Inhibition of the tissue factor-thrombin pathway limits infarct size after myocardial ischemia-reperfusion injury by reducing inflammation.

Functional inhibition of tissue factor (TF) has been shown to improve coronary blood flow after myocardial ischemia/reperfusion (I/R) injury. TF initiates the coagulation protease cascade, resulting in the generation of the serine protease thrombin and fibrin deposition. Thrombin can also contribute to an inflammatory response by activating various cell types, including vascular endothelial cells. We used a rabbit coronary ligation model to investigate the role of TF in acute myocardial I/R injury. At-risk areas of myocardium showed increased TF expression in the sarcolemma of cardiomyocytes, which was associated with a low level of extravascular fibrin deposition. Functional inhibition of TF activity with an anti-rabbit TF monoclonal antibody administered either 15 minutes before or 30 minutes after coronary ligation reduced infarct size by 61% (P = 0.004) and 44% (P = 0.014), respectively. Similarly, we found that inhibition of thrombin with hirudin reduced infarct size by 59% (P = 0.014). In contrast, defibrinogenating the rabbits with ancrod had no effect on infarct size, suggesting that fibrin deposition does not significantly contribute to infarct size. Functional inhibition of thrombin reduced chemokine expression and inhibition of either TF or thrombin reduced leukocyte infiltration. We propose that cardiomyocyte TF initiates extravascular thrombin generation, which enhances inflammation and injury during myocardial I/R.

Adaptive responses of the endothelium to stress.

It is now established that endothelial cells acquire several functional properties in response to a diverse array of extracellular stimuli. This expression of an altered phenotype is referred to as endothelial cell activation, and it includes several activities that promote inflammation and coagulation. While it is recognized that endothelial cell activation has a principal role in host defense, recent studies also demonstrate that endothelial cells are capable of complex molecular responses that protect the endothelium against various forms of stress including heat shock, hypoxia, oxidative stress, shock, ischemia-reperfusion injury, toxins, wounds, and mechanical stress. In this review, we examine endothelial cell genotypic and phenotypic responses to stress. Also, we highlight important cellular stress responses that, although not yet demonstrated directly in endothelial cells, likely exist as part of the repertoire of stress responses in endothelium. A detailed understanding of the molecular mechanisms mediating the adaptive responses of endothelial cells to stress should facilitate the development of novel therapeutics to aid in the management of diverse surgical diseases and their complications.

Treating myocardial ischemia-reperfusion injury by targeting endothelial cell transcription.

Exacerbation of, rather than improvement in, a hypoxic injury after reperfusion of ischemic tissues is recognized as the specific clinicopathologic entity referred to as ischemia/reperfusion (I/R) injury. Arguably, one of the most common forms of I/R injury occurs during cardiac surgery, which has a mandatory period of myocardial ischemia required to allow surgery in a bloodless, motionless field, followed by coronary artery reperfusion after removal of the aortic cross-clamp. In this review, we examine the endothelial cell activation phenotype that initiates and propagates myocardial I/R injury. Emphasis is given to the biology of one transcription factor, NF-kappaB, that has the principal role in the regulation of many endothelial cell genes expressed in activated endothelium. NF-kappaB-dependent transcription of endothelial cell genes that are transcribed in response to I/R injury may be a favorable approach to preventing tissue injury in the setting of I/R. Elucidating safe and effective therapy to inhibit transcription of endothelial cell genes involved in promoting injury after I/R injury may have wide applicability to the patients with heart disease and other forms of I/R injury.

Oxidative stress induces NF-kappaB nuclear translocation without degradation of IkappaBalpha.

