Endothelial dysfunction enhances vasoconstriction due to scavenging of nitric oxide by a hemoglobin-based oxygen carrier.

BACKGROUND: To date, there is no safe and effective hemoglobin-based oxygen carrier (HBOC) to substitute for erythrocyte transfusion. It is uncertain whether a deficiency of endothelial nitric oxide bioavailability (endothelial dysfunction) prevents or augments HBOC-induced vasoconstriction. METHODS: Hemodynamic effects of infusion of PolyHeme (1.08 g hemoglobin/kg; Northfield Laboratories, Evanston, IL) or murine tetrameric hemoglobin (0.48 g hemoglobin/kg) were determined in awake healthy lambs, awake mice, and anesthetized mice. In vitro, a cumulative dose-tension response was obtained by sequential addition of PolyHeme or tetrameric hemoglobin to phenylephrine-precontracted murine aortic rings. RESULTS: Infusion of PolyHeme did not cause systemic hypertension in awake lambs but produced acute systemic and pulmonary vasoconstriction. Infusion of PolyHeme did not cause systemic hypertension in healthy wild-type mice but induced severe systemic vasoconstriction in mice with endothelial dysfunction (either db/db mice or high-fat fed wild-type mice for 4-6 weeks). The db/db mice were more sensitive to systemic vasoconstriction than wild-type mice after the infusion of either tetrameric hemoglobin or PolyHeme. Murine aortic ring studies confirmed that db/db mice have an impaired response to an endothelial-dependent vasodilator and an enhanced vasoconstrictor response to HBOC. CONCLUSIONS: Reduction in low molecular weight hemoglobin concentrations to less than 1% is insufficient to abrogate the vasoconstrictor effects of HBOC infusion in healthy awake sheep or in mice with reduced vascular nitric oxide levels associated with endothelial dysfunction. These findings suggest that testing HBOCs in animals with endothelial dysfunction can provide a more sensitive indication of their potential vasoconstrictor effects.

Soluble guanylate cyclase agonists inhibit expression and procoagulant activity of tissue factor.

OBJECTIVE: Tissue factor (TF), a major initiator of blood coagulation, contributes to inflammation, atherosclerosis, angiogenesis, and vascular remodeling. Pharmacological agonists of soluble guanylate cyclase (sGC) attenuate systemic and pulmonary hypertension, vascular remodeling, and platelet aggregation. However, the influence of these novel pharmacophores on TF is unknown. METHODS AND RESULTS: We evaluated effects of BAY 41-2272 and BAY 58-2667 on expression and activity of TF in human monocytes and umbilical vein endothelial cells (HUVECs). Both compounds reduced expression of active TF protein in monocytes stimulated with lipopolysaccharide, as demonstrated by immunoblotting and a TF procoagulant activity assay. In-cell Western assay revealed that this effect was associated with a marked reduction of total and surface TF presentation. Furthermore, BAY 41-2272 and BAY 58-2667 decreased TF protein expression and the TF-dependent procoagulant activity in HUVECs stimulated with TNF-alpha. The sGC agonists also suppressed transcriptional activity of NF-kappaB. A siRNA-mediated knockdown of the alpha1-subunit of sGC in monocytes and HUVECs confirmed that the inhibitory effect of BAY 41-2272 and BAY 58-2667 on TF expression is mediated through the sGC-dependent mechanisms. CONCLUSIONS: Inhibition of TF expression and activity by sGC agonists might provide therapeutic benefits in cardiovascular diseases associated with enhanced procoagulant and inflammatory response.

Regulation of tissue factor procoagulant activity by post-translational modifications.

Post-translational modification of amino acid residues is a common way to regulate localization, stability and ultimately the function of the protein. Tissue factor (TF), the major initiator of blood coagulation cascade, receives several post-translational modifications, like glycosylation, phosphorylation, palmitoylation and nitrosylation. Recent studies have demonstrated that these processes play important roles in modulating biological functions of TF. The present review highlights the mechanisms of several common protein post-translational modifications of TF with the special reference on the recent knowledge about their roles in regulation of trafficking, stability as well as procoagulant and signaling functions of TF.

