In vitro endothelial hyperpermeability occurs early following traumatic hemorrhagic shock

Abstract

Background: Endothelial hyperpermeability is suggested to play a role in the development of microcirculatory perfusion disturbances and organ failure following hemorrhagic shock, but evidence is limited.

Objective: To study the effect of plasma from traumatic hemorrhagic shock patients on in vitro endothelial barrier function.

Methods: Plasma from traumatic hemorrhagic shock patients was obtained at the emergency department (ED), the intensive care unit (ICU), 24 h after ICU admission and from controls (n = 8). Sublingual microcirculatory perfusion was measured using incident dark field videomicroscopy at matching time points. Using electric cell-substrate impedance sensing, the effects of plasma exposure on in vitro endothelial barrier function of human endothelial cells were assessed.

Results: Plasma from traumatic hemorrhagic shock patients collected at ED admission induced a 19% loss of in vitro endothelial resistance compared to plasma from controls (p < 0.001). This loss was due to reduced cell-cell contacts (p < 0.01). Plasma withdrawn at later time points did not affect endothelial barrier function (p > 0.99). Interestingly, in vitro endothelial resistance showed a positive association with in vivo microcirculatory perfusion (r = 0.56, p < 0.01).

Conclusions: Plasma from traumatic hemorrhagic shock patients obtained following ED admission, but not at later stages, induced in vitro endothelial hyperpermeability. This coincided with in vivo microcirculatory perfusion disturbances.

Keywords: Hemorrhagic shock; endothelial barrier function; endothelial permeability; microcirculation; plasma.

Fig.1

Study protocol and in vitro experimental set-up. (A) Blood was collected (for plasma and serum samples respectively) and sublingual non-invasive incident dark field videomicroscopy was performed at three different time points: after arrival at the emergency department (ED; n = 8), after admission at the intensive care unit (ICU; n = 8) and 24 hours later (ICU+24 h; n = 8). (B) Human endothelial cells were seeded and culture until a monolayer was formed. After starvation in 1% human serum albumin (HSA), endothelial cells were exposed to plasma from traumatic hemorrhagic shock patients collected at ED admission, ICU admission or 24 h later. Measurements were performed using electric cell-substrate impedance sensing (ECIS; experiment I.) or via immunofluorescence imaging (IF imaging; experiment II.).

Fig.2

Loss of in vitro endothelial barrier function. Human endothelial cells were exposed to plasma from traumatic hemorrhagic shock patients collected at admission at the emergency department (ED), intensive care unit (ICU), 24 hours (ICU+24 h) after admission at the ICU and from controls (all n = 8). Endothelial resistance after plasma exposure over time (A) and after 2 hours of plasma exposure (B). A: Data represent mean and were tested with a two-way ANOVA with Bonferroni post-hoc analysis. B: Data represent mean±SD and were tested with a one-way ANOVA with Bonferroni post-hoc analysis. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared to ED admission.

Fig.3

Impaired in vitro endothelial cell-cell integrity. Rb and alpha can be modelled from endothelial resistance measurements, which distinguish between cell-cell (Rb) and cell-matrix (alpha) integrity (A). Human endothelial cells were exposed to plasma from traumatic hemorrhagic shock patients collected at admission at the emergency department (ED), intensive care unit (ICU), 24 hours (ICU+24 h) after admission at the ICU and from controls (all n = 8). Cell-cell integrity (Rb; B), cell-matrix integrity (alpha; C) and representative images of endothelial cells stained for VE cadherin (adherents junctions; green), F-actin (stress fibers; white) and DAPI (nuclei; blue) (D) after 2 hours of plasma exposure. Data represent mean±SD and were tested with a one-way ANOVA with Bonferroni post-hoc analysis, ^**p < 0.01 compared to ED admission.

Fig.4

Microcirculatory perfusion associated with in vitro endothelial barrier function. Association between proportion of perfused vessels and in vitro endothelial resistance determined after exposure to plasma collected at corresponding time points. Data are presented with a linear regression with 95% CI and tested with a Pearson’s correlation test.