BACKGROUND: Rel/NF-kappaB, an oxidative stress-responsive transcription factor, participates transiently in the control of gene expression. The cellular mechanisms that mediate NF-kappaB activation during ischemia (and during reperfusion in the course of treating ischemia) are not known. METHODS AND RESULTS: To investigate the NF-kappaB activation induced during oxidative stress, we examined human cardiac tissue obtained during surgical procedures requiring cardiopulmonary bypass. In vitro, we examined human umbilical vein endothelial cells (HUVECs) exposed to hypoxia, reoxygenation after hypoxia, or a reactive oxygen intermediate (H(2)O(2)). Electrophoretic mobility shift assays performed on right atrial tissue revealed prominent NF-kappaB activation after hearts had been exposed to ischemia and reperfusion. The assays also showed that NF-kappaB activation was observed in hypoxic HUVECs after reoxygenation and in cultures treated with H(2)O(2) (500 micromol/L). Pervanadate (200 micromol/L) also induced marked NF-kappaB activation in HUVECs, indicating that H(2)O(2)-induced NF-kappaB activation is potentiated by the inhibition of tyrosine phosphatases. Western blotting of cytoplasmic IkappaBalpha demonstrated that NF-kappaB activation induced by oxidative stress was not associated with IkappaBalpha degradation. In contrast, tumor necrosis factor-alpha-induced NF-kappaB activation occurred in concert with degradation of IkappaBalpha. Inhibition of IkappaBalpha degradation with a proteasome inhibitor, MG-115, blocked NF-kappaB activation induced by tumor necrosis factor-alpha; however, MG-115 had no effect on NF-kappaB activation during oxidative stress. CONCLUSIONS: This study demonstrated a stimulus-specific mechanism of NF-kappaB activation in endothelial cells that acts independently of IkappaBalpha degradation and may require tyrosine phosphorylation.

Platelet-activating factor acetylhydrolase prevents myocardial ischemia-reperfusion injury.

BACKGROUND: Platelet-activating factor (PAF) is one of the most potent biological mediators of tissue injury. PAF acetylhydrolase (PAF-AH) is a recently isolated naturally occurring enzyme that hydrolyzes PAF and renders it inactive. We hypothesize that inhibition of PAF with PAF-AH will reduce myocardial ischemia-reperfusion (I/R) injury in vivo. METHODS AND RESULTS: The coronary ligation model was used in New Zealand white rabbits. The large branch of the marginal coronary artery was occluded for 45 minutes, followed by 2 hours of reperfusion. Fifteen minutes before reperfusion, animals were given either 2 mg/kg of vehicle or of PAF-AH. At the completion of 120 minutes of reperfusion, percentage of necrosis, degree of neutrophil infiltration, and measurements of regional contractility were assessed. Data are expressed as the mean+/-SEM and compared by Student's t test or Mann-Whitney ANOVA. Both groups of animals showed an equivalent area at risk; however, 46.7+/-11% was necrotic in the animal treated with vehicle. In contrast, 20.9+/-7.0% was necrotic in the animals treated with PAF-AH (P<0.05). Systolic shortening and wall thickness were significantly greater in those animals treated with PAF-AH at 15, 30, 60, and 120 minutes of reperfusion (P<0.05). Quantification of neutrophil infiltration showed a 62% reduction in the PAF-AH treated animals compared with those treated with vehicle alone. CONCLUSIONS: PAF-AH is a potent cardioprotective agent in an in vivo model of I/R injury.

An essential role for NF-kappaB in the cardioadaptive response to ischemia.

BACKGROUND: Ischemic preconditioning (IP) is the phenomenon whereby brief episodes of ischemia protect the heart against a subsequent ischemic stress. We hypothesize that activation of the transcription factor NF-kappaB mediates IP. METHODS: Rabbits were randomly allocated to one of three groups: (1) 45 minutes of myocardial ischemia followed by 2 hours of reperfusion (I/R); (2) three cycles of 5-minute ischemia and 5 minutes of reperfusion followed by I/R (IP + I/R); or (3) IP in the presence of ProDTC, a specific NF-kappaB inhibitor, followed by I/R (IPProDTC + I/R). Infarct size, indices of regional contractility, and NF-kappaB activation were determined. RESULTS: In preconditioned rabbits (IP + I/R), infarct size was reduced 83% compared with both I/R alone and IPProDTC + I/R groups (p < 0.05). Throughout reperfusion, preconditioned myocardium showed enhanced regional contractile function compared with I/R and IPProDTC + I/R groups (p < 0.05). Gel shift analysis showed NF-kappaB activation with IP that was blocked by ProDTC. I/R and IPProDTC + I/R groups showed NF-kappaB activation with I/R that was absent in preconditioned animals. CONCLUSIONS: The cytoprotective effects induced by IP require activation of NF-kappaB.

Inhibition of the transcriptional activator protein nuclear factor kappaB prevents hemodynamic instability associated with the whole-body inflammatory response syndrome.

