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Ann Thorac Surg 2008;86:1576-1583. doi:10.1016/j.athoracsur.2008.06.076
© 2008 The Society of Thoracic Surgeons
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Original Articles: Adult Cardiac

Endothelin-A Receptor Inhibition After Cardiopulmonary Bypass: Cytokines and Receptor Activation

Rachael L. Ford, BS, Ira M. Mains, BS, Ebony J. Hilton, BS, Scott T. Reeves, MD, Robert E. Stroud, MS, Fred A. Crawford, Jr, MD, John S. Ikonomidis, MD, PhD, Francis G. Spinale, MD, PhD*

Division of Cardiothoracic Surgery and Anesthesia, Medical University of South Carolina, and Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina

Accepted for publication June 23, 2008.

* Address correspondence to Dr Spinale, Cardiothoracic Surgery, Strom Thurmond Research Bldg, 114 Doughty St, Room 625, Medical University of South Carolina, Charleston, SC 29403 (Email: wilburnm{at}musc.edu).

Presented at the Basic Science Forum of the Fifty-fourth Annual Meeting of the Southern Thoracic Surgical Association, Bonita Springs, FL, Nov 7–10, 2007.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Background: Basic studies have suggested that cross-talk exists between the endothelin-A receptor (ET-AR) and tumor necrosis factor signaling pathway. This study tested the hypothesis that administration of an ET-AR antagonist at the separation from cardiopulmonary bypass would alter the tumor necrosis factor activation in the early postoperative period.

Methods: Patients (n = 44) were randomly allocated to receive bolus infusion of vehicle, 0.1, 0.5, 1, or 2 mg/kg of the ET-AR antagonist (sitaxsentan), at the separation from cardiopulmonary bypass (n = 9, 9, 9, 9, and 8, respectively). Plasma levels of tumor necrosis factor-{alpha} and soluble tumor necrosis factor receptor 1 and 2 were measured.

Results: Compared with the vehicle group at 24 hours, plasma levels of tumor necrosis factor-{alpha} and tumor necrosis factor receptor 2 (indicative of receptor activation) were reduced in the 1 mg/kg ET-AR antagonist group (by approximately 13 pg/mL and approximately 0.5 ng/mL, respectively; p < 0.05). Plasma tumor necrosis factor receptor I levels also decreased (by approximately 1 ng/mL) after infusion of the higher doses of the ET-AR antagonist and remained lower (by approximately 3 ng/mL) at 24 hours after infusion (p < 0.05). In addition, a dose effect was observed between the ET-AR antagonist and these indices of tumor necrosis factor activation (p < 0.01).

Conclusions: This study demonstrated a mechanistic relationship between the ET-AR and tumor necrosis factor receptor activation in the post–cardiac surgery period. Thus, in addition to the potential cardiovascular effects, a selective ET-AR antagonist can modify other biological processes relevant to the post–cardiac surgery setting.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Coronary revascularization is one of the most common adult cardiac surgical procedures, and can often require cardiopulmonary bypass (CPB). While CPB provides for myocardial quiescence and a bloodless operative field, myocardial reperfusion and separation from CPB can be associated with transient cardiovascular compromise in the postoperative setting. Coincident with the separation from CPB is the release of bioactive molecules including endothelin (ET) and cytokines [1–9]. While mechanistic links remain to be established, ET release and cytokine activation have been associated with a protracted postoperative course after CPB [6–9].

Studies have demonstrated that ET has diverse biological effects on myocyte contractility and upon the vasculature [10–12]. The cardiovascular effects of ET are mediated through two unique receptor subtypes: ET-A receptor (ET-AR) and ET-B receptor (ET-BR). The ET-AR mediates local and systemic vasoconstriction and has negative inotropic effects [10–12]. In contradistinction, the ET-BR mediates the release of vasodilatory factors, such as nitric oxide, as well as facilitates ET clearance [13, 14]. Accordingly, a number of selective ET-AR antagonists have been developed and are under clinical investigation for pulmonary hypertension [15]. One such selective ET-AR antagonist, sitaxsentan [16], has been utilized by this laboratory in a dose ranging and safety study in patients undergoing coronary revascularization requiring CPB [17]. However, several unanswered questions remained from this initial dose ranging study, and they include the potential interactions that exist between ET-AR and the cytokine cascade.

