Leukocyte-mediated Injury Research Articles

Abstract 161: Impact Of Leukocyte Filtration And Leukocyte Modulation On Myocardial Injury After Prolonged Cardiac Arrest Treated With Extracorporeal Cardiopulmonary Resuscitation In A Swine Model

Background: Post-arrest myocardial dysfunction is one of the major causes of rearrest within early period after return of spontaneous circulation (ROSC). Multiple interacting processes including leukocyte-mediated injury is suspected to contribute to deterioration of cardiac function after cardiac arrest. Unlike the standard leukocyte filter (LF), the leukocyte modulation (L-MOD) device is expected to alter the natural inflammatory process by preferentially binding and inactivating the activated leukocytes. We tested the hypothesis that either a standard LF system or a L-MOD device will decrease myocardial injury during extracorporeal cardiopulmonary resuscitation (ECPR). Methods: Total 34 swine (45.5±2.5 kg, male=18, female=16) underwent 8 minutes of untreated ventricular fibrillation cardiac arrest followed by mechanical CPR for 30 minutes. Immediately followed by 8 hours of ECPR with randomly assigned leukocyte treatment protocol; ECPR with standard care (n=10), LF (n=12), and L-MOD (n=12). Left ventricular ejection fraction (LVEF) was evaluated hourly by transthoracic echocardiography. At the end of the study, the heart was removed, fixed and embedded in paraffin. 5- μm sections off the left and right ventricular (RV) free wall were H & E stained for analysis. Five views each of LV and RV under 20хobjective lens were randomly selected with Leica LAS X software. Myofiber degeneration and hemorrhage were graded as 0 (absence) to 3 (severe). Grade was average of 5 views. Results: The mean LVEF at baseline was 65±6%. There were no significantly difference in rate of achieving ROSC (70% vs. 75% vs. 58.3%, p=0.671) and LVEF at 8 hours after initiating ECPR (35±27% vs. 36±31% vs. 28±30%, p=0.776) between the group with control, LF, and LMOD respectively. There were no significant difference in myofiber degeneration grade (LV, 1.5±0.5 vs. 1.1±0.6 vs. 1.4±0.4, p=0.334; and RV, 1.6±0.6 vs. 1.8±0.7 vs. 1.5±0.6, p=0.525) and hemorrhage grade (LV, 0.0±0.1 vs. 0.0±0.1 vs. 0.0±0.1, p=0.841; and RV, 0.0±0.0 vs. 0.0±0.0 vs. 0.0±0.0) between control, LF, and L-MOD groups. Discussion: In swine model with prolonged cardiac arrest treated with ECPR, a standard LF system and a L-MOD device did not decrease early myocardial injury.

Anti-vascular endothelial growth factor therapy for diabetic macular edema.

Diabetes mellitus is a serious health problem that affects over 350 million individuals worldwide. Diabetic retinopathy (DR), which is the most common microvascular complication of diabetes, is the leading cause of new cases of blindness in working-aged adults. Diabetic macular edema (DME) is an advanced, vision-limiting complication of DR that affects nearly 30% of patients who have had diabetes for at least 20 years and is responsible for much of the vision loss due to DR. The historic standard of care for DME has been macular laser photocoagulation, which has been shown to stabilize vision and reduce the rate of further vision loss by 50%; however, macular laser leads to significant vision recovery in only 15% of treated patients. Mechanisms contributing to the microvascular damage in DR and DME include the direct toxic effects of hyperglycemia, sustained alterations in cell signaling pathways, and chronic microvascular inflammation with leukocyte-mediated injury. Chronic retinal microvascular damage results in elevation of intraocular levels of vascular endothelial growth factor A (VEGF), a potent, diffusible, endothelial-specific mitogen that mediates many important physiologic processes, including but not limited to the development and permeability of the vasculature. The identification of VEGF as an important pathophysiologic mediator of DME suggested that anti-VEGF therapy delivered to the eye might lead to improved visual outcomes in this disease. To date, four different inhibitors of VEGF, each administered by intraocular injection, have been tested in prospective, randomized phase II or phase III clinical trials in patients with DME. The results from these trials demonstrate that treatment with anti-VEGF agents results in substantially improved visual and anatomic outcomes compared with laser photocoagulation, and avoid the ocular side effects associated with laser treatment. Thus, anti-VEGF therapy has become the preferred treatment option for the management of DME in many patients.