BACKGROUND: The transcription factor nuclear factor kappaB mediates the expression of a number of inflammatory genes involved in the whole-body inflammatory response to injury. We and others have found that dithiocarbamates specifically inhibit nuclear factor kappaB-mediated transcriptional activation in vitro. OBJECTIVE: We hypothesized that inhibition of nuclear factor kappaB with dithiocarbamate treatment in vivo would attenuate interleukin 1 alpha-mediated hypotension in a rabbit model of systemic inflammation. METHODS: New Zealand White rabbits were anesthetized and cannulated for continuous hemodynamic monitoring during 240 minutes. Rabbits were treated intravenously with either phosphate-buffered saline solution or 15 mg/kg of a dithiocarbamate, either pyrrolidine dithiocarbamate or proline dithiocarbamate, 60 minutes before the intravenous infusion of 5 micrograms/kg interleukin 1 alpha. Nuclear factor kappaB activation was evaluated by electrophoretic gel mobility shift assay of whole-tissue homogenates. RESULTS: Infusion of interleukin 1 alpha resulted in significant decreases in mean arterial pressure and systemic vascular resistance, both of which were prevented by treatment with dithiocarbamate. Pyrrolidine dithiocarbamate induced a significant metabolic acidosis, whereas proline dithiocarbamate did not. Nuclear factor kappaB-binding activity was increased within heart, lung, and liver tissue 4 hours after interleukin 1 alpha infusion. Treatment with dithiocarbamate resulted in decreased nuclear factor kappaB activation in lung and liver tissue with respect to that in control animals. CONCLUSIONS: These results demonstrate that nuclear factor kappaB is systemically activated during whole-body inflammation and that inhibition of nuclear factor kappaB in vivo attenuates interleukin 1 alpha-induced hypotension. Nuclear factor kappaB thus represents a potential therapeutic target in the treatment of hemodynamic instability associated with the whole-body inflammatory response.

Transcriptional arrest of the human E-selectin gene.

BACKGROUND: E-selectin transcription requires binding of transcription factors, NF-kappaB, ATF-2, and HMG-I(Y). Here we characterize the mechanism responsible for the transcriptional downregulation of E-selectin expression. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha for 24 h. HUVEC E-selectin expression was measured by enzyme-linked immunosorbent assay, Northern blotting, and nuclear run-on assays, and NF-kappa B was assessed by electrophoretic gel mobility shift assays (EMSAs). RESULTS: (1) E-selectin surface expression peaked at 4 h and then diminished over the next 20 h. (2) Transcription of E-selectin began within 1 h of TNF-alpha exposure and ceased by 8 h, despite continuous stimulation of HUVECs with TNF-alpha. (3) EMSAs revealed persistent binding activity of NF-kappa B proteins to two NF-kappa B-binding sites during 24 h of continuous stimulation with TNF-alpha. However, binding activity of proteins that recognize a third NF-kappa B element, -126 to -116 bp from the transcription start site, was lost after 4 h during 24 h of continuous stimulation with TNF-alpha; ATF-2 binding was unchanged over 24 h stimulation with TNF-alpha. CONCLUSION: The termination of E-selectin expression is controlled at the level of transcription, with loss of protein-DNA interactions at only one of three NF-kappa B-binding sites in the E-selectin promoter.

Inhibition of nuclear factor-kappa B nuclear localization reduces human E-selectin expression and the systemic inflammatory response.

BACKGROUND: One proinflammatory property observed during endothelial cell activation is the expression of the neutrophil adhesion molecule E-selectin on the surface of endothelial cells. An important regulatory element in endothelial cell E-selectin expression is the nuclear localization of the transcription factor nuclear factor (NK)-kappa B, which binds to and affects the function of several genes encoding proteins mediating inflammation. METHODS AND RESULTS: In this study, we investigated the ability of pyrrolidine dithiocarbamate (PDTC), an agent that inhibits the nuclear localization of NF-kappa B, to (1) block endothelial cell E-selectin expression in vitro in response to tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and lipopolysaccharide (LPS) and (2) reduce neutrophil infiltration in a rabbit model of systemic inflammation. As measured with the use of an enzyme-linked immunosorbent assay, TNF-alpha, IL-1, and LPS each induced a significant increase in surface expression of E-selectin in cultured human umbilical vein endothelial cells (HUVECs) compared with HUVECs treated with medium alone. In contrast, E-selectin surface expression was blocked in HUVECs pretreated with PDTC before TNF-alpha, IL-1, or LPS stimulation. NF-kappa B was present in HUVEC nuclei treated with TNF-alpha, whereas translocation of NF-kappa B to the nucleus was absent in TNF-alpha-treated HUVECs pretreated with PDTC. In vivo, rabbits pretreated with PDTC before LPS infusion showed significantly less neutrophil infiltration in the lungs, liver, and heart compared with animals infused with LPS alone. This correlated with a reduction in E-selectin expression in vivo. CONCLUSIONS: Our data suggest that NF-kappa B regulation of gene expression in the vascular endothelium may be an important cellular mechanism in endothelial cell activation.

