Basic Science Aspects
Enalapril Does Not Alter Adhesion Molecule Levels in Human Endotoxemia
Graninger, Monika*†; Marsik, Claudia*; Dukic, Tanja*; Wagner, Oswald F.‡; Blann, Andrew D.§; Jilma, Bernd*
*Department of Clinical Pharmacology and †Division of Angiology, and ‡Clinical Institute of Medical and Chemical Laboratory Diagnostics, Vienna University, Vienna, Austria; and §Department of Medicine, Thrombosis and Vascular Biology Unit, University of Birmingham, Birmingham, United Kingdom
Received 16 Sep 2002;
first review completed 15 Oct 2002; accepted in final form 6 Nov 2002
Address reprint requests to Dr. Bernd Jilma, Department of Clinical Pharmacology, Division of Hematology and Immunology, Vienna University, Waehringer Guertel 18-20, A-1090 Wien, Austria.
This study was supported by the Austrian Nationalbank (grant no. 8917).
The angiotensin-converting enzyme inhibitor (ACE-I) enalapril has been shown to lower elevated levels of circulating adhesion molecules (cAM) in critically ill patients. To delineate the mechanisms of this possibly beneficial effect of enalapril, we studied the acute effects of enalapril in a well-defined model of endotoxin-triggered, cytokine-mediated cAM up-regulation. In a randomized, controlled trial, 30 healthy male volunteers received 2 ng/kg lipopolysaccharide (LPS) after pretreatment with placebo or 20 mg/day enalapril for 5 days or with a single dose of 20 mg of enalapril 2 h before LPS infusion. LPS infusion increased TNF levels 300-fold above normal, circulating (c) E-selectin levels by 425% (CI, 359%–492%), and P-selectin, VCAM-1, ICAM-1, and von Willebrand factor levels by 47%–74%. LPS infusion also enhanced ICAM-1 and CD11b expression 2- to 3-fold on monocytes. However, no differences were seen between treatment groups (P > 0.05), despite 95% inhibition of ACE activity by enalapril. Inhibition of ACE activity by enalapril does not influence plasma indices of endothelial activation after endotoxin infusion in healthy individuals. Our results do not support the concept of a beneficial clinical effect of enalaprilat in septicemia.
During the development and the propagation of septic shock, the interactions of the endothelium with platelets and leukocytes play a crucial role (1). The recruitment of circulating blood cells to sites of endothelial activation is mediated by the expression of adhesion molecules. Among other bacterial mediators lipopolysaccharide (LPS, endotoxin) contributes to gram-negative sepsis. LPS strongly stimulates the expression of adhesion molecules on endothelial cells (1), which is followed by the release of soluble E-selectin into the bloodstream in vivo (2,3). As a consequence, plasma levels of circulating (c) E-selectin are augmented in sepsis (4,5). The increase in cE-selectin appears to depend on the dose of LPS (2,3), and is determined by LPS-enhanced tumor necrosis factor (TNF) production (3,6). Interestingly, cE-selectin levels correlate with cardiovascular compromise and clinical outcome in septic patients (5,7). Similarly, levels of cP-selectin (8), circulating intercellular adhesion molecule-1 (cICAM-1) (7,9), and vascular cell adhesion molecule-1 (cVCAM-1) are increased in sepsis and may predict organ dysfunction in patients with sepsis (4,10,11).
Angiotensin-converting enzyme inhibitors (ACE-I) including enalapril lower cAM levels in patients with chronic heart failure, diabetic late complications, and hypertension (12–16). Interestingly, infusion of enalaprilat over 5 days prevented a further rise in cE-selectin, cVCAM-1, or cICAM-1 levels in critically ill patients (17). In addition, high enalapril concentrations inhibit in vitro activation of nuclear factor κB (NF-κB), (18) which regulates the expression of AM among other immediate early genes (19).
Based on these clinical and in vitro findings, we hypothesized that ACE-I may decrease cytokine-inducible expression of AM, which could be exploited in a variety of indications. To test this hypothesis, we studied the effects of enalapril on LPS-induced, TNF-mediated endothelial activation (3,20) in a well-standardized human endotoxemia model (12,21,22).
MATERIALS AND METHODS
Study design and study subjects
The protocol was approved by the Institutional Ethics Committee. Written informed consent was obtained from all participants. A block randomized, double-blind, placebo-controlled study was performed in three parallel groups (n = 10 in each group). Blinding was achieved by encapsulation of study drugs into opaque gelatin capsules filled with starch. Healthy male volunteers were included, aged 31 ± 5 (SD) years, with a body mass index of 22 ± 2 (SD) kg/m2. Before inclusion into this trial, health status was determined by medical history, physical examination, laboratory parameters, virological and drug screening, and testing for hereditary thrombophilia.