Recombinant human activated protein C attenuates endotoxin-induced lung injury in awake sheep.

INTRODUCTION: Acute lung injury often complicates severe sepsis. In gram-negative sepsis, bacterial endotoxin activates both coagulation and inflammation. Enhanced lung vascular pressures and permeability, increased extravascular lung water content and deteriorated gas exchange characterize ovine endotoxin-induced lung injury, a frequently used model of acute lung injury. Recombinant human activated protein C (rhAPC), with its anticoagulant, anti-inflammatory, fibrinolytic and antiapoptotic effects, reportedly reduces the respirator-dependent days and the mortality of patients with severe sepsis. We speculate whether rhAPC antagonizes endotoxin-induced lung injury in sheep. METHODS: Two groups of sheep were exposed to Escherichia coli endotoxin (lipopolysaccharide) 15 ng/kg/minute intravenously from 0 to 24 hours; one group received only lipopolysaccharide throughout (n = 8), and the other group received lipopolysaccharide in combination with rhAPC 24 microg/kg/hour from 4 to 24 hours (n = 9). In addition, one group received rhAPC as above as the only intervention (n = 4), and four sham-operated sheep were used for determination of the alpha and epsilon isoforms of protein kinase C in pulmonary tissue. Data were assessed by one-way analysis of variance for repeated measurements. Biochemical data were analyzed using Student's t test, or using the Mann-Whitney U test when appropriate. RESULTS: Infusion of endotoxin caused lung injury, manifested by increments in pulmonary artery pressure, in pulmonary micro-occlusion pressure, in pulmonary vascular downstream resistance, in pulmonary vascular permeability index, in extravascular lung water index and in deterioration of oxygenation that were all attenuated by infusion of rhAPC. Endotoxemia led to changes in inflammation and coagulation, including pulmonary neutrophil accumulation paralleled by increased TNFalpha and decreased protein C and fibrinogen in animal plasma, which all improved following infusion of rhAPC. Moreover, rhAPC prevented the translocation of protein kinase C alpha and epsilon isoforms from the cytosolic fraction of lung tissue extracts. CONCLUSION: In awake sheep, rhAPC alleviates endotoxin-induced lung injury--as characterized by improvements of oxygenation, coagulation and inflammation, as well as by reversal of pulmonary hemodynamic and volumetric changes.

Induction of monocytic tissue factor expression after rewarming from hypothermia in vivo is counteracted by heat shock in c-Jun-dependent manner.

OBJECTIVE: Triggering of tissue factor (TF)-mediated blood coagulation leads to the development of disseminated intravascular coagulation during rewarming from hypothermia. We studied post-rewarming TF levels, activity, and surface redistribution, along with the regulation of TF gene transcription in mononuclear cells (MNCs) obtained from an in vivo rat model. METHODS AND RESULTS: Rewarming after a 5-hour episode of 15 degrees C hypothermia caused an increase in TF activity, protein levels, and externalization of TF antigen in rat MNCs. This was accompanied by a dramatic elevation of c-Jun and JNK phosphorylation, and the absence of EGR-1 and NF-kappaB activation. To search for a stimulus to counteract c-Jun-mediated induction of TF activity in MNCs from rewarmed rats, we applied heat shock pretreatment one day before the hypothermia/rewarming experiment. This restored post-rewarming TF activity, protein levels, and surface-to-total TF ratio in rat MNCs to normothermic levels. Furthermore, in heat shock-pretreated animals, rewarming failed to increase phosphorylated c-Jun and JNK levels. We attribute this to the profound overexpression of heat shock protein 70 and inhibition of JNK. CONCLUSIONS: MNCs respond to rewarming from hypothermia by an induction of active TF antigen. This effect is dependent on c-Jun activation and is abolished by heat shock pretreatment.