Cytokines are a diverse group of molecules that form a part of the immune response and affect the cardiovascular system by regulating inflammation, myocardial function, and cell apoptosis [18, 19]. Tumor necrosis factor-{alpha} (TNF) is a proinflammatory cytokine that has been reported to increase after CPB [3, 9]. Tumor necrosis factor-{alpha} is a membrane-bound molecule, which is cleaved by a protease and released, resulting in binding to the TNF receptors TNFRI and TNFRII [20]. Subsequent to TNF binding, the extracellular TNF/TNFRI and TNF/TNFRII complexes are released into the interstitial space and can be detected in the systemic circulation [4, 21]. Thus, measurements of relative changes in TNF/TNFRI and TNF/TNFRII within the systemic circulation provide an index of TNFR activation. Past in vitro and in vivo studies have demonstrated ET can induce the biosynthesis and release of TNF [22, 23]. Therefore, the hypothesis of this study is that administration of a selective ET-AR antagonist immediately after separation from CPB would alter the TNF receptor profile in a dose-dependent manner.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
Overview
This study utilized plasma samples obtained from a previous dose ranging and safety study in which a selective ET-AR antagonist was administered after CPB [17]. The patient demographics, hemodymanics, and operative statistics were fully described in that study and accordingly are only briefly described below.

Patients
This study obtained full approval by the Human Subjects Review Committee of the Medical University of South Carolina (HR11122). Patients undergoing elective coronary artery bypass surgery requiring CPB provided informed consent to participate in the substudy. Exclusion criteria included emergent revascularization, stroke or thromboembolic event within 3 months preceding surgery, previous myocardial infarction, documented coagulopathy; hepatic dysfunction as defined by aspartate transaminase or alanine transaminase greater than 1.5 times the upper limit of normal, and chronic renal insufficiency as defined by a creatinine greater than 2.5 mg/dL or requirement for dialysis. All patients were male and averaged 62 ± 1 years of age.

Operative Procedure
Standard induction and maintenance of anesthesia was accomplished with a combination of sufentanil, midazolam, and isoflurane. All patients received the full Hammersmith dose of aprotinin before surgery. No patients were administered corticosteroids during surgery. Before CPB, systemic heparinization was accomplished with a heparin dose of 400 units/kg and was administered to maintain an activated clotting time of more than 400 s. Cardiopulmonary bypass was accomplished with a Sarns membrane oxygenator (Terumo/Sarns, Ann Arbor, Michigan) and a hemoconcentration pack with Smart Tubing circuits (Sorin Group, Arvada, Colorado). A cell-saving device was utilized during CPB (Haemonetics, Brainetree, Massachusetts), and wound aspirate was returned after the separation from CPB. Cardiopulmonary bypass was maintained at a cardiac index of 2.0 to 2.4 L · min–1 · m–2, and cardioplegia was accomplished with antrograde normothermic administration of a blood crystalloid solution as previously described. Cardiac arrest was maintained with routine intervals of administration of cardioplegic solution in a retrograde fashion. Patients were not actively cooled during CPB and were rewarmed to a rectal temperature of 36.5°C before CPB separation. At the termination of CPB, heparin was neutralized with protamine in a 1:1 ratio.

Endothelin-A Receptor Antagonist, Randomization, and Sampling Times
The selective ET-AR antagonist utilized in this study was sitaxsentan sodium (TBC11251Na), which reaches a steady-state level within 30 minutes after intravenous administration and has a half-life of approximately 6 hours [24]. This study was performed under FDA IND#52,527.

Patients (n = 44) were assigned to treatment groups by an independent clinical research nurse the night before surgery by a predetermined randomized table. The treatment groups included administration of vehicle (saline bolus, n = 9), 0.1 mg/kg (n = 9), 0.5 mg/kg (n = 9), 1.0 mg/kg (n = 9), or 2.0 mg/kg (n = 8) of the selective ET-AR antagonist. The ET-AR antagonist was infused at the separation of CPB to correspond with the peak release of ET from the myocardial interstitium and to avoid confounding effects of hemoconcentration during CPB [1]. The average duration of infusion was 7 ± 1 minutes. The following time points were used: baseline (after placement of arterial and pulmonary catheters, but before the onset of CPB), immediately after cross-clamp release, time 0 (immediately at cessation of CPB and after vehicle/ET-AR antagonist infusion), 0.5, 6, and 24 hours after CPB. Blood samples were collected at the designated time points for the determination of plasma ET and cytokine levels.