Strategies for the analysis of chlorinated lipids in biological systems

Myeloperoxidase-derived HOCl reacts with the vinyl ether bond of plasmalogens yielding α-chlorofatty aldehydes. These chlorinated aldehydes can be purified using thin-layer chromatography, which is essential for subsequent analysis of extracts from some tissues such as myocardium. The α-chlorofatty aldehyde 2-chlorohexadecanal (2-ClHDA) is quantified after conversion to its pentafluorobenzyl oxime derivative using gas chromatography–mass spectrometry and negative-ion chemical ionization detection. 2-ClHDA accumulates in activated human neutrophils and monocytes, as well as in atherosclerotic lesions and infarcted myocardium. Metabolites of 2-ClHDA have also been identified, including the oxidation product, 2-chlorohexadecanoic acid (2-ClHA), and the reduction product, 2-chlorohexadecanol. 2-ClHA can be quantified using LC–MS with selected reaction monitoring (SRM) detection. 2-ClHA can be ω-oxidized by hepatocytes and subsequently β-oxidized from the ω-end, leading to the production of the dicarboxylic acid, 2-chloroadipic acid. This dicarboxylic acid is excreted in the urine and can also be quantified using LC–MS methods with SRM detection. Quantitative analyses of these novel chlorinated lipids are essential to identify the role of these lipids in leukocyte-mediated injury and disease.

Pretreatment with perfluorohexane vapor attenuates fMLP-induced lung injury in isolated perfused rabbit lungs

ABSTRACTThe authors investigated the protective effects and dose dependency of perfluorohexane (PFH) vapor on leukocyte-mediated lung injury in isolated, perfused, and ventilated rabbit lungs. Lungs received either 18 vol.% (n = 7), 9 vol.% (n = 7), or 4.5 vol.% (n = 7) PFH. Fifteen minutes after beginning of PFH application, lung injury was induced with formyl-Met-Leu-Phe (fMLP). Control lungs (n = 7) received fMLP only. In addition 5 lungs (PFH-sham) remained uninjured receiving 18 vol.% PFH only. Pulmonary artery pressure (mPAP), peak inspiratory pressure (Pmax), and lung weight were monitored for 90 minutes. Perfusate samples were taken at regular intervals for analysis and representative lungs were fixed for histological analysis. In the control, fMLP application led to a significant increase of mPAP, Pmax, lung weight, and lipid mediators. Pretreatment with PFH attenuated the rise in these parameters. This was accompanied by preservation of the structural integrity of the alveolar architecture and air-blood barrier. In uninjured lungs, mPAP, Pmax, lung weight, and lipid mediator formation remained uneffected in the presence of PFH. The authors concluded that pretreatment with PFH vapor leads to an attenuation of leukocyte-mediated lung injury. Vaporization of perfluorocarbons (PFCs) offers new therapeutic options, making use of their protective and anti-inflammatory properties in prophylaxis or in early treatment of acute lung injury.

Shiga toxin-associated hemolytic uremic syndrome: pathophysiology of endothelial dysfunction

Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli O157:H7 has become a global threat to public health, as a primary cause of a worldwide spread of hemorrhagic colitis complicated by diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure that mainly affects early childhood. Endothelial dysfunction has been recognized as the trigger event in the development of microangiopathic processes. Endothelial cells, mainly those located in the renal microvasculature, are primary targets of the toxic effects of Stx1 and 2. Stxs bound to their specific globotriaosylceramide (Gb3Cer) receptor on the cell surface trigger a cascade of signaling events, involving NF-κB activation, that induce expression of genes encoding for adhesion molecules and chemokines, and culminate in the adhesion of leukocytes to endothelial cells, thereby increasing the endothelial susceptibility to leukocyte-mediated injury. Activated endothelial cells in response to Stxs lose the normal thromboresistance phenotype and become thrombogenic, initiating microvascular thrombus formation. Evidence is emerging that complement activation in response to Stxs favors platelet thrombus formation on endothelial cells, which may play a role in amplifying the inflammation-thrombosis circuit in Stx-associated HUS.