Inhibition of interleukin-8 blocks myocardial ischemia-reperfusion injury.

INTRODUCTION: Interleukin-8 is thought to play a role in neutrophil activation and transcapillary migration into the interstitium. Because neutrophils are principal effector cells in acute myocardial ischemia-reperfusion injury, we postulated that the inhibition of interleukin-8 activity with a neutralizing monoclonal antibody directed against rabbit interleukin-8 (ARIL8.2) would attenuate the degree of myocardial injury encountered during reperfusion. METHODS: In New Zealand White rabbits, the large branch of the marginal coronary artery supplying most of the left ventricle was occluded for 45 minutes, followed by 2 hours of reperfusion. Fifteen minutes before reperfusion, animals were given an intravenous bolus of either 2 mg/kg of ARIL8.2 or 2 mg/kg anti-glycoprotein-120, an isotype control antibody that does not recognize interleukin-8. At the completion of the 120-minute reperfusion period, infarct size was determined. RESULTS: In the area at risk for infarction, 44.3% +/- 4% of the myocardium was infarcted in the anti-glycoprotein-120 group compared with 24.8% +/- 9% in the ARIL8.2 group (p < 0.005). In control animals, edema and diffuse infiltration of neutrophils were observed predominantly in the infarct zone and the surrounding area at risk. Tissue myeloperoxidase determinations did not differ significantly between groups, indicating that the cardioprotective effect of ARIL8.2 was independent of an effect on neutrophil infiltration. CONCLUSIONS: A specific monoclonal antibody that neutralizes interleukin-8 significantly reduces the degree of necrosis in a rabbit model of myocardial ischemia-reperfusion injury.

CD14-dependent activation of human endothelial cells by Bacteroides fragilis outer membrane.

We studied the capacity of isolated Bacteriodes fragilis outer membrane, B. fragilis NCTC9343 lipopolysaccharide (LPS; endotoxin), and B. fragilis NCTC9343 capsular polysaccharides to activate human umbilical vein endothelial cell (HUVEC) monolayers. To assess HUVEC activation, E-selectin expression was measured by enzyme-linked immunosorbent assay (ELISA), Northern blot analysis for E-selectin-specific mRNA, and electrophoretic gel mobility shift assay (EMSA) for NF-kappa B, a transcription factor necessary for E-selectin gene activation. Exposure of HUVECs to B. fragilis outer membrane fractions, separated from other components of the B. fragilis cell wall by isopycnic, sucrose gradient centrifugation, significantly increased surface expression of E-selectin and induced functional endothelial cell-dependent leukocyte adhesion. B. fragilis outer membranes induced translocation of NF-kappa B to HUVEC nuclei and accumulation of E-selectin mRNA in HUVEC cytoplasm. E-selectin expression induced by B. fragilis outer membranes was not blocked by polymixin B. In contrast, E-selectin expression induced by outer membrane fractions purified from E. coli was competitively inhibited by polymixin B. Neither purified B. fragilis LPS, a prominent constituent of the outer membrane, nor purified B. fragilis capsular polysaccharides induced HUVEC activation. Two different monoclonal antibodies directed against human CD14 completely inhibited B. fragilis outer membrane-induced NF-kappa B activation, E-selectin transcription, and E-selectin surface expression. We conclude that the outer membrane component of the B. fragilis cell wall contains a proinflammatory factor(s), that is not LPS, which induces human endothelial cell activation by a soluble CD14-dependent mechanism.

Endothelial cell injury in cardiovascular surgery: the systemic inflammatory response.

Many of the components currently used to perform cardiovascular operations lead to systemic insults that result from cardiopulmonary bypass circuit-induced contact activation, circulatory shock, and resuscitation, and a syndrome similar to endotoxemia. Experimental observations have demonstrated that these events have profound effects on activating endothelial cells to recruit neutrophils from the circulation. Once adherent to the endothelium, neutrophils release cytotoxic proteases and oxygen-derived free radicals, which are responsible for much of the end-organ damage seen after cardiovascular operations. Recently the cellular and molecular mechanisms of endothelial cell activation have become increasingly understood. It is conceivable that once the molecular mechanisms of endothelial cell activation are better defined, therapies will be developed allowing the selective or collective inhibition of vascular endothelial activation during the perioperative period.

Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion.