Intracellular and surface distribution of monocyte tissue factor: application to intersubject variability.

OBJECTIVE: The high and low responder phenomenon describes individual differences in lipopolysaccharide (LPS)-induced monocyte tissue factor (TF) activity. We characterized patterns of intracellular accumulation, externalization, and shedding of TF in response to LPS in mononuclear cells (MNCs) from high responders (HRs) and low responders (LRs). METHODS AND RESULTS: After 2 hours of LPS stimulation of whole blood, flow cytometry analyses revealed a larger population of TF-positive monocytes in HRs (32.0+/-3.5%) versus LRs (11.2+/-1.2%; P< or =0.05), along with a stronger mean fluorescence intensity of TF signal in HRs (7.1+/-0.5 arbitrary units [AU]) compared with LRs (5.4+/-0.4 AU; P< or =0.05). The LPS-treated blood of the HR group contained 2-fold more TF-positive microparticles than LRs. In-cell Western assay demonstrated higher intracellular accumulation of TF in mononuclear cells (MNCs) from LRs because LPS induced a 3.7-fold increase of total TF levels in LRs versus a 1.5-fold increase in HRs. In contrast, in response to LPS stimulation, MNCs from HRs exhibited a 4-fold induction of surface TF, whereas MNCs from LRs only had a minor increase in surface TF levels. CONCLUSIONS: The higher availability of surface TF antigen on MNCs from HRs and TF-containing microparticles might make these individuals more susceptible to hypercoagulation.

Tezosentan-induced attenuation of lung injury in endotoxemic sheep is associated with reduced activation of protein kinase C.

INTRODUCTION: Studies in vitro reveal that endothelin-1 (ET-1) activates the alpha isoform of protein kinase C (PKC-alpha) in cultures of endothelial cells, thereby deranging cellular integrity. Sepsis and endotoxemia are associated with increased plasma concentrations of ET-1 that induce acute lung injury (ALI). We recently reported that non-selective ET-1 receptor blockade attenuates ALI in sheep by reducing the endotoxin-induced increase in extravascular lung water index (EVLWI). The aim of this study was to find out whether this attenuation is associated with reduced translocation of PKC-alpha from the cytosolic to the membrane fraction of lung tissue homogenate. METHODS: Seventeen awake, instrumented sheep were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide (LPS) group (n = 7) receiving an intravenous infusion of Escherichia coli 15 ng/kg per min for 24 hours, and a tezosentan group (n = 7) subjected to LPS and, from 4 hours, an intravenous injection of tezosentan 3 mg/kg followed by infusion at 1 mg/kg per hour for the reminder of the experiment. Pulmonary micro-occlusion pressure (Pmo), EVLWI, plasma concentrations of ET-1, tumor necrosis factor-a (TNF-a), and interleukin-8 (IL-8) were determined every 4 hours. Western blotting was used to assess PKC-alpha. RESULTS: In non-treated sheep a positive correlation was found between the plasma concentration of ET-1 and Pmo in the late phase of endotoxemia (12 to 24 hours). A positive correlation was also noticed between Pmo and EVLWI in the LPS and the LPS plus tezosentan groups, although the latter was significantly reduced in comparison with LPS alone. In both endotoxemic groups, plasma concentrations of ET-1, TNF-alpha, and IL-8 increased. In the LPS group, the cytosolic fraction of PKC-alpha decreased by 75% whereas the membrane fraction increased by 40% in comparison with the sham-operated animals. Tezosentan completely prevented the changes in PKC-alpha in both the cytosolic and the membrane fractions, concomitantly causing a further increase in the plasma concentrations of ET-1, TNF-alpha, and IL-8. CONCLUSION: In endotoxemic sheep, ET-1 receptor blockade alleviates lung injury as assessed by a decrease in EVLWI paralleled by a reduction in Pmo and the prevention of activation of PKC-alpha.

Tezosentan reduces the microvascular filtration coefficient in isolated lungs from rats subjected to cecum ligation and puncture.