The contributions of aspirin and microbial oxygenase to the biosynthesis of anti-inflammatory resolvins: Novel oxygenase products from ω-3 polyunsaturated fatty acids

Resolvins (Rvs) are oxygenated products derived from ω-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid that carry potent protective bioactions present in resolving inflammatory exudates. Resolvin E1 (RvE1) is biosynthesized in vivo from EPA via transcellular biosynthetic routes during cell–cell interactions, and thus RvE1 is formed in vivo during multicellular responses such as inflammation and microbial infections. RvE1 protects tissues from leukocyte-mediated injury and counterregulates proinflammatory gene expression. These newly identified Rvs may underlie the beneficial actions of ω-3 PUFAs especially in chronic disorders where unresolved inflammation is a key mechanism of pathogenesis. Here, we present an overview of the biosynthesis of RvE1, with a focus on the aspirin-triggered and microbial P450-initiated pathways. The generation of RvE1 and its actions appear to dampen acute leukocyte responses and facilitate the resolution of inflammation.

Novel lipid mediator aspirin-triggered lipoxin A4 induces heme oxygenase-1 in endothelial cells

Lipoxins (LX) and aspirin-triggered LX (ATL) are eicosanoids generated during inflammation via transcellular biosynthetic routes that elicit distinct anti-inflammatory and proresolution bioactions, including inhibition of leukocyte-mediated injury, stimulation of macrophage clearance of apoptotic neutrophils, repression of proinflammatory cytokine production, and inhibition of cell proliferation and migration. Recently, it was reported that aspirin induces heme oxygenase-1 (HO-1) expression on endothelial cells (EC) in a COX-independent manner, what confers protection against prooxidant insults. However, the underlying mechanisms remain unclear. In this study, we investigated whether an aspirin-triggered lipoxin A(4) stable analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A(4) (ATL-1) was able to induce endothelial HO-1. Western blot analysis showed that ATL-1 increased HO-1 protein expression associated with increased mRNA levels on EC in a time- and concentration-dependent fashion. This phenomenon appears to be mediated by the activation of the G protein-coupled LXA(4) receptor because pertussis toxin and Boc-2, a receptor antagonist, significantly inhibited ATL-1-induced HO-1 expression. We demonstrate that treatment of EC with ATL-1 inhibited VCAM and E-selectin expression induced by TNF-alpha or IL-1beta. This inhibitory effect of the analog is modulated by HO-1 because it was blocked by SnPPIX, a competitive inhibitor that blocks HO-1 activity. Our results establish that ATL-1 induces HO-1 in human EC, revealing an undescribed mechanism for the anti-inflammatory activity of these lipid mediators.

Causes of severe chronic venous insufficiency

A large number of adults in this country have some form of chronic venous insufficiency and a significant percentage of these have venous ulcers. The past decade has refined understanding of leukocyte-mediated injury and has elucidated the role of inflammatory processes in the dermal pathology of chronic venous insufficiency. Understanding of these pathologic cellular functions and molecular regulation of these processes is increasing.

Lipoxins: endogenous regulators of inflammation.

Over the past decade, compelling in vivo and in vitro studies have highlighted lipoxins (LXs) and aspirin-triggered LXs (ATLs) as endogenously produced anti-inflammatory eicosanoids. LXs and ATLs elicit distinct anti-inflammatory and proresolution bioactions that include inhibition of leukocyte-mediated injury, stimulation of macrophage clearance of apoptotic neutrophils, repression of proinflammatory cytokine production, modulation of cytokine-stimulated metalloproteinase activity, and inhibition of cell proliferation and migration. An overview of recent advances in LX physiology is provided, with particular emphasis on the cellular and molecular processes involved. These data coupled with in vivo models of inflammatory diseases suggest that LX bioactions may be amenable to pharmacological mimicry for therapeutic gain.

Novel Docosanoids Inhibit Brain Ischemia-Reperfusion-mediated Leukocyte Infiltration and Pro-inflammatory Gene Expression

Ischemic stroke triggers lipid peroxidation and neuronal injury. Docosahexaenoic acid released from membrane phospholipids during brain ischemia is a major source of lipid peroxides. Leukocyte infiltration and pro-inflammatory gene expression also contribute to stroke damage. In this study using lipidomic analysis, we have identified stereospecific messengers from docosahexaenoate-oxygenation pathways in a mouse stroke model. Aspirin, widely used to prevent cerebrovascular disease, activates an additional pathway, which includes the 17R-resolvins. The newly discovered brain messenger 10,17S-docosatriene potently inhibited leukocyte infiltration, NFkappaB, and cyclooxygenase-2 induction in experimental stroke and elicited neuroprotection. In addition, in neural cells in culture, this lipid messenger also inhibited both interleukin 1-beta-induced NFkappaB activation and cyclooxygenase-2 expression. Thus, the specific novel bioactive docosanoids generated in vivo counteract leukocyte-mediated injury as well as pro-inflammatory gene induction. These results challenge the view that docosahexaenoate only participates in brain damage and demonstrate that this fatty acid is also the endogenous precursor to a neuroprotective signaling response to ischemia-reperfusion.