Endothelin and Cytokine Assays
Plasma levels of ET were analyzed by a radioimmunoassay (RPA 545; Amersham, Piscataway, New Jersey), which has been previously validated by this laboratory [1, 5, 6]. Analysis of plasma TNF, TNFRI, and TNFRII levels was performed using multiplex suspension array technology [25]. To determine the potential effects of the ET-AR antagonist on other classical cytokines previously observed with CPB, the proinflammatory cytokine interleukin-6 and the anti-inflammatory cytokine interleukin-10 were also measured by a multiplex suspension array. Undiluted plasma was incubated for 2 hours at room temperature with analyte-specific monoclonal antibodies conjugated to polystyrene beads in a 96-well filter plate. The wells were washed three times and incubated for 1 hour at room temperature with secondary biotinylated antisera (Invitrogen, Camarillo, California). The wells were washed again before incubation for 30 minutes with streptavidin R-phycoerythin. Finally, the wells were washed and the analyte/bead complexes were resuspended within the filter plate. The identification and quantification of the analyte/bead complexes were determined by flow cytometry with dual excitation lasers (Bio-Plex Suspension Array Workstation; Bio-Rad, Hercules, California). Using this approach, the interassay and intra-assay variation was 10% or less, and the level of detection was 0.1 pg/mL. Analyte concentrations were corrected for hemodilution.

Data Analysis
Categorical analysis of patient medications and transfusions were evaluated by Pearson's {chi}2 analysis to determine differences between treatment groups. The duration of hypothermia, CPB, and cross-clamp time were compared between treatment groups by Bonferroni adjusted analysis of variance. Before and immediately after infusion of the ET-AR antagonist, plasma levels of ET and cytokines were evaluated by a two-way analysis of variance to determine the effects of time or ET-AR antagonist treatment. Potential correlations between plasma levels before vehicle/ET-AR antagonist infusion of ET and TNFRI, or TNFRII were analyzed by Spearman's rank correlation. The absolute changes of plasma levels ET and cytokine levels were computed at 0.5, 6, and 24 hours after infusion of the ET-AR antagonist. If the analyte value was within 25% of the detection limits, the analyte value was assigned a numerical value of zero for the purposes of computing the absolute change. A one-sided t test was used to determine if an analyte level significantly changed from the 0 hour/infusion value. The absolute changes of plasma ET and cytokines levels after the infusion of the ET-AR antagonist were evaluated by a two-way analysis of variance to determine the effects of time, ET-AR antagonist dose, or potential interactions. If any time or dose effects were observed after the initial two-way analysis of variance, the effect was further examined with the Wilcoxon signed-rank test. All analysis was performed with the statistical package Intercooled Stata V 8.0 (StataCorp, College Station, Texas). Values are reported as mean ± SEM, and statistical significance was determined at p less than 0.05.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
Clinical Results
Forty-four patients were enrolled and successfully completed the study. Preoperative medications were not different across the treatment groups for the following: nonsteroidal anti-inflammatory drugs (ie, aspirin: 73%, p = 0.20), nitrates (27%, p = 0.97), diuretics (ie, hydrochlorothiazide: 18%, p = 0.94), renin angiotension aldosterone system antagonists (64%, p = 0.27), calcium-channel antagonists (23%, p = 0.30), and β-adrenergic antagonists (73%, p = 0.28). The duration of hypothermia (below 35°C: 37 ± 4 minutes, p = 0.33), CPB (86 ± 4 minutes, p = 0.75), and cross-clamp time (68 ± 3 minutes, p = 0.52) were not different across treatment groups. Patient medications during the intraoperative period also did not differ across treatment groups for positive inotropic agents (ie, epinephrine and dopamine: 37%, p = 0.95), -{alpha}-adrenergic agonists (ie, phenylephrine: 78%, p = 0.93), and nitrates (12%, p = 0.21). The frequency of patients receiving transfusions after the separation from CPB (defined as whole blood, packed red blood cells, fresh frozen plasma, or platelets, 55%, p = 0.70) was not different across treatment groups.

