Angiopoietin-2 is a potential mediator of endothelial barrier dysfunction following cardiopulmonary bypass

Abstract

Introduction: Endothelial activation leading to vascular barrier dysfunction and organ failure is a well-recognized complication of cardiovascular surgery with cardiopulmonary bypass (CPB). The endothelial-specific angiopoietin-Tie2 ligand-receptor system has been identified as a non-redundant regulator of endothelial activation. Binding of angiopoietin-2 (Ang-2) to the Tie2 receptor antagonizes Tie2 signaling and renders the endothelial barrier responsive to pro-inflammatory cytokines. We aimed to study the time course and potential triggering factors of Ang-2 release after CPB, as well as the association of Ang-2 changes with surrogates of increased vascular permeability, organ dysfunction, and outcome.

Methods: Serum levels of Ang-2 from 25 adult patients (140 screened) were measured before and at 0, 12, and 24h following CPB procedure by in-house immuno-luminometric assay (ILMA), and compared with indices of organ dysfunction, duration of mechanical ventilation (MV), length of stay (LOS) in the intensive care unit (ICU), and hospital mortality. The effect of Ang-2 was studied in vitro by incubating high Ang-2 patient serum with endothelial cells (EC).

Results: Ang-2 levels steadily increased from 2.6 ± 2.4 ng/mL at 0 h up to 7.3 ± 4.6 ng/mL at 24h following CPB (P<0.001). The release of Ang-2 correlated with the duration of CPB, aortic cross-clamp time, and post-CPB lactate levels. Changes in Ang-2 during follow-up correlated with partial pressure of oxygen in arterial blood (PaO(2))/fraction of inspired oxygen (FiO(2)) ratio, alveolar-arterial oxygen tension difference (AaDO(2)), hemodynamics, fluid balance, and disease severity measures. Ang-2 levels at 12h predicted the duration of MV, ICU-LOS, and hospital mortality. High Ang-2 patient sera disrupted EC architecture in vitro, an effect reversed by treatment with the competitive Tie2 ligand angiopoietin-1 (Ang-1).

Conclusions: Collectively, our results suggest that Ang-2 is a putative mediator of endothelial barrier dysfunction after CPB. These findings suggest that targeting the Ang/Tie2 pathway may mitigate organ dysfunction and improve outcome in patients undergoing CPB.

Fig. 1

Bar charts showing (A) absolute and (B) relative (n-fold vs. 0 h) changes of Ang-2 after CPB during the 24 h study period (SD + SEM). Individual changes of Ang-2 are provided in Supplementary data file 1.

Fig. 2

Scatter plots showing Pearson’s product-moment correlation of Ang-2 (12 h after CPB) with (A) duration of CPB, (B) duration of aortic cross-clamp duration, and (C) post-CPB lactate levels. Dots indicate individual cases.

Fig. 3

Kaplan–Meier curves showing (A) the duration of mechanical ventilation and (B) ICU-LOS in patients stratified to Ang-2≤ vs. >median at 12 h after CPB.

Fig. 4

Confocal microscopy images showing immunofluorescence staining for VE-cadherin (green), F-actin (red), and DAPI (blue) was performed on 100% confluent P5 HMVECs. Cells were treated with 500 ng/mL recombinant human Ang-1 or PBS 90 min prior to challenge with EBM-2 media supplemented with 5% pre-filtered human serum. Serum was collected from a patient at two time points, 0 h (Ang-2: 3.2 ng/mL) and 24 h (Ang-2: 12.2 ng/mL), after CPB surgery, respectively. White arrows point to inter-endothelial gaps (n = 2 independent experiments per condition). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)