Lipoxins in chronic inflammation.

The discovery of endogenous molecules involved in counterregulation of inflammatory responses that may lead to tissue injury provides an opportunity to explore new therapeutic approaches based on manipulation of new pathways. Natural counterregulatory pathways may reduce the possibility of unwanted toxic side-effects. Lipoxins are trihydroxytetraene-containing eicosanoids that are generated within the vascular lumen during platelet-leukocyte interactions and at mucosal surfaces via leukocyte-epithelial cell interactions. During cell-cell interactions, transcellular biosynthetic pathways are the major lipoxin biosynthetic routes, and thus, in humans, lipoxins are formed in vivo during multicellular responses, such as inflammation and asthma. This branch of the eicosanoid cascade generates specific tetraene-containing products that serve as "stop signals" for neutrophils that regulate key steps in leukocyte trafficking and prevent neutrophil-mediated tissue injury. These novel anti-inflammatory lipid mediators also appear to facilitate the resolution of the acute inflammatory response. In this review, recent findings and new concepts pertaining to the generation of lipoxins and their impact on the resolution of acute inflammation, and organ protection from leukocyte-mediated injury, are presented. The parallels and possible associations with periodontal diseases are discussed.

Glucocorticoids reduce ischemia-reperfusion-induced myocardial apoptosis in immature hearts

Background. Transient myocardial dysfunction often occurs after ischemia-reperfusion with immature myocardium appearing particularly susceptible. Neutrophil adhesion and activation contribute to ischemia-reperfusion injury after cardiopulmonary bypass (CPB), possibly resulting in cell death. The hypothesis was that glucocorticoids could prevent reperfusion-induced myocardial dysfunction by blunting leukocyte-mediated injury. Methods. Neonatal piglets were cooled with CPB followed by 2 hours of circulatory arrest. Animals were rewarmed, removed from CPB, and allowed to recover for 2 hours. Methylprednisolone (60 mg/kg) was administered in the CPB priming solution to one group (intraoperative glucocorticoids). In another group (preoperative glucocorticoids), 30 mg/kg methylprednisolone was administered 6 hours before CPB in addition to the intraoperative dose (30 mg/kg). Control animals received no glucocorticoids. Results. Apoptotic myocardial cells measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay and caspase-3 activity were reduced in animals administered glucocorticoids compared with controls ( p < 0.05). Animals receiving either intraoperative or preoperative glucocorticoids had 0.10 ± 0.07 and 0.13 ± 0.05 apoptotic cells per high-power field, respectively, whereas 0.33 ± 0.15 apoptotic cells were detected with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling in control animals. Glucocorticoid administration reduced myocardial intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 mRNA expression compared with control piglets. Maximum rate of increase of left ventricular pressure was 62% ± 9% of baseline in control animals at 120 minutes of recovery compared with 96% ± 6% and 95% ± 10% of baseline in animals receiving intraoperative and preoperative glucocorticoids, respectively ( p < 0.05). Conclusions. The reduction of neutrophil adhesion and activation proteins in neonatal myocardium was associated with less apoptotic cell death after glucocorticoid administration. The blunting of apoptosis in glucocorticoid-treated animals was also associated with improved recovery of left ventricular systolic function in neonatal animals after CPB and circulatory arrest. Glucocorticoid attenuation of myocardial apoptosis might have important implications for maintaining long-term ventricular function after ischemia and reperfusion.

Lipoxins and aspirin-triggered 15-epi-lipoxin biosynthesis: an update and role in anti-inflammation and pro-resolution

Lipoxins (LX) are trihydroxytetraene-containing eicosanoids that are generated within the vascular lumen during platelet–leukocyte interactions and at mucosal surfaces via leukocyte-epithelial cell interactions. Recent findings have given several new concepts that are reviewed here regarding the generation of LX and 15 epi-LX and their impact in the resolution of acute inflammation and organ protection from leukocyte-mediated injury. During cell–cell interactions, transcellular biosynthetic pathways are used as major LX biosynthetic routes, and thus, in humans, LX are formed in vivo during multicellular responses such as inflammation, and asthma. This branch of the eicosanoid cascade generates specific tetraene-containing products that serve as neutrophil “stop signals,” in that they regulate key steps in leukocyte trafficking and prevent neutrophil-mediated acute tissue injury. In addition, aspirin’s mechanism of action also involves the triggering of carbon 15 epimers of lipoxins or 15-epi-lipoxins that mimic the bioactions of native LX. An overview of these recent developments is presented with a focus on the cellular and molecular interactions of these novel anti-inflammatory lipid mediators that also appear to facilitate the resolution of acute inflammatory responses.