INTRODUCTION: We recently demonstrated that the non-selective endothelin-1 (ET-1) receptor blocker tezosentan antagonizes ovine acute lung injury (ALI) following infusion of endotoxin or ET-1 by reducing the enhanced lung microvascular pressure, although we could not exclude the possibility of a simultaneous decline in microvascular permeability. In the present study, our aim was to find out if tezosentan reverses the rise in microvascular filtration coefficient (Kfc) in rat lungs that have been isolated and perfused 12 h after cecum ligation and puncture (CLP) or infusion of ET-1. METHODS: Wistar rats (n = 42) were subjected to CLP. Postoperatively, rats were randomized to a CLP group (n = 7) and a CLP + tezosentan group (n = 7); the latter received tezosentan 30 mg/kg. A sham-operated group (n = 5) underwent laparotomy without CLP. Twelve hours postoperatively, the lungs were isolated and perfused with blood from similarly treated rats that also were used to assess plasma concentration of ET-1 and protein kinase Calpha (PKCalpha) in lung tissue. Additionally, isolated blood perfused lungs from healthy rats were randomized to a control group (n = 8), an ET-1 group (n = 7) subjected to pulmonary arterial injection of ET-1 10 nM, and an ET-1 + tezosentan group (n = 7) that received tezosentan 30 mg/kg. All lung preparations received papaverine 0.1 microg/kg added to the perfusate for vasoplegia. Pulmonary hemodynamic variables, Kfc and lung compliance (CL) were assessed. RESULTS: After CLP, the plasma concentration of ET-1 increased. Papaverine abolished the vasoconstrictor response to ET-1 and the pulmonary vascular pressures remained close to baseline throughout the experiments. Both CLP and injection of ET-1 caused significant changes in Kfc and CL that were prevented in tezosentan-treated rats. Compared to sham-operated animals, CLP increased the content of PKCalpha by 50% and 70% in the cytosolic and the membrane fractions of lung tissue homogenates, respectively. Tezosentan prevented the upregulation of PKCalpha in the membrane fraction. CONCLUSION: In rat lungs isolated and perfused after CLP, tezosentan precludes both the increase in Kfc and the upregulation of PKCalpha in the membrane fraction of lung tissue.

Differential expression of plasma miRNAs in patients with unprovoked venous thromboembolism and healthy control individuals.

BACKGROUND: Venous thromboembolism (VTE) remains the third most common cardiovascular disease with a vague pathogenesis. Circulating miRNAs are small regulatory RNAs found in plasma, serum and other body fluids in an apparently stable form. Although circulating miRNAs, a novel family of regulatory molecules, emerge as a promising class of biomarkers in many cardiovascular diseases and malignancies, knowledge on plasma miRNA levels in VTE remains sparse. AIMS: The present work was conducted as a pilot study in order to estimate the plasma levels of miRNAs in patients with unprovoked VTE and to assess miRNAs as potential novel biomarkers of VTE. METHODS: Twenty patients with a history of unprovoked VTE 1-5 years prior to inclusion in the study and twenty age- and sex-matched healthy control participants were enrolled in a case-control study (Tromsø IV). Plasma levels of 742 miRNAs were assessed after RNA extraction and reverse transcription. Profiling of miRNA was conducted on the Universal RT microRNA PCR Human panels I and II (Exiqon, Denmark). For normalization of the data, the average of the assays detected in all samples (n=40 samples) was applied. RESULTS: Ninety-seven miRNAs were detected throughout all samples. Of these, miR-10b-5p, -320a, -320b, -424-5p, and -423-5p were upregulated, whereas miR-103a-3p, -191-5p, -301a-3p, and 199b-3p were downregulated in plasmas of VTE patients versus controls (P≤0.05). These miRNAs were confined to the extracellular vesicles-depleted plasma fraction, and yielded clear clustering distinguishing samples from the VTE and control groups. CONCLUSIONS: The results of this pilot study indicate that plasma miRNAs profiling can provide novel biomarkers of unprovoked VTE.