Myocardial ischemia and reperfusion is a common occurrence in cardiovascular surgery patients. Acute ischemia results in a spectrum of derangements, which range from transient reversible stunning of the myocardium to severe irreversible abnormalities such as infarction. Many of these abnormalities are accentuated upon reperfusion with oxygenated blood. Recently, the endothelium has been shown to play a key role in the injury suffered after ischemia and reperfusion. When rendered hypoxic and then reoxygenated, endothelial cells become activated to express proinflammatory properties that include the induction of leukocyte-adhesion molecules, procoagulant factors and vasoconstrictive agents that increase vasomotor tone. These changes may contribute to the no-reflow phenomenon by promoting endothelial edema, neutrophil and platelet plugging, microthrombosis, and enhanced vasomotor tone. An increased understanding of the role that hypoxic endothelial cell activation plays in myocardial dysfunction after ischemia/reperfusion may allow therapies to be designed to further attenuate this response.

Hypothermia inhibits human E-selectin transcription.

During endothelial cell activation, the formation and expression of E-selectin require transcriptional activation of the E-selectin gene, mediated by the coordinated action of several transcription factors and cis-acting elements in its 5'-flanking region. It is reported that in vitro hypothermia (25 degrees C) transiently inhibits transcriptional activation and surface expression of E-selectin as well as neutrophil adherence to cultured human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS), interleukin-1 (IL-1), or tumor necrosis factor (TNF). Rewarming HUVECs treated with LPS, IL-1, or TNF to 37 degrees C restores E-selectin transcript accumulation, E-selectin surface expression, and neutrophil adherence to HUVECs at levels equivalent to similarly treated HUVECs maintained at 37 degrees C continuously. Despite the absence of detectable E-selectin transcription at 25 degrees C, activation of the transcription factor NF-kappaB still occurred in HUVECs treated with LPS, IL-1, or TNF, indicating that signal transduction was not blocked by hypothermia. It is concluded that neutrophil adherence to activated endothelium mediated by E-selectin is reversibly inhibited by hypothermia. The protective effect of hypothermia clinically (e.g., cardiopulmonary bypass) may, in part, be mediated by transiently inhibiting the expression of an endothelial cell activation phenotype.

Activation of human endothelial cells by viable or heat-killed gram-negative bacteria requires soluble CD14.

In response to bacterial lipopolysaccharides (LPS; endotoxin), endothelial cells are converted to an activation phenotype expressing both proinflammatory and procoagulant properties that include the induction of leukocyte adhesion molecules and tissue factor expression. LPS-induced endothelial cell activation requires a soluble form of the monocyte LPS receptor, sCD14. We evaluated the capacity of multiple strains of gram-negative and gram-positive bacteria to induce endothelial E-selectin and tissue factor expression through sCD14-dependent pathways with cultured human umbilical vein endothelial cells (HUVE). Both viable and heat-killed gram-negative bacteria (Bacteroides fragilis, Enterobacter cloacae, Haemophilus influenzae, and Klebsiella pneumoniae) but not viable or heat-killed gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae) induced prominent E-selectin surface expression detected by enzyme-linked immunosorbent assay. Tissue factor activity on HUVE, indicated by factor X activation, was induced in response to gram-negative bacteria but not in response to gram-positive bacteria. Gram-negative bacteria induced transcriptional activation in HUVE, indicated by the appearance of E-selectin-specific mRNA and by the demonstration of activation of NF-kappa B, a trans-activating factor necessary for E-selectin and tissue factor gene transcription. In contrast, neither E-selectin mRNA nor activation of NF-kappa B was detected in HUVE treated with gram-positive bacteria. Endothelial cell activation by gram-negative bacteria in each of these assays was inhibited with a monoclonal antibody (60bd) against CD14. Furthermore, CHO-K1 cells, transfected with human recombinant CD14, responded to all strains of gram-negative bacteria (viable or heat killed), indicated by CHO-K1 NF-kappa B activation. We conclude that gram-negative bacteria induce endothelial cell activation through a common sCD14-dependent pathway.

Expression of cytochrome P450s and microsomal epoxide hydrolase in primary cultures of human umbilical vein endothelial cells.