Endothelin receptor blockade reduces ventricular dysfunction and injury after reoxygenation

Background. Reoxygenation of hypoxic myocardium during repair of congenital heart defects results in poor ventricular function and cellular injury. Endothelin-1 (ET-1), a potent vasoconstrictor that increases during hypoxia, may suppress myocardial function and activate leukocytes. The objective was to determine whether administration of an endothelin receptor antagonist could improve ventricular function and decrease cardiac injury after hypoxia and reoxygenation. Methods. Fourteen piglets underwent 90 minutes of ventilator hypoxia, 1 hour of reoxygenation on cardiopulmonary bypass, and 2 hours of recovery (controls). Nine additional animals received an infusion of Bosentan, an ET(A/B) receptor antagonist, (5 mg/kg per hour) during hypoxia and reoxygenation. Results. Right and left ventricular dP/dt in controls decreased to 78% and 52% of baseline, respectively, after recovery ( p < 0.05). In contrast, Bosentan-treated animals had complete preservation of RV dP/dt and less depression of LV dP/dt. Bosentan reduced the hypoxia and reoxygenation–induced elevation of ET-1 and iNOS mRNA at the end of recovery ( p < 0.05). Bosentan-treated animals had diminished myocardial myeloperoxidase activity and lipid peroxidation compared with controls ( p < 0.05). Myocardial apoptotic index, elevated by hypoxia and reoxygenation, was lower in the Bosentan-treated animals ( p < 0.05). Conclusions. Endothelin-1 receptor antagonism improved functional recovery and decreased leukocyte-mediated injury after reoxygenation. The reduction in cardiac cell death might also improve long-term outcome after reoxygenation injury.

Pentoxifylline reduces coronary leukocyte accumulation early in reperfusion after cold ischemia

Background. Ischemia/reperfusion injury can complicate recovery in cardiac operations. Ischemia induces endothelial dysfunction, which may contribute to leukocyte accumulation during reperfusion. Leukocyte-mediated injury may then occur. Using intravital microscopy we previously reported increased leukocyte retention in coronary capillaries and venules during early reperfusion during warm ischemia/reperfusion. In this study we investigated whether cold cardioplegic protection would limit leukocyte sequestration in coronary microvessels early in reperfusion. Pentoxifylline (PTX) has antiinflammatory effects and may limit endothelial dysfunction during ischemia/reperfusion. The effect of cardioplegia modification with PTX was also examined. Methods. Isolated rat hearts were subjected to 90 minutes of 4°C ischemia after arrest with cardioplegia. Hearts were reperfused with diluted whole blood containing fluorescent-labeled leukocytes. Leukocyte retention in coronary microvessels was observed with intravital microscopy. Three groups were studied, nonischemic control, cold ischemia, and PTX-modified cold ischemia. Results. In coronary capillaries, leukocyte trapping was nearly doubled in unmodified cold ischemia versus control. PTX modification significantly reduced leukocyte accumulation. In coronary venules, greater leukocyte adhesion was observed in unmodified cold ischemia compared to nonischemic controls. PTX modification significantly reduced leukocyte adhesion. Conclusions. Cold cardioplegia did not prevent leukocyte retention in the coronary microcirculation early in reperfusion. PTX modification of cardioplegia significantly reduced leukocyte sequestration in coronary capillaries and venules. Preserving endothelial function during ischemia may limit leukocyte accumulation and ischemia/reperfusion injury after cardiac operation.

Inhibition by leukocyte depletion of neointima formation after balloon angioplasty in a rabbit model of restenosis.