Evidence for direct transfer of tissue factor from monocytes to platelets in whole blood.

Varying specificity of anti-tissue factor (anti-TF) antibodies gives rise to erroneous conclusions on TF positivity of platelets. Although monocytes are a well established source of TF in whole blood, there is no consensus whether platelets express or acquire TF from external sources. To test whether platelets can acquire TF expressed in monocytes, we studied a transfer of TF-yellow fluorescent protein (TF-YFP) from monocytes nucleofected with TF-YFP to platelets in a whole blood model. Platelets isolated from whole blood were found positive for TF when immunostained with anti-TF antibody from one supplier, whereas no platelet TF antigen was found in whole blood immunostained with anti-TF antibody from another supplier. Both antibodies recognized TF in monocytes. Platelets isolated from whole blood reconstituted with monocytes expressing TF-YFP fusion protein were found positive for TF-YFP only after stimulation with lipopolysaccharide (LPS). Taken together, TF protein could be transferred from monocytes upon stimulation with LPS.

Circulating monocytes mirror the imbalance in TF and TFPI expression in carotid atherosclerotic plaques with lipid-rich and calcified morphology.

BACKGROUND: Thrombogenicity of atherosclerotic plaque largely depends on plaque morphology and their content of tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The relationship between morphological composition of plaque (lipid-rich or calcified) and expression of TF and TFPI in circulating blood monocytes and within the plaques is not characterized. OBJECTIVE: To investigate whether lipid-rich (echolucent) or calcified (echogenic) morphology of carotid atherosclerotic plaques is associated with differences in TF and TFPI expression in circulating blood monocytes and within carotid atherosclerotic plaques. METHODS: We studied levels of monocyte TF and TFPI mRNA and protein expression and association with traditional risk factors for atherosclerosis in asymptomatic subjects with echolucent (n=20) or echogenic (n=20) carotid plaques, or controls without carotid atherosclerosis (n=20) determined by ultrasonography. Sections of calcified or lipid-rich carotid plaques obtained from symptomatic patients were assessed for TF and TFPI antigen expression. RESULTS: TF and TFPI surface presentation, surface TF/TFPI ratio, and TF activity were higher in monocytes obtained from subjects with echolucent than with echogenic plaques or controls without carotid atherosclerosis. Multiple regression analyses revealed inverse association between serum apoA1 and monocyte surface TF antigen expression (p=0.007), and positive association between serum apoB and monocyte surface TFPI expression (p=0.028). Sections from lipid-rich carotid plaques contained 2.5-fold more TF and 1.5-fold more TFPI antigens relative to calcified lesions, also yielding a higher TF/TFPI ratio. CONCLUSIONS: Our findings indicate that circulating monocytes of asymptomatic individuals with echolucent lipid-rich carotid atherosclerosis express an imbalance between TF and TFPI expression cohering with changes found within advanced carotid atherosclerotic plaques obtained from symptomatic patients.

The role of tissue factor in systemic inflammatory response syndrome.

Tissue factor (TF) is a major initiator of extrinsic pathway of blood coagulation. A dual role of TF in the extensive crosstalk between blood coagulation and inflammation has recently become apparent. The majority of the cases of systemic inflammatory response syndrome, disseminated intravascular coagulation, and sepsis are accompanied by hyperactivation of TF in circulating monocytes and damaged tissue. Systemic Gram-negative infection induces expression of TF by vascular cells. In addition to extrinsic coagulation pathway, TF induces proinflammatory signaling cascade originating from activation of protease-activated receptors. Because TF-activated proteolytic cascade is placed in a nexus between coagulation and inflammation, early modulation of TF activity presently becomes a tempting experimental therapeutic strategy in systemic inflammatory response syndrome patients.

Low thrombogenicity of calcified atherosclerotic plaques is associated with bone morphogenetic protein-2-dependent inhibition of tissue factor expression.

