Time course of angiopoietin-2 release during experimental human endotoxemia and sepsis

Abstract

Introduction: Endothelial activation leading to vascular barrier breakdown denotes a devastating event in sepsis. Angiopoietin (Ang)-2, a circulating antagonistic ligand of the endothelial specific Tie2 receptor, is rapidly released from Weibel-Palade and has been identified as a non-redundant gatekeeper of endothelial activation. We aimed to study: the time course of Ang-2 release during human experimental endotoxemia; the association of Ang-2 with soluble adhesion molecules and inflammatory cytokines; and the early time course of Ang-2 release during sepsis in critically ill patients.

Methods: In 22 healthy volunteers during a 24-hour period after a single intravenous injection of lipopolysaccharide (LPS; 4 ng/kg) the following measurement were taken by immuno luminometric assay (ILMA), ELISA, and bead-based multiplex technology: circulating Ang-1, Ang-2, soluble Tie2 receptor, the inflammatory molecules TNF-alpha, IL-6, IL-8 and C-reactive protein, and the soluble endothelial adhesion molecules inter-cellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin. A single oral dose of placebo or the p38 mitogen activated protein (MAP) kinase inhibitor drug, RWJ-67657, was administered 30 minutes before the endotoxin infusion. In addition, the course of circulating Ang-2 was analyzed in 21 septic patients at intensive care unit (ICU) admission and after 24 and 72 hours, respectively.

Results: During endotoxemia, circulating Ang-2 levels were significantly elevated, reaching peak levels 4.5 hours after LPS infusion. Ang-2 exhibited a kinetic profile similar to early pro-inflammatory cytokines TNF-alpha, IL-6, and IL-8. Ang-2 levels peaked prior to soluble endothelial-specific adhesion molecules. Finally, Ang-2 correlated with TNF-alpha levels (r = 0.61, P = 0.003), soluble E-selectin levels (r = 0.64, P < 0.002), and the heart rate/mean arterial pressure index (r = 0.75, P < 0.0001). In septic patients, Ang-2 increased in non-survivors only, and was significantly higher compared with survivors at baseline, 24 hours, and 72 hours.

Conclusions: LPS is a triggering factor for Ang-2 release in men. Circulating Ang-2 appears in the systemic circulation during experimental human endotoxemia in a distinctive temporal sequence and correlates with TNF-alpha and E-selectin levels. In addition, not only higher baseline Ang-2 concentrations, but also a persistent increase in Ang-2 during the early course identifies septic patients with unfavorable outcome.

Figure 1

Time course of Ang-2, cytokines, and adhesion molecules after LPS challenge in healthy subjects. (a) Concentrations of circulating angiopoietin (Ang)-2 compared with plasma levels of TNF-alpha. IL-6, IL-8, and C-reactive protein (CRP) after lipopolysaccharide (LPS) challenge in six healthy volunteers. (b) Concentrations of circulating Ang-2 compared with plasma levels of endothelial adhesion molecules E-selectin, P-selectin, and inter-cellular adhesion molecule-1 (ICAM-1) after LPS challenge in six healthy volunteers. Non-parametric analysis of variance (Friedman's test) with Dunn's test for multiple comparison (two-sided) was used to demonstrate statistical changes in Ang-2, cytokines, and adhesion molecules (y-axes denote percentage increase; baseline = 100%).

Figure 2

Correlation of Ang-2 with TNF-α, E-selectin and heart rate/mean arterial pressure index after LPS challenge in healthy subjects. Dot blots showing the correlation between circulating angiopoietin (Ang)-2 and (a) plasma levels of TNF-alpha, (b) plasma levels of the soluble endothelial specific adhesion molecule E-selectin, and (c) the heart rate/mean arterial pressure index (HR/MAP index) at 6.5 hours after lipopolysaccharide (LPS) challenge in 21 subjects (placebo (n = 6), and medication groups: 350 mg (n = 5). 700 mg (n = 6), and 1400 mg (n = 4), respectively). Pearsons correlation coefficient was used after logarithmic transformation of variables (axes denote percentage increase after logarithmic transformation; baseline = 100%).

Figure 3

Time course of Ang-2 in critically ill patients with sepsis. Dot blots showing the concentration of angiopoietin (Ang)-2 (ng/ml) in 21 septic on intensive care unit (ICU) admission, 24 hours and 72 hours after admission, respectively. Of note, median Ang-2 levels increased in non-survivors (P = 0.019) (continuous line), but remained unchanged in survivors (P = 0.83) (dotted line) during the time course. Mean Ang-2 level was higher in non-survivors (filled circles, n = 11) compared with survivors (open circles, n = 10) on admission (P = 0.032), after 24 hours (P = 0.027), and 72 hours (P = 0.008) (two-sided Mann-Whitney test).