: The objectives of this study were to determine whether a signal-averaged electrocardiogram (SAECG) or measurement of interlead variability of QT intervals on an electrocardiogram (ECG) obtained at the time of wait-listing could provide prognostic value with respect to cardiac death during the waiting period. BACKGROUND: Because heart transplantation is a life-saving but limited resource, there remains an urgent need to identify those patients at greatest risk of dying while awaiting heart transplantation as part of the strategy to optimize the allocation of donor organs to those in greatest need. This study was undertaken to prospectively identify clinical, ECG or SAECG variables that might predict mortality during the waiting period. METHODS: Of 108 consecutive patients referred for heart transplant evaluation, 80 were placed on a waiting list, at which time a standard 12-lead ECG and a SAECG were recorded. In this cohort of 80 patients, QT dispersion was characterized from the 12-lead ECG as either the maximal-minimal QT interval (QTDISP) or as the coefficient of variation of all QT intervals (QTCV). RESULTS: During the 25-month follow-up period (mean time on waiting list, 201 days), the mortality rate was 27%/year, divided equally between heart failure and sudden deaths. No clinical variable identified at entry predicted mortality. QTDISP and QTCV were strong mortality predictors, with a 4.1-fold increase in mortality in patients with QTDISP > 140 ms compared with those patients with QTDISP < or = 140 ms (95% CI 1.1 to 14.9), whereas a QTCV > or = 9% also predicted a 4.1-fold increased risk of death (95% CI 1.4 to 11.8). Although 88% of all SAECGs were abnormal, no patient with a normal SAECG died suddenly during the waiting period. CONCLUSIONS: Indexes of QT dispersion provide a means of stratifying a patient's risk of dying while awaiting heart transplantation and may help to establish priority on a heart transplant waiting list.

Extinguishing Egr-1-dependent inflammatory and thrombotic cascades after lung transplantation.

Hypoxic induction of the early growth response-1 (Egr-1) transcription factor initiates proinflammatory and procoagulant gene expression. Orthotopic/isogeneic rat lung transplantation triggers Egr-1 expression and nuclear DNA binding activity corresponding to Egr-1, which leads to increased expression of downstream target genes such as interleukin-1b, tissue factor, and plasminogen activator inhibitor-1. The devastating functional consequences of Egr-1 up-regulation in this setting are prevented by treating donor lungs with a phosphorothioate antisense oligodeoxyribonucleotide directed against the Egr-1 translation initiation site, which blocks expression of Egr-1 and its gene targets. Post-transplant graft leukostasis, inflammation, and thrombosis are consequently diminished, with marked improvement in graft function and recipient survival. Blocking expression of a proximal transcription factor, which activates deleterious inflammatory and coagulant effector mechanisms, is an effective molecular strategy to improve organ preservation.

A modified transorbital baboon model of reperfused stroke.

BACKGROUND AND PURPOSE: Although pathophysiological studies of focal cerebral ischemia in nonhuman primates can provide important information not obtainable in rodent models, primate experimentation is limited by considerations of cost, availability, effort, and ethics. A reproducible and quantitative model that minimizes the number of animals necessary to detect differences between treatment groups is therefore crucial. METHODS: Eight male baboons (weight, 22+/-2 kg) underwent left transorbital craniectomy followed by 1 hour of temporary ipsilateral internal carotid artery occlusion at the level of the anterior choroidal artery together with bilateral temporary occlusion of both anterior cerebral arteries (A1) proximal to the anterior communicating artery. A tightly controlled nitrous oxide-narcotic anesthetic allowed for intraoperative motor evoked potential confirmation of middle cerebral artery (MCA) territory ischemia. Animals survived to 72 hours or 10 days if successfully self-caring. Outcomes were assessed with a 100-point neurological grading system, and infarct volume was quantified by planimetric analysis of both MRI and triphenyltetrazolium chloride-stained sections. RESULTS: Infarction volumes (on T2-weighted images) were 32+/-7% (mean+/-SEM) of the ipsilateral hemisphere, and neurological scores averaged 29+/-9. All animals demonstrated evidence of hemispheric infarction, with damage evident in both cortical and subcortical regions in the MCA vascular territory. Histologically determined infarction volumes differed by <3% and correlated with absolute neurological scores (r=0.9, P:=0.003). CONCLUSIONS: Transorbital temporary occlusion of the entire anterior cerebral circulation with strict control of physiological parameters can reliably produce reperfused MCA territory infarction. The magnitude of the resultant infarct with little interanimal variability diminishes the potential number of animals required to distinguish between 2 treatment regimens. The anatomic distribution of the infarct and associated functional deficits offer comparability to human hemispheric strokes.

Postischemic cerebrovascular E-selectin expression mediates tissue injury in murine stroke.