Morphology of atherosclerotic plaque is a major determinant of plaque thrombogenicity. Calcified atherosclerotic lesions are less prone to thrombosis and contain less tissue factor (TF) than lipid-rich plaques. Although bone morphogenetic protein (BMP)-2 is a known mediator of vascular calcification, the role of BMP-2 in the regulation of plaque thrombogenicity has not been established. We hypothesized that the expression of BMP-2 within highly calcified atherosclerotic plaques inhibits TF expression and reduces thrombogenicity of calcified lesions. In the present study, we measured levels of TF and BMP-2 in human calcified and lipid-rich carotid plaques and studied the effects of BMP-2 on TF expression in human monocytes in vitro. Quantitative immunohistochemical analysis of endarterectomy specimens for TF and BMP-2 revealed that calcified plaques contained nearly three-times less TF antigen than lipid-rich ones. In contrast, calcified plaques expressed two-times more BMP-2 antigen than lipid-rich lesions. BMP-2 markedly decreased protein expression and surface redistribution of TF in activated human monocytes in vitro. BMP-2-mediated inhibition of TF expression in monocytes/macrophages could contribute to reduced thrombogenicity of calcified atherosclerotic plaques.

The role of TFPI in regulation of TF-induced thrombogenicity on the surface of human monocytes.

INTRODUCTION: Although the procoagulant reactivity of monocytes largely depends on expression and cell surface presentation of tissue factor (TF), little is known about the impact of tissue factor pathway inhibitor (TFPI) on regulation of TF function on the monocyte surface. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy subjects and cryopreserved. We investigated TF and TFPI mRNA expression by reverse transcription-quantitative real-time PCR (RT-qPCR), surface presentation by flow cytometry and confocal microscopy, and TFPI-mediated regulation of TF functional activity on the surface of resting and LPS-stimulated PBMCs by TF activity assay and Calibrated Automated Thrombogram (CAT) assay. RESULTS: Unstimulated PBMCs contained nearly no TF, but detectable TFPI protein levels. TFPI mRNA levels were 2-fold higher than TF, and the TFPIα mRNA isoform expression was higher than TFPIß. LPS stimulation caused a parallel and sustained upregulation of both TFPI isoforms, concomitant with increased surface presentation of TFPI antigen. Stronger, but transient upregulation of TF mRNA and surface antigen was observed at 6hrs of LPS stimulation. After LPS stimulation TF and TFPI were co-localized in the same areas of the monocyte membrane. Pre-incubation of PBMCs with anti-TFPI IgG significantly enhanced TF activity, shortened Lag-time, and increased thrombin generation. TFPI-dependent inhibition of TF was more prominent in resting than in LPS-stimulated cells. CONCLUSIONS: Our results support the concept that surface TFPI is an important regulator of procoagulant reactivity of human monocytes.

Granulocytes do not express but acquire monocyte-derived tissue factor in whole blood: evidence for a direct transfer.

Unlike unanimous opinion on tissue factor (TF) expression in monocytes, the quest for TF presence in granulocytes has been going on for decades. To study the cell origin and track the blood-borne TF, we assessed TF activity and protein levels, knocked-down endogenous TF expression with small interfering RNA (siRNA), and overexpressed TF-yellow fluorescent protein (TF-YFP) fusion in immunologically isolated human monocytes and granulocytes. Monocytes and, to a much lesser extent, granulocytes isolated from lipopolysaccharide (LPS)/phorbol 12-myristate-13-acetate (PMA)-stimulated whole blood contained active TF antigen. However, only monocytes possessed significant TF activity and protein levels when stimulated with LPS/PMA in suspension. Reintroduction of TF-silenced monocytes to whole blood led to a profound reduction of LPS/PMA-stimulated TF activity in both mononuclear cell (MNC) and granulocyte fractions. No reduction in TF activity in MNC and granulocyte fractions was observed when TF-silenced granulocytes were reintroduced to whole blood. As shown by immunoblotting, flow cytometry, and confocal microscopy, granulocytes became positive for TF-YFP when isolated from whole blood reconstituted with TF-YFP-expressing monocytes. Together, we pinpoint monocytes as a major source of TF and provide solid experimental evidence for a direct transfer of TF protein from the monocytes to granulocytes in the blood.