Individuals in group A (active) took 20 mg of enalapril every morning for 5 days and 2 h before LPS challenge. Group B received placebo for 5 days and a single dose of 20 mg of enalapril 2 h before LPS infusion, whereas group C (controls) received only placebo. In the main study day, fasting volunteers reported to the study ward at 8 AM, and study set-up was as previously described (23). Ninety minutes after subjects had received their last tablet, they ingested 1000 mg of acetaminophen (= paracetamol; Paracetamol, Genericon Pharma) to alleviate subjective symptoms without compromising the host response; uncompromised response in innate immunity in this model was demonstrated by no effect of paracetamol on any of the adhesion molecules or cytokines measured (23,24). After 30 min, LPS (2 ng/kg) was infused (National Reference Endotoxin, Escherichia coli; lot g1; USP Convention, Rockville, MD).
Blood samples were collected into Vacutainer tubes by venipuncture at times indicated in Figure 2. Citrated plasma samples were processed immediately by centrifugation at 2000 g at 4°C for 15 min and were stored at −80°C before analysis, while EDTA anticoagulated blood was used for flow cytometric analysis.
Circulating adhesion molecules were measured by enzyme immunoassays (EIAs; R&D Systems, Oxon, UK) for soluble E-selectin, P-selectin, ICAM-1 and VCAM-1, TNF, and IL-6 with high-sensitivity EIAs (25,26). Von Willebrand factor (vWF) was determined by EIA using polyclonal antisera (Dako-Patts, High Wycombe, UK) (27). ACE activity was measured with ACE reagent (Sigma Diagnostics, St. Louis, MO) on a Hitachi 911 analyzer. Flow cytometric analyses were performed as previously described (17) using phycoerythrin-labeled anti-CD11b and anti-CD54 monoclonal antibodies (Becton Dickinson, Vienna, Austria). Neutrophils and monocytes were identified by gates set according to forward/sideward scatter properties, and gates were regularly cross-checked by double staining of leukocytes with CD14/CD45.
Leukocyte and platelet counts
Differential blood counts were obtained with Sysmex cell counter (Sysmex, Milton Keynes, UK), except for monocyte counts (spuriously high when measured with cell counters), which were calculated from flow cytometric analyses.
Data are expressed as mean and 95% CI. Owing to non-normal distribution, comparisons within groups were done by Friedman analysis of variance (ANOVA) and Wilcoxon signed-rank test for post hoc comparisons. For comparisons between groups, the Kruskal-Wallis ANOVA was applied, followed by Mann-Whitney U test. cE-selectin was determined a priori as the main outcome variable because its increase is TNF dependent (3) and most pronounced (17). Post hoc comparisons were restricted to times of peak values, whereas all other data are presented in a descriptive manner (95% CI).
TNF-levels, IL-6-levels, and leukocyte and platelet counts
TNF and IL-6 levels are depicted in Figure 1. Peak TNF levels (at 90 min) averaged 319 pg/mL (CI: 198–440) after placebo, 345 pg/mL (CI: 191–499) after single-dose enalapril, and 224 pg/mL (CI: 174–275) after six doses of enalapril (n.s. between groups).
Peak IL-6 levels averaged 512 pg/mL (CI: 264–760) after placebo, 560 pg/mL (CI: 250–869) after single-dose enalapril, and 548 pg/mL (CI:360–735) after six doses of enalapril (n.s. between groups). As expected, nadir neutrophil counts were seen at 90 min (1.2 × 109/L; CI:1.0–1.4; n.s. between groups), and platelet counts decreased by 20% in all groups (data not shown).
Endothelial/platelet activation markers
As depicted in Figure 2, cE-selectin levels increased by a maximum of 425% (CI: 359%– 492%), cICAM-1 by 74% (CI: 62%–87%), cVCAM-1 by 54% (CI: 42%–66%), and vWF by 67% (CI: 47%–86%). However, no differences were seen between groups (P > 0.05). Absolute plasma levels of the platelet activation marker cP-selectin (27) were different between groups even at baseline (P = 0.02), likely due to chance. Yet, the relative increase in cP-selectin was not different between treatment arms after LPS infusion (47%; CI: 38%–56%; n = 30).