BACKGROUND AND PURPOSE: Although the deleterious role of several proinflammatory mediators, including P-selectin, in reperfused stroke is well established, the role of E-selectin has not been fully characterized. METHODS: E-selectin mRNA expression was studied at 4, 10, and 24 hours after reperfusion with reverse transcription and polymerase chain reaction in mice (n=18) subjected to transient intraluminal middle cerebral artery occlusion (MCAO). Mice received intravenous injection with anti-E-selectin monoclonal antibody (10, 35, or 50 microg), nonimmune IgG, or vehicle immediately before MCAO and 90 minutes later (n=85). Others received anti-E-selectin antibody 3 or 6 hours after MCAO (n=32). Myeloperoxidase activity was measured in sham-operated mice and after 10 hours of reperfusion in saline-, nonimmune IgG-, or anti-E-selectin IgG-treated cohorts (n=17). Serial cerebral blood flow was measured with laser-Doppler flowmetry, and outcomes were assessed by neurological deficits and infarct volumes with the use of planimetric analysis of triphenyltetrazolium chloride-stained sections. RESULTS: Upregulated E-selectin expression occurred in the ischemic cerebral vasculature within 4 hours of reperfusion and persisted for 24 hours. Anti-E-selectin antibody increased ischemic cortical cerebral blood flow up to 2.6-fold (P:<0.05). In addition to dose-dependent reductions in neurological deficits (P:<0.05), mortality, and infarct volumes (P:<0.01 for 35 and 50 microg), anti-E-selectin treatment reduced cerebral neutrophil accumulation (P:<0.05) and was neuroprotective even if delayed until 3 hours after ischemia (P:<0. 05). CONCLUSIONS: These findings establish a functional role for E-selectin in the pathogenesis of tissue injury after cerebral ischemia and reperfusion and suggest that E-selectin blockade may be clinically useful in the treatment of reperfused stroke.

Heterologous cell-cell interactions: thromboregulation, cerebroprotection and cardioprotection by CD39 (NTPDase-1).

Blood platelets maintain vascular integrity and promote primary and secondary hemostasis following interruption of vessel continuity. Biochemical or physical damage to the coronary, carotid or peripheral arteries is followed by excessive platelet activation and recruitment culminating in vascular occlusion and tissue ischemia. Currently inadequate therapeutic approaches to stroke and coronary artery disease are a public health issue. Following our demonstration of neutrophil leukotriene production from arachidonate released from activated aspirin-treated platelets, we studied interactions between platelets and other blood cells, leading to concepts of transcellular metabolism and thromboregulation. Thrombosis has a proinflammatory component whereby biologically active substances are synthesized by interactions between different cell types that could not individually synthesize the product(s). Endothelial cells control platelet reactivity via three biochemical systems-autacoids leading to production of prostacyclin and nitric oxide, and endothelial ecto-ADPase/CD39/NTPDase-1. The autacoids are fluid-phase reactants, not produced by tissues in the basal state. They are only synthesized intracellularly and released upon interactions of cells with an agonist. When released, autacoids exert fleeting actions in the immediate milieu, and are rapidly inactivated. CD39 is an integral component of the endothelial cell surface and is substrate-activated. It maintains vascular fluidity in the complete absence of prostacyclin and nitric oxide, indicating that they are ancillary components of hemostasis. Therapeutic implications for the autacoids have not been compelling because of their transient, local and fleeting action, and limited potency. Conversely, CD39, acting solely on the platelet releasate, is efficacious in three different animal models. It metabolically neutralizes a prothrombotic platelet releasate via deletion of ADP--the major recruiting agent responsible for formation of an occlusive thrombus. In addition, solCD39 reduced ATP- and ischemia-induced norepinephrine release in the heart. This reduction can prevent fatal arrhythmia. Moreover, solCD39 ameliorated the sequelae of stroke in CD39 null mice. CD39 represents the next generation of cardioprotective and cerebroprotective molecules.

Complement activation as a cause for primary graft failure in an isogeneic rat model of hypothermic lung preservation and transplantation.

Although agents that inhibit complement activation may be beneficial in discordant xenotransplantation, it is not known whether local complement activation occurs and is deleterious after isogeneic lung transplantation. Lungs were harvested from Lewis rats subjected to 4 degrees C 6-hr preservation followed by transplantation into strain-, gender-, and weight-matched recipients. Transplanted lungs demonstrated increased immunostaining for C5b-9 compared with nontransplanted controls, confirming local complement activation in this isograft model. To investigate the physiologic relevance of complement activation in the transplanted lung, the native pulmonary artery was ligated after transplantation, and pulmonary vascular resistance (mmHg/ml/min), arterial oxygenation (mmHg), graft neutrophil infiltration (myeloperoxidase activity, deltaAbs 460 nm/min), and recipient survival were measured at 30 min. Animals received either saline (control; n=22) or soluble complement receptor type-1 (sCR1, 15 mg/kg; n=19) 2 min before reperfusion. Animals treated with sCR1 showed a marked reduction in serum complement hemolytic activity (CH50; 90% lower than that of control animals, P<0.001). Compared with controls, sCR1-treated animals showed reduced pulmonary vascular resistance (2.9+/-1.1 vs. 8.5+/-1.5 mmHg/ml/min, P<0.05), improved arterial oxygenation (194+/-34 vs. 91+/-17 mmHg, P<0.05), decreased neutrophil infiltration (35% decrease, P<0.005), and improved recipient survival (74% vs. 23%, P<0.005). In parallel with the reduction in complement hemolytic activity in sCR1-treated animals, immunohistology of the transplanted lung revealed decreased C5b-9 deposition compared with controls. Taken together, these data indicate that complement activation occurs after lung preservation and transplantation in an isograft model, and that inhibiting complement activation improves outcome after transplantation.