Is oxygen supply a limiting factor for survival during rewarming from profound hypothermia?

It has been postulated that unsuccessful resuscitation of victims of accidental hypothermia is caused by insufficient tissue oxygenation. The aim of this study was to test whether inadequate O2 supply and/or malfunctioning O2 extraction occur during rewarming from deep/profound hypothermia of different duration. Three groups of rats (n = 7 each) were used: group 1 served as normothermic control for 5 h; groups 2 and 3 were core cooled to 15 degrees C, kept at 15 degrees C for 1 and 5 h, respectively, and then rewarmed. In both hypothermic groups, cardiac output (CO) decreased spontaneously by > 50% in response to cooling. O2 consumption fell to less than one-third during cooling but recovered completely in both groups during rewarming. During hypothermia, circulating blood volume in both groups was reduced to approximately one-third of baseline, indicating that some vascular beds were critically perfused during hypothermia. CO recovered completely in animals rewarmed after 1 h (group 2) but recovered to only 60% in those rewarmed after 5 h (group 3), whereas blood volume increased to approximately three-fourths of baseline in both groups. Metabolic acidosis was observed only after 5 h of hypothermia (15 degrees C). A significant increase in myocardial tissue heat shock protein 70 after rewarming in group 3, but not in group 2, indicates an association with the duration of hypothermia. Thus mechanisms facilitating O2 extraction function well during deep/profound hypothermia, and, despite low CO, O2 supply was not a limiting factor for survival in the present experiments.

Preconditioning by 17beta-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS.

To study the cell signaling events leading to 17beta-estradiol (E(2))-induced acute cardioprotection, we subjected isolated rat hearts to three 5-min cycles of 10 microM E(2) before 30 min of regional ischemia, followed by 2 h of reperfusion. Protection was judged by changes in infarct size in percentage of risk zone volume. To test the importance of phosphoinositide 3-kinase (PI3-K), protein kinase C (PKC), or reactive oxygen species (ROS) in E(2)-induced protection, we combined wortmannin (1 microM), chelerythrine (2 microM), and 2-mercaptopropionylglycine (300 microM), respectively, with E(2) exposure. Changes in phosphorylation of protein kinase B (PKB) and selected PKC isoforms were tested by immunoblotting of total lysates and subcellular fractions, along with assessment of PKC translocation from soluble to membrane fraction of heart tissue homogenates. Intracellular ROS levels induced by E(2) preconditioning were investigated. E(2) preconditioning led to significant reduction in infarct size from 31.8 +/- 5.3 to 20.2 +/- 2.6% in male hearts and from 42.7 +/- 4.7 to 17.1 +/- 3.4% in female hearts (P < 0.05). Protection was abolished by wortmannin (30.0 +/- 3.2%), chelerythrine (45.1 +/- 4.4%), and 2-mercaptopropionylglycine (36.8 +/- 4.7%). E(2) preconditioning induced phosphorylation of PKB, PKCalpha, and PKCepsilon and membrane translocation of PKCepsilon and PKCdelta. Intracellular ROS levels were found elevated after transient treatment with hormone. Therefore, our data demonstrate the ability of E(2) to induce preconditioning-like cardioprotection via cell signaling events shared by classic ischemic preconditioning.

Extravascular lung water determined with single transpulmonary thermodilution correlates with the severity of sepsis-induced acute lung injury.