CD11b and ICAM-1 (CD54) expression on monocytes and neutrophils
Although mean fluorescence intensity (MFI)of CD11b expression peaked 3-fold over baseline on monocytes at 4 h (data not shown), ICAM-1 expression increased 2-fold on monocytes at 24 h (Fig. 2; CI: 120–140 and CI: 230–281 at 0 and 24 h, respectively; n.s. between groups). Basal binding of the anti-CD54 antibody to neutrophils (MFI;Fig. 3) was considerably lower as compared with monocytes (Fig. 2), which was also reflected by the lower CD54 response to LPS.
The percentage of CD54+ neutrophils significantly decreased after 2 h (Fig. 3), a phenomenon likely related to changing neutrophil populations because in vitro incubation of whole blood with 50 pg/mL LPS for 2 h increased rather than decreased CD54 expression (data not shown). MFI of CD54 on neutrophils increased from 29 (CI: 24–37) to 41 (CI: 36–46) at 24 h (P < 0.001 vs. baseline; n.s. between groups;Fig. 3). MFI of CD 11b on neutrophils increased from 661 (CI: 556–765) to 1272 (CI: 1112–1432) at 6 h (P < 0.001 vs. baseline; n.s. between groups;Fig. 3).
Plasma ACE activity and hemodynamic effects
Subjects pretreated with enalapril presented with 80% lower baseline ACE activity on the study day (Fig. 4). A single dose of 20 mg of enalapril decreased ACE activity by 95% (CI:91%–99%; to 2 U/L, CI: 1.3–2.7) 4 h after intake, i.e., 2 h after LPS infusion. As enalaprilat attenuated the systemic cardiovascular response in a hemorrhagic shock model in dogs (28), it was also of interest to examine its effects in endotoxin-induced hemodynamic changes. Overall, the effects of LPS with and without enalapril on blood pressure and heart rate were mild to moderate and are depicted in Figure 4 (P = n.s. between groups).
Several lines of in vitro and clinical evidence (14–16) led us to hypothesize that enalapril may reduce the LPS-induced, TNF-mediated increase in endothelial activation (3), as measured by cE-selectin. However, we did not observe an inhibitory effect of enalapril on cE-selectin release or on any other outcome parameter (Figs. 1–4), although we had an 80% power to detect 30% lower cE-selectin levels (29). This result is in contrast to the clinical trial in critically ill patients (17), although peak cE-selectin levels were comparable between the placebo groups of both studies.
This discrepancy may be explained by several issues. The duration of treatment was identical in both trials, and Boldt et al. (16,17) observed a decrease in cAM levels already after 1 day. We administered a standard dose of 20 mg/day enalapril (bioavailability >60%), and achieved 95% inhibition of ACE activity. It can be estimated from published pharmacokinetic data that we reached peak enalapril concentrations of about 100 μg/L at 1 h. (30) In contrast, Boldt et al. (17) infused 6 mg/day enalaprilat, but unfortunately provided no data on the ACE activity or renal function of their patients, so that it is difficult to estimate whether drug accumulation may have occurred.
Although suprapharmacological captopril concentrations (10–40 mg/L) inhibit LPS-induced NF-κB activation in vitro, clinically relevant concentrations were ineffective in vitro (18). NF-κB transcriptionally regulates several “immediate early genes” such as TNF or E-selectin. In agreement with unaltered TNF and IL-6 levels in our study (Fig. 1), intake of 80 mg of captopril did not blunt ex vivo cytokine production by LPS, whereas ∼220 mg/L captopril suppressed LPS-induced cytokine production in vitro (31). In our current in vivo trial, enalapril also lacked an effect on downstream events because enalapril did not blunt E-selectin shedding or neutrophilia, both of which are critically dependent on TNF (3).
Differences in endothelial function or in the duration of inflammation between young, male, healthy subjects and older, mixed-sex, critically ill patients could provide an alternative explanation for the different effects of enalapril on cAM-levels in critically ill patients. It is also possible that the degree of systemic inflammation induced in our model acutely exceeds that observed in critically ill patients. Without doubt, infusion of endotoxin is by no means the same as clinical sepsis.
Finally, the stimulus for systemic inflammation, although not specified, is probably different from endotoxemia in many of Boldt's surgical patients. Thus, further studies on the beneficial effects of ACE-I in other well-defined groups of critically ill patients or in in vivo models of septicemia will be necessary to delineate possible protective mechanisms of ACE-I on the endothelium.
In summary, short-term inhibition of ACE activity by enalapril had no effects on LPS-induced, cytokine-mediated indices of endothelial, leukocyte, or platelet activation.
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