Endothelin and Cytokine Plasma Levels
The plasma levels of ET and cytokines are shown in Table 1 and have been segmented with respect to treatment and time: baseline, cross-clamp release, and immediately after the separation from CPB and the infusion of the ET-AR antagonist (0 hour). There was not a significant time effect with respect to plasma ET levels; however, a secondary treatment effect was observed with the infusion of the ET-AR antagonist. Specifically, immediately after the ET-AR antagonist infusion, an increase in plasma ET concentrations occurred. This effect is likely due to the displacement of ET from the ET-AR and is consistent with a ligand-receptor-antagonist interaction [26]. It has been previously reported that an acute infusion of an ET receptor antagonist can cause an increase in plasma ET [27]. Plasma interleukin-6 levels increased in a time dependent manner, but there was no significant treatment effect. There was no significant time or treatment effect with respect to interleukin-10 or TNF. Plasma TNFRI and TNFRII levels increased with respect to time, but not treatment. To examine whether an interrelationship existed between plasma ET and TNFR levels, a correlation analysis was performed. This analysis was performed on the time points before the ET-AR antagonist infusion, to obviate the confounding effects of these later time points with respect to antagonist-receptor interactions. A significant correlation was observed between plasma ET and TNFRI levels (r = 0.25, p = 0.02), and a positive correlation, although not reaching statistical significance, was observed between plasma ET and TNFRII levels (r = 0.19, p = 0.08). In summary, while cytokine levels changed from baseline as a function of CPB, the changes were equivalent across the randomized groups. Thus, cytokine levels were equivalent and uniform for all treatment groups up to, and immediately after, the separation of CPB and the infusion of the ET-AR antagonist.


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  		Table 1 Plasma Concentrations of Endothelin and Cytokines at Baseline, Cross-Clamp Release, (CCR), and 0 Hour
 
Endothelin and Cytokine Plasma Levels After ET-AR Antagonist Infusion
The absolute changes in plasma levels of ET and cytokines were computed at 0.5, 6, and 24 hours after the infusion of increasing doses of the ET-AR antagonist and vehicle (Figs 1 and 2). Go At 24 hours after infusion, plasma ET levels were increased, except in the high-dose ET-AR antagonist group. Plasma interleukin-6 levels were increased at 6 hours after infusion in all of the ET-AR antagonist groups. In contrast, the plasma interleukin-10 levels were decreased after infusion of the higher doses of the ET-AR antagonist, and this effect persisted at longer time points. At 24 hours after infusion, plasma TNF levels were reduced in the higher dose of the ET-AR antagonist when compared with the vehicle group. Plasma TNFRI levels decreased immediately after infusion of the higher doses of the ET-AR antagonist, and this effect persisted at longer time points. At 24 hours after infusion, plasma TNFRII levels were reduced in the higher doses of the ET-AR antagonist when compared with the vehicle group.


Figure 1
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  		Fig 1. The absolute changes in plasma (A) endothelin, (B) interleukin-6, and (C) interleukin-10 concentrations were computed at 0.5, 6, and 24 hours after infusion of increasing doses of the endothelin-A receptor (ET-AR) antagonist (0.1 to 2.0 mg/kg [black bars]) or vehicle (0 mg/kg [white bars]). At 24 hours after infusion, plasma endothelin levels were increased, except in the high-dose ET-AR group. Plasma interleukin-6 levels were increased at 6 hours after infusion in all of the ET-AR antagonist groups. In contrast, interleukin-10 levels were reduced after infusion of the higher doses of the ET-AR antagonist, and this effect persisted at longer time points. (*p < 0.05 versus immediate postinfusion time point; #p < 0.05 versus respective 0.5-hour time point.)

 

Figure 2
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  		Fig 2. The absolute changes in plasma (A) tumor necrosis factor-{alpha} (TNF), (B) TNF receptor I, and (C) TNF receptor II concentrations were computed at 0.5, 6, and 24 hours after infusion of increasing doses of the endothelin-A receptor (ET-AR) antagonist (0.1 to 2.0 mg/kg [black bars]) or vehicle (0 mg/kg [white bars]). At 24 hours after infusion, plasma TNF levels were reduced in the higher dose of the ET-AR antagonist when compared with the vehicle group. Plasma TNF receptor I levels decreased immediately after infusion of the higher doses of the ET-AR antagonist, and this effect persisted at longer time points. Similarly, at 24 hours after infusion, plasma TNF receptor II levels were reduced in the higher doses of the ET-AR antagonist when compared with the vehicle group. (*p < 0.05 versus immediate postinfusion time point; #p < 0.05 versus respective vehicle group.)