OBJECTIVE: To find out if the extravascular lung water index (EVLWI) and the derived permeability indexes determined by the single transpulmonary thermodilution technique are associated with markers of acute lung injury in human septic shock. DESIGN: Prospective, observational study. SETTING: Mixed intensive care unit of a 900-bed university hospital. PATIENTS: Thirty-eight consecutive adult patients with septic shock and acute lung injury. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The variables were assessed over a 72-hr period and included hemodynamics, EVLWI, and pulmonary vascular permeability indexes determined with the single indicator transpulmonary thermodilution technique, lung compliance, oxygenation ratio (Pao2/Fio2), lung injury score, cell counts, and the plasma concentration of endothelin-1. At day 1, EVLWI was elevated (>or=7 mL/kg) in 28 (74%) patients and correlated with lung compliance (r=-.48, p=.002), Pao2/Fio2 (r=-.50, p=.001), lung injury score (r=.46, p=.004), roentgenogram quadrants (r=.39, p=.02), and platelet count (r=-.43, p=.007). At day 3, EVLWI correlated with compliance (r=-.51, p=.002), Pao2/Fio2 (r=-.49, p = .006), and lung injury score (r=.53, p=.003). At day 3, EVLWI and pulmonary vascular permeability indexes were higher in nonsurvivors (p<.05). The plasma concentration of endothelin-1 (mean+/-sd) was significantly higher in patients with elevated EVLWI (>or=7 mL/kg) (3.85+/-1.40 vs. 2.07+/-0.38 pg/mL, respectively). Twenty-two (59%) patients died before day 28. CONCLUSIONS: In human septic shock, EVLWI demonstrated moderate correlation with markers of acute lung injury, such as lung compliance, oxygenation ratio, roentgenogram quadrants, and lung injury score. In nonsurvivors, EVLWI and permeability indexes were significantly increased at day 3. Thus, EVLWI might be of value as an indicator of prognosis and severity of sepsis-induced acute lung injury.

Novel endothelin receptor antagonist attenuates endotoxin-induced lung injury in sheep.

OBJECTIVE: To evaluate the cardiopulmonary effects of the novel endothelin receptor antagonist tezosentan in endotoxin-induced lung injury in sheep and to assess the dose response to tezosentan and endothelin-1 in healthy sheep. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University animal laboratory. SUBJECTS: Twenty-one yearling sheep. INTERVENTIONS: Seventeen awake, chronically instrumented sheep were subjected to intravenous infusion of Ringer's lactate for 24 hrs. The animals were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide group (n = 7) receiving an intravenous infusion of Escherichia coli lipopolysaccharide 15 ng x kg x min, and a tezosentan group (n = 7) subjected to lipopolysaccharide and, from 4 hrs, an intravenous injection of tezosentan 3 mg/kg followed by infusion of 1 mg x kg x hr. In addition, four healthy sheep, exposed to an intravenous infusion of endothelin-1 at 20 ng x kg x min, after 1 hr received tezosentan in stepwise increasing doses of 0.5, 1, and 2 mg x kg x hr that were maintained for 1 hr each. After a 4-hr recovery, the sheep received infusions of tezosentan at the same dose rates as a pretreatment to endothelin-1. MEASUREMENTS AND MAIN RESULTS: In the sham-operated sheep, all cardiopulmonary variables remained unchanged. Lipopolysaccharide caused pulmonary hypertension, increased extravascular lung water index, and induced arterial hypoxemia. Tezosentan decreased the increments in pulmonary vascular resistance and extravascular lung water index by as much as 60% and 70%, respectively. In parallel, tezosentan ameliorated arterial hypoxemia, increased cardiac index, attenuated the decrease in stroke volume index, and reduced systemic vascular resistance. Compared with the lipopolysaccharide group, tezosentan further increased plasma concentrations of endothelin-1. In healthy animals, the administration of endothelin-1 induced systemic and pulmonary hypertension, increased extravascular lung water index, and evoked bradycardia and a decrease in cardiac index. These changes were attenuated by tezosentan infused at 1 and 2 mg x kg x hr. CONCLUSIONS: In an ovine model of endotoxin-induced lung injury, tezosentan ameliorates pulmonary hypertension, lung edema, cardiac dysfunction, and arterial hypoxemia. Tezosentan counteracts the hemodynamic effects of endothelin-1 in a dose-dependent manner.