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.
1. Yan W, Zhao K, Jiang Y, Huang Q, Wang J, Kan W, Wang S: Role of p38 MAPK in ICAM-1 expression of vascular endothelial cells induced by lipopolysaccharide. Shock 17:433–438, 2002.
2. Kuhns DB, Alvord WG, Gallin JI: Increased circulating cytokines, cytokine antagonists, and E-selectin after intravenous administration of endotoxin in humans. J Infect Dis 171:145–152, 1995.
3. van Der Poll T, Coyle SM, Levi M, Jansen PM, Dentener M, Barbosa K, Buurman WA, Hack CE, ten Cate JW, Agosti JM, Lowry SF: Effect of a recombinant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans. Blood 89:3727–3734, 1997.
4. Cowley HC, Heney D, Gearing AJ, Hemingway I, Webster NR: Increased circulating adhesion molecule concentrations in patients with the systemic inflammatory response syndrome: a prospective cohort study. Crit Care Med 22:651–657, 1994.
5. Cummings CJ, Sessler CN, Beall LD, Fisher BJ, Best AM, Fowler AA: Soluble E-selectin levels in sepsis and critical illness. Correlation with infection and hemodynamic dysfunction. Am J Respir Crit Care Med 156:431–437, 1997.
6. Suffredini AF, Reda D, Banks SM, Tropea M, Agosti JM, Miller R: Effects of recombinant dimeric TNF receptor on human inflammatory responses following intravenous endotoxin administration. J Immunol 155:5038–5045, 1995.
7. Kayal S, Jais JP, Chaudiere J, Labrousse J: Elevated circulating E-selectin, intercellular adhesion molecule-1, and von Willebrand Factor in patients with severe infection. Am J Respir Crit Care Med 157:776–784, 1998.
8. Fijnheer R, Frijns CJ, Korteweg J, Rommes H, Peters JH, Sixma JJ, Nieuwenhuis HK: The origin of P-selectin as a circulating plasma protein. Thromb Haemost 77:1081–1085, 1997.
9. Moss M, Gillespie MK, Ackerson L, Moore FA, Moore EE, Parsons PE: Endothelial cell activity varies in patients at risk for the adult respiratory distress syndrome. Crit Care Med 24:1782–1786, 1996.
10. Sakamaki F, Ishizaka A, Handa M, Fujishima S, Urano T, Sayama K, Nakamura H, Kanazawa M, Kawashiro T, Katayama M, et al.: Soluble form of P-selectin in plasma is elevated in acute lung injury. Am J Respir Crit Care Med 151:1821–1826, 1995.
11. Sessler CN, Windsor AC, Schwartz M, Watson L, Fisher BJ, Sugerman HJ, Fowler AA: Circulating ICAM-1 is increased in septic shock. Am J Respir Crit Care Med 151:1420–1427, 1995.
12. Hollenstein U, Homoncik M, Knobl P, Pernerstorfer T, Graggaber J, Eichler HG, Handler S, Jilma B: Acenocoumarol decreases tissue factor-dependent coagulation during systemic inflammation in humans. Clin Pharmacol Ther 71:368–374, 2002.
13. Jilma B, Li-Saw-Hee Fl, Wagner OF, Beevers DG, Lip GYH: Effects of enalapril and losartan on circulating adhesion molecules and monocyte chemotactic protein-1 (MCP-1) in hypertensive patients. Clin Sci 103:131–136, 2002.
14. Drexler H, Kurz S, Jeserich M, Munzel T, Hornig B: Effect of chronic angiotensin-converting enzyme inhibition on endothelial function in patients with chronic heart failure. Am J Cardiol 76:13E–18E, 1995.
15. Andersen S, Schalkwijk CG, Stehouwer CD, Parving HH: Angiotensin II blockade is associated with decreased plasma leukocyte adhesion molecule levels in diabetic nephropathy [letter]. Diabetes Care 23:1031–1032, 2000.
16. Gasic S, Wagner OF, Fasching P, Ludwig C, Veitl M, Kapiotis S, Jilma B: Fosinopril decreases levels of soluble vascular cell adhesion molecule-1 in borderline hypertensive type II diabetic patients with microalbuminuria. Am J Hypertens 12:217–222, 1999.
17. Boldt J, Papsdorf M, Kumle B, Piper S, Hempelmann G: Influence of angiotensin-converting enzyme inhibitor enalaprilat on endothelial-derived substances in the critically ill. Crit Care Med 26:1663–1670, 1998.
18. Napoleone E, Di SA, Camera M, Tremoli E, Lorenzet R: Angiotensin-converting enzyme inhibitors downregulate tissue factor synthesis in monocytes. Circ Res 86:139–143, 2000.
19. Sun Z, Andersson R: NF-κB activation and inhibition: a review. Shock 18:99–106, 2002.
20. Kohn G, Wong HR, Bshesh K, Zhao B, Vasi N, Denenberg A, Morris C, Stark J, Shanley TP: Heat shock inhibits TNF-induced ICAM-1 expression in human endothelial cells via Iκ kinase inhibition. Shock 17:91–97, 2002.
21. Suffredini AF, Hochstein HD, McMahon FG: Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli
O:113 endotoxin. J Infect Dis 179:1278–1282, 1999.
22. Santos AA, Wilmore DW: The systemic inflammatory response: perspective of human endotoxemia. Shock 6(Suppl 1):S50–S56, 1996.
23. Pernerstorfer T, Hollenstein U, Hansen J, Knechtelsdorfer M, Stohlawetz P, Graninger W, Eichler HG, Speiser W, Jilma B: Heparin blunts endotoxin-induced coagulation activation. Circulation 100:2485–2490, 1999.
24. Pernerstorfer T, Schmid R, Bieglmayer C, Eichler HG, Kapiotis S, Jilma B: Acetaminophen has greater antipyretic efficacy than aspirin in endotoxemia: a randomized, double-blind, placebo-controlled trial. Clin Pharmacol Ther 66:51–57, 1999.
25. Jilma B, Blann A, Pernerstorfer T, Stohlawetz P, Eichler HG, Vondrovec B, Amiral J, Richter V, Wagner OF: Regulation of adhesion molecules during human endotoxemia. No acute effects of aspirin. Am J Respir Crit Care Med 159:857–863, 1999.
26. Jilma B, Marsik C, Mayr F, Graninger MT, Taylor FBJ, Ribel MC, Erhardtsen E, Handler S, Eichler HG: Pharmacodynamics of active site inhibited factor VIIa (ASIS, FFR-rFVIIa) in endotoxin induced coagulation in humans. Clin Pharmacol Ther 72:403–410, 2002.
27. Blann AD, Lip GY, Beevers DG, McCollum CN: Soluble P-selectin in atherosclerosis: a comparison with endothelial cell and platelet markers. Thromb Haemost 77:1077–1080, 1997.
28. Wall P, Buising C, Henderson L, Rickers T, Cardenas A, Owens L, Timberlake G, Paradise N: Enalaprilat improves systemic cardiovascular parameters and mesenteric blood flow during hypotensive resuscitation from hemorrhagic shock in dogs. Shock 17:228–233, 2002.
29. Stolley PD, Strom BL: Sample size calculations for clinical pharmacology studies. Clin Pharmacol Ther 39:489–490, 1986.
30. Gomez HJ, Cirillo VJ, Irvin JD: Enalapril: a review of human pharmacology. Drugs 30 (Suppl 1):13–24, 1985.
31. Peeters AC, Netea MG, Kullberg BJ, Thien T, van der Meer JW: The effect of renin-angiotensin system inhibitors on pro- and anti-inflammatory cytokine production. Immunology 94:376–379, 1998.