 
After the infusion of the ET-AR antagonist, the effects of time and dose on plasma ET levels and cytokine levels were determined by a two-way analysis of variance (Table 2). In this analysis, there were two main treatment effects: time after infusion of vehicle/ET-AR antagonist and dose (0 to 2.0 mg/kg). Plasma ET levels were significantly affected by both time and dose, but no interaction occurred. Plasma levels of interleukin-6 and interleukin-10 were affected with time, but not by dose. In contrast, the plasma levels of TNF, TNFRI, and TNFRII were not affected by time, but a significant dose effect was observed. There was no interaction between time and dose with respect to the indices of TNFR activation. In conclusion, the ET-AR antagonist dose significantly affected the TNF signaling pathway without altering the other cytokines.


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  		Table 2 Absolute Changes of Endothelin and Cytokine Levels From Infusion of Endothelin-A Receptor (ET-AR) Antagonist (Vehicle or Sitaxsentan 0.1, 0.5, 1, and 2 mg/kg) was Evaluated by Two-Way Analysis of Variance to Determine Effects of Time, Dose, and Potential Interactions
 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
Separation from CPB is often associated with the release of bioactive molecules, which include ET and cytokines [1–9]. Endothelin causes systemic and coronary vasoconstriction as well as negative inotropic effects primarily mediated through the ET-A receptor subtype (ET-AR) [10–13]. The proinflammatory cytokine, tumor necrosis factor-{alpha} (TNF), has been associated with negative inotropic effects and cell apoptosis [18–20]. Accordingly, the overall goal of this study was to examine the potential influence of ET-AR activation upon indices of TNF activation after coronary revascularization requiring CPB. After the administration of a selective ET-AR antagonist at the separation from CPB, plasma levels of TNF and soluble TNF receptors (indicative of TNF activation) were significantly lower in the early post-CPB period. The ET-AR antagonist had a significant dose effect on the indices of the TNF signaling pathway, which was not observed with the other cytokines released in the post-CPB period. In addition to the established vasoconstrictive and negative inotropic effects of the ET-AR, this study provides clinical evidence for an additional biological role of modification of the TNF activation pathway.

Endothelin antagonists have been utilized in cardiovascular diseases such as pulmonary hypertension and congestive heart failure [15, 16]. Nonselective ET antagonist, in other words, equally inhibiting ET-AR and the ET-B receptor subtype (ET-BR), have demonstrated transient hemodynamic improvements, with long-term treatment being associated with liver toxicity and worsening of clinical status [28]. One potential explanation for the undesirable effects of nonselective ET antagonists is the inhibition of the beneficial vasodilatory and ET clearance mechanisms of the ET-BR. Accordingly, a number of selective ET-AR antagonists have been developed and are under clinical investigation for pulmonary hypertension [15]. This laboratory has previously shown that the selective ET-AR antagonist, sitaxsentan, reduced pulmonary vascular resistance after separation from CPB in an initial dose ranging and safety study [17]. However, several unanswered questions remain and include the potential interactions that exist between ET-AR and the TNF signaling pathway.

Tumor necrosis factor-{alpha} is released in the post-CPB period and has been associated with a protracted postoperative course including longer intensive care unit stay, greater inotropic support, and multiple organ dysfunction [8, 9]. In this study, plasma TNF levels were lower with the infusion of the ET-AR antagonist in the post-CPB period. TNF is synthesized and released from a variety of cell types which results in the binding and activation of the TNF receptors (TNFR). After activation, the TNFR complexes are cleaved and released into the circulation. In this study, there was a reduction in the TNFR activation with the infusion of the ET-AR antagonist, indicated by reduced plasma levels of soluble TNFR. The reduction of TNF and TNFR activation with the ET-AR antagonist signifies attenuation of the inflammatory response which may be beneficial in the post-CPB period. Based upon the findings of past reports and the present study, a prospective study designed to determine the relationship between ET-AR inhibition, postoperative outcome, and cytokine profile is warranted.