The aim of the current study was to examine neointima formation in balloon injured left subclavian artery of rabbits subjected to two different methods of leukocyte depletion at the time of injury. Angioplasty of the left subclavian artery was performed in leukopenic male New Zealand White rabbits. Depletion of circulating leukocytes was induced by either mustine hydrochloride or an antibody against leukocyte common antigen (anti-LCA) before angioplasty. Left and right subclavian arteries were removed 28 days after injury for morphological analysis and measurement of neointimal size. At the same time, leukocytes were isolated from autologous rabbit blood for 51Cr-labelling for assessment of leukocyte adhesion to injured and non-injured artery segments. Leukopenia decreased neointima formation in injured arteries (neointimal area was 0.09+/-0.03 mm(2) in mustine-treated arteries, n=8, vs. 0.56+/-0.07 mm(2) in control arteries, n=7; P<0.001 and 0.07+/-0.01 mm(2) in anti-LCA treated arteries, n=9, vs. 0.22+/-0.04 mm(2) in non immune serum-treated arteries, n=9; P<0.001). Adventitial fibrosis was also significantly (P<0.05) decreased by both leukopenic interventions. Neither medial nor adventitial area was modified in any of the groups. No differences in leukocyte adhesion were observed between injured and non-injured arteries in any of the experimental groups at the 28 day time point. These results suggest that leukocytes play a major role in the development of two of the major characteristics of the response to balloon injury, namely formation of neointima and adventitial fibrosis, that currently limit the success of clinical angioplasty. Elucidation of the fine mechanisms involved in leukocyte-mediated injury may lead to the discovery of novel therapeutic targets for the prevention of restenosis.

Myocardial protection with leukocyte depletion in cardiac surgery.

A role of neutrophils in ischemia-reperfusion injury has been focused on as one of the mediating factors of inflammatory reactions. Current studies have reported the efficacy of leukocyte-depletion in reperfusion by using leukocyte removal filter to attenuate reperfusion injury during open heart surgeries. For clinical application, we have introduced leukocyte-depleted terminal blood cardioplegia (LDTC) in adult patients and leukocyte-depleted blood cardioplegia in pediatric patients. The results of elective surgery in noncompromised LDTC did not significantly alter the results in terms of leakage of creatine kinase (CK)-MB, production of malondialdehyde from myocardium, and dopamine dose required at the weaning from cardiopulmonary bypass compared with the whole-blood reperfusion or with terminal cardioplegia alone. In contrast, the results in emergency coronary artery bypass graft (CABG) patients differed significantly between the LDTC group and the other two groups. Leukocyte-depleted reperfusion was also effective in a similar fashion for patients with severe left ventricular hypertrophy caused by chronic aortic valve disease. Leukocyte-depleted blood cardioplegia was useful in pediatric patients. Thus, leukocyte depletion may be beneficial as an adjunct to terminal blood cardioplegia or blood cardioplegia during cardiac surgery to attenuate leukocyte-mediated ischemia-reperfusion injury in patients with compromised hearts, such as those with preoperative ischemic insults, severe left ventricular hypertrophy, and in pediatric patients.

Acetylcholine prevents intercellular adhesion molecule 1 (CD54)-induced focal adhesion complex assembly in endothelial cells via a nitric oxide-cGMP-dependent pathway.

Nitric oxide (NO) is induced by exposure of endothelial cells (EC) to acetylcholine, where it acts in a paracrine manner to relax smooth muscle and as a defensive molecule to inhibit the adhesion of leukocytes to EC. The mechanism(s) of the antiadhesive properties of constitutive NO are poorly understood. In these studies, we found that NO induced by acetylcholine exerts autocrine effects, which interfere with normal adhesion mechanisms. The function of the adhesion molecule intercellular adhesion molecule 1 (CD54) of EC was measured using latex beads coated with antibody to CD54 as a model for CD54 ligation by the leukocyte beta2 integrin. Recruitment of filamentous actin (F-actin) and of the signaling molecule vasodilator-stimulated phosphoprotein (VASP) was measured by immunofluorescence microscopy. Exposure of EC to anti-CD54 beads induced the subplasmalemmal assembly of F-actin and VASP. Acetylcholine blocked the anti-CD54 bead-induced translocation of F-actin and VASP; this effect was reversed by inhibition of NO production. The NO action did not interfere with binding, but completely inhibited the assembly of the focal activation complex, which we believe is necessary for firm heterotypic adhesion between leukocyte and EC. Further studies indicated that the NO effect was due to its capacity to raise cGMP. Platelet endothelial cell adhesion molecule 1 (CD31, also implicated in leukocyte adhesion) did not mimic CD54 responses. These results indicate that the ligation of endothelial cell CD54 induces the assembly of subplasmalemmal F-actin and the recruitment of VASP. NO derived from constitutive nitric oxide synthase acts to disrupt these CD54-elicited endothelial cell responses. This action may protect vascular endothelium from leukocyte-mediated injury.