In view of the potential role of the cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) biotransformation enzymes in the metabolism of protoxicants in the circulatory system, we examined CYP and mEH expression in several primary cultures of human umbilical vein endothelial cells (HUVEC), each established from a different individual. Total RNA was isolated from untreated cells and cells 72 hr after exposure to dimethyl sulfoxide (DMSO), Arochlor 1254 (PCB), and beta-naphthoflavone (beta NF). Specific mRNA transcripts were examined by Northern blotting and reverse transcriptase-coupled polymerase chain reaction (RT/PCR) analyses. CYP2E1, CYP3A, and CYP1A2 mRNAs were not detectable in any of the cultures by Northern blot analysis with radiolabeled oligomer probes; however, CYP1A1 mRNA was detected using this procedure in HUVEC cultures exposed to beta NF for 72 hr. Using RT/PCR, constitutive levels of CYP1A1, CYP1A2, CYP2E1, and CYP3A gene expression in HUVEC cultures were evident; however, constitutive CYP2B6 mRNA was not detected. Constitutive CYP1A2 transcript levels were detected in four of six HUVEC cultures, but levels varied between individual cultures. CYP1A2 mRNA levels were also increased in HUVEC cultures exposed to PCB and beta NF. No increases in the levels of CYP2E1 and CYP3A mRNAs were observed in HUVEC cells subsequent to PCB or beta NF exposures. Constitutive CYP2E1 transcript levels were present in all HUVEC cultures examined and varied among individuals. All HUVEC cultures examined for mEH activity exhibited constitutive levels of mEH which varied 40% between individual cultures and produced on average, 1.51 pmol benzo[a]pyrene 4,5-dihydrodiol per milligram protein per minute of reaction. Thus, these results demonstrate that human endothelial cells express CYP and mEH gene products and suggest that these enzymes may play important roles in determining metabolic fates for circulating protoxicants.

The role of protein kinase C in the induction of VCAM-1 expression on human umbilical vein endothelial cells.

The role of protein kinase C (PKC) in interleukin-1 beta- (II-1 beta)-, tumor necrosis factor-alpha- (TNF-alpha)-, and lipopolysaccharide- (LPS)-induced vascular cell adhesion molecule-1 (VCAM-1) expression on human umbilical vein endothelial cells (HUVEC) was studied. PKC inhibition or downregulation diminished VCAM-1 mRNA accumulation and protein expression. Interleukin-1 beta, TNF-alpha, and LPS induce nuclear factor (NF)-kappa B-like binding activity, which precedes VCAM-1 transcription. PKC inhibition did not prevent NF-kappa B-like binding activity, indicating that this is PKC-independent, and NF-kappa B-like binding activity is insufficient for transcription of VCAM-1.

A protein kinase C agonist, selective for the beta I isozyme, induces E-selectin and VCAM-1 expression on HUVEC but does not translocate PKC.

A protein kinase C (PKC) agonist selective for the beta I isozyme, 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), induced NF-kappa B-like binding activity and surface expression of E-selectin and VCAM-1 in human umbilical vein endothelial cells (HUVEC), similar to the effects of tumor necrosis factor-alpha (TNF-alpha). Induction of E-selectin and VCAM-1 expression by dPPA was completely inhibited by the PKC inhibitors staurosporine and Ro31-7549. The PKC inhibitors also reduce TNF-alpha-induced VCAM-1 expression. However, neither dPPA nor TNF-alpha translocated PKC from the cytosolic to the plasma or nuclear membrane particulate fractions in HUVEC. These results indicate that activation of the beta I PKC isozyme is sufficient for expression of E-selectin and VCAM-1, and suggest that PKC may mediate the effects of TNF-alpha and dPPA without requiring the translocation normally associated with activation of PKC.

Liposome-encapsulated hemoglobin inhibits tumor necrosis factor release from rabbit alveolar macrophages by a posttranscriptional mechanism.

Macrophages contribute to the systemic inflammatory response that characterizes the sepsis syndrome through the production of inflammatory cytokines such as tumor necrosis factor (TNF). Liposome-encapsulated hemoglobin (LEH), a potential red cell substitute, is cleared by fixed tissue macrophages. In these studies, in vitro incubation of alveolar macrophages with stored LEH was shown to inhibit the expression of TNF induced by endotoxin (lipopolysaccharide, LPS) stimulation. This effect was dependent on LEH dose but independent of the period of exposure to the LEH. Despite inhibition of TNF expression, Northern blot analysis of total cellular RNA from LPS-stimulated macrophages revealed accumulations of TNF-specific transcripts in cells treated with or without LEH. Thus the mechanism of LEH inhibition of TNF expression appears to involve a posttranscriptional event. Although these results suggest a potential advantage of resuscitation with LEH when sepsis complicates hemorrhagic shock, immunomodulation in vivo remains to be defined.