The immune response in the post-CPB period also includes the release of other cytokines such as the proinflammatory interleukin-6 and the anti-inflammatory interleukin-10 [3, 4]. Interleukin-10 has been shown to have beneficial effects by suppressing production of TNF and interleukin-6 [29]. This study demonstrated that plasma interleukin-6 and interleukin-10 levels after infusion were significantly affected by time, but both cytokines were independent of effects by the ET-AR antagonist dose. In this study, the ET-AR antagonist selectively altered the TNF signaling pathway without significantly affecting a potentially beneficial cytokine, interleukin-10.

Previous pharmacologic interventions for the attenuation of proinflammatory cytokines released in the post-CPB period have included corticosteroids [30, 31]. The infusion of corticosteroids reduced circulating levels of TNF to a similar degree found in this study [31]. However, corticosteroids can be associated with nonspecific and undesired effects including postoperative hyperglycemia, pulmonary dysfunction, and prolonged ventilatory time [30]. In contrast, the infusion of a selective ET-AR antagonist in a previous dose ranging and safety study demonstrated beneficial hemodynamic effects with no significant differences in adverse events in the perioperative period when compared with the vehicle [17]. Taken together, these results suggest that reducing ET-AR activation may possess a favorable profile with respect to reducing the deleterious effects of TNF.

Study Limitations and Summary
Past studies demonstrated that circulating levels of ET and TNF can be associated with a protracted postoperative course [5–9]. The present study design (duration and sample size) prevented the examination of potential relationships between the postoperative outcome, plasma ET levels, and the cytokine profile. The duration of the present study extended to 24 hours after CPB; however, the effects of ET-AR inhibition on plasma levels of ET, cytokines, and TNF receptors throughout the remaining hospitalization remains unknown.

The present study focused upon the potential interrelationship between ET-AR activation and cytokine release. However, multiple biological pathways are activated after separation from CPB, and it is unlikely that the ET signaling pathway is mutually exclusive. For example, the inflammatory response includes complement activation in the early post-CPB period [32]. The involvement of ET-AR activation in other biological signaling cascades relevant to cytokine release remains to be established.

Past in vitro and in vivo experimental models have established that ET-AR activation can induce the synthesis and release of TNF [22, 23]. Binding of ET to the ET-AR can result in the induction of an intracellular cascade that culminates in both pretranscriptional and transcriptional events (Fig 3) [22, 33]. Specifically, ET-AR can result in the formation of phosphorylation intermediates, which in turn could cause phosphorylation and mobilization of TNF convertase to the cell membrane [33, 34]. The TNF convertase in turn would cause solubilization of membrane-bound TNF and ultimately binding to the TNFR complex [20]. The binding of TNF to the TNFR will ultimately be proteolytically processed and yield soluble TNFR complexes [21]. Stimulation of both the ET-AR and TNFR will cause binding to transcription factor sites such as nuclear factor {kappa}-B and activating protein-1, which can cause the formation of ET and TNF [20, 35–38]. As such, it is possible that ET-AR and TNFR signaling results in amplification and a positive feedback loop of the ET and TNF cascade. In the present study, the index of TNFR activation was attenuated in the ET-AR antagonist group in the post-CPB period. The present study provides clinical evidence of an interrelationship between the ET-AR and TNF release and activation in the postoperative period after CPB.


Figure 3
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  		Fig 3. Schematic of the potential interaction of the endothelin-A receptor (ET-AR) and the tumor necrosis factor-{alpha} (TNF) pathway. Binding of endothelin (ET) to the ET-AR caused the induction of an intracellular cascade which culminates in both pretranscriptional and transcriptional events. Specifically, ET-AR can result in the formation of phosphorylation intermediates, which in turn could cause phosphorylation and mobilization of TNF convertase (TACE) to the cell membrane. In turn, TACE would cause solubilization of membrane-bound TNF and ultimately binding to the TNF receptor complex (TNFR). The binding of TNF to the TNFR will ultimately be proteolytically processed and yield soluble TNFR complexes, which can be quantified in the plasma. Stimulation of both the ET-AR and TNFR will cause binding to transcription factor sites such as nuclear factor {kappa}-B (NF{kappa}B) and activating protein 1 (AP-1), which can cause the formation of ET and TNF. Thus, stimulation of the ET-AR and TNFR thereby form an amplification loop. The present study demonstrated that selective ET-AR can directly modify TNFR activation.

 


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This study was supported by an unrestricted grant from Encysive Pharmaceuticals, Houston, Texas; National Institutes of Health Grants HL075488, HL056603, HL057952; and NIH Minority Supplement Research Grant.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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