Bosentan prevents hypoxia-reoxygenation–induced pulmonary hypertension and improves pulmonary function

Background. Acute hypoxia results in increased pulmonary vascular resistance. Despite reoxygenation, pulmonary vascular resistance remains elevated and pulmonary function is altered. Endothelin-1 might contribute to hypoxia-reoxygenation–induced pulmonary hypertension and to reoxygenation injury by stimulating leukocytes. This study was carried out using an established model of hypoxia and reoxygenation to determine whether endothelin-1 blockade with Bosentan could prevent hypoxia-reoxygenation–induced pulmonary hypertension and reoxygenation injury.Methods. Twenty neonatal piglets underwent 90 minutes of hypoxia, 60 minutes of reoxygenation on cardiopulmonary bypass, and 2 hours of recovery. Control animals (n = 12) received no drug treatment, whereas the treatment group (n = 8) received the endothelin-1 receptor antagonist, Bosentan, throughout hypoxia.Results. In controls, pulmonary vascular resistance increased during hypoxia to 491% of baseline and remained elevated after reoxygenation; however in the Bosentan group, it increased to only 160% of baseline by end-hypoxia, then decreased to 76% at end-recovery. Arterial endothelin-1 levels in controls increased to 591% of baseline after reoxygenation. Arterial nitrite levels decreased during hypoxia in controls but were maintained in the Bosentan group. Consequently, animals in the Bosentan group had better postreoxygenation pulmonary vascular resistance, A-a gradient, and airway resistance along with lower myeloperoxidase levels than controls.Conclusions. Acute hypoxia and postreoxygenation pulmonary hypertension was attenuated by Bosentan, which maintained nitric oxide levels during hypoxia, decreased leukocyte-mediated injury, and improved pulmonary function.

Effect of modified ultrafiltration on plasma thromboxane B2, leukotriene B4, and endothelin-1 in infants undergoing cardiopulmonary bypass

Background. Plasma thromboxane B2 (TXB2), leukotriene B4 (LTB4), and endothelin-1 (ET-1) levels increase on cardiopulmonary bypass (CPB). Elevated levels of TXB2 and ET-1 have been correlated with postoperative pulmonary hypertension in infants undergoing repair of congenital heart defects. LTB4 is a potent chemotactic cytokine whose levels correlate with leukocyte-mediated injury. Modified ultrafiltration (MUF) has been associated with improved hemodynamics and pulmonary function, in addition to its beneficial effects on fluid balance and blood conservation. Recent investigations have suggested that removal of cytokines may be the cause of the improved cardiopulmonary function seen with MUF. Methods. Plasma TXB2, ET-1, and LTB4 levels were measured in 34 infants undergoing CPB: 22 underwent MUF (group 1), and 12 did not (group 2). Samples were obtained at various time points. All patients underwent conventional ultrafiltration during the rewarming phase of cardiopulmonary bypass. Results. In group 1, mean end-CPB TXB2 level was 101.2 pg/mL versus 46.9 pg/mL post-MUF ( p < 0.05). The mean TXB2 level 1 hour post-CPB (54.1 pg/mL) was not significantly different from the post-MUF level. In group 2, the mean end-CPB TXB2 level was 123.6 pg/mL versus 53.2 pg/mL 1 hour post-CPB. Hence, TXB2 levels decreased by similar amounts and to similar levels by 1 hour post-CPB in both groups. ET-1 levels increased after CPB and were unaffected by MUF: 1.45, 1.80, 2.55 pg/mL at end-CPB, post-MUF, and 1 hour post-CPB, respectively, in group 1; and 1.51, and 2.73 pg/mL at end-CPB and 1 hour post-CPB in group 2. LTB4 levels post-MUF were 119% of pre-MUF values, and were similar at 1 hour post-CPB in both groups. Conclusions. Despite reduction in TXB2 by MUF, values were similar and approached baseline 1 hour post-CPB in both groups. LTB4 levels increased slightly with MUF. ET-1 levels increased during and post-CPB and were unaffected by MUF. MUF does not appear to have a significant effect on post-CPB levels of TXB2, ET-1, and LTB4. Therefore, the improved hemodynamics observed with MUF do not appear to be related to removal of these cytokines.