Effects of therapeutic plasma exchange on the endothelial glycocalyx in septic shock

Klaus Stahl, Uta Carola Hillebrand, Yulia Kiyan, Benjamin Seeliger, Julius J Schmidt, Heiko Schenk, Thorben Pape, Bernhard M W Schmidt, Tobias Welte, Marius M Hoeper, Agnes Sauer, Malgorzata Wygrecka, Christian Bode, Heiner Wedemeyer, Hermann Haller, Sascha David, Klaus Stahl, Uta Carola Hillebrand, Yulia Kiyan, Benjamin Seeliger, Julius J Schmidt, Heiko Schenk, Thorben Pape, Bernhard M W Schmidt, Tobias Welte, Marius M Hoeper, Agnes Sauer, Malgorzata Wygrecka, Christian Bode, Heiner Wedemeyer, Hermann Haller, Sascha David

Abstract

Background: Disruption of the endothelial glycocalyx (eGC) is observed in septic patients and its injury is associated with multiple-organ failure and inferior outcomes. Besides this biomarker function, increased blood concentrations of shedded eGC constituents might play a mechanistic role in septic organ failure. We hypothesized that therapeutic plasma exchange (TPE) using fresh frozen plasma might influence eGC-related pathology by removing injurious mediators of eGC breakdown while at the time replacing eGC protective factors.

Methods: We enrolled 20 norepinephrine-dependent (NE > 0.4 μg/kg/min) patients with early septic shock (onset < 12 h). Sublingual assessment of the eGC via sublingual sidestream darkfield (SDF) imaging was performed. Plasma eGC degradation products, such as heparan sulfate (HS) and the eGC-regulating enzymes, heparanase (Hpa)-1 and Hpa-2, were obtained before and after TPE. A 3D microfluidic flow assay was performed to examine the effect of TPE on eGC ex vivo. Results were compared to healthy controls.

Results: SDF demonstrated a decrease in eGC thickness in septic patients compared to healthy individuals (p = 0.001). Circulating HS levels were increased more than sixfold compared to controls and decreased significantly following TPE [controls: 16.9 (8-18.6) vs. septic patients before TPE: 105.8 (30.8-143.4) μg/ml, p < 0.001; vs. after TPE: 70.7 (36.9-109.5) μg/ml, p < 0.001]. The Hpa-2 /Hpa-1 ratio was reduced in septic patients before TPE but normalized after TPE [controls: 13.6 (6.2-21.2) vs. septic patients at inclusion: 2.9 (2.1-5.7), p = 0.001; vs. septic patients after TPE: 13.2 (11.2-31.8), p < 0.001]. Ex vivo stimulation of endothelial cells with serum from a septic patient induced eGC damage that could be attenuated with serum from the same patient following TPE.

Conclusions: Septic shock results in profound degradation of the eGC and an acquired deficiency of the protective regulator Hpa-2. TPE removed potentially injurious eGC degradation products and partially attenuated Hpa-2 deficiency. Trial registration clinicaltrials.gov NCT04231994, retrospectively registered 18 January 2020.

Keywords: DAMP; Extracorporeal treatment; Heparan sulfate; Heparanase; Plasmapheresis.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2021. The Author(s).

Figures

Fig. 1
Fig. 1
Flowchart of study participants. NE means norepinephrine and TPE therapeutic plasma exchange
Fig. 2
Fig. 2
Injury to the endothelial glycocalyx (eGC) in vivo. Sublingual dark field imaging in patients allows quantification of the eGC thickness as indicated by the perfused boundary region (PBR). Box and whisker plots showing results for healthy controls (n = 10) as well as patients with septic shock before commencement of therapeutic plasma exchange (TPE) (n = 11)
Fig. 3
Fig. 3
Effect of therapeutic plasma exchange (TPE) on glycocalyx shedding products. Box and whisker plots showing blood concentrations of hyaluronic acid (HA) (A), chondroitin sulfate (CS) (B) and heparan sulfate (HS) (C) for healthy controls (n = 20) as well as patients with septic shock before (pre) (n = 20) and directly after (post) (n = 20) TPE
Fig. 4
Fig. 4
Effect of therapeutic plasma exchange (TPE) on key regulating enzymes of the endothelial glycocalyx (eGC). Box and whisker plots showing blood concentrations of circulating Heparanase-1 (Hpa-1) (A), Heparanase-2 (Hpa-2) (B) as well as the ratio of Hpa-2 to Hpa-1 (C) for healthy controls (n = 18) as well as patients with septic shock before (pre) (n = 20) and directly after (post) (n = 20) TPE
Fig. 5
Fig. 5
Ex vivo stimulation of 3D microvessels with septic serum before and after therapeutic plasma exchange (TPE). Exemplary 3D reconstruction of the heparan sulfate (HS) layer images of naive endothelial cells in a microfluidic chip (HS in red, DAPI nuclei staining in blue) after perfusion with serum and incubation overnight of a representative septic patient both before and after TPE compared to healthy control serum (pooled out of four control patients) (A) and quantification of the HS positive surface area as box and whisker plots (B)

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