Bioassay for Endothelial Damage Mediators Retrieved by Hemoadsorption

Abstract

Hemoadsorption devices are used to treat septic shock by adsorbing inflammatory cytokines and as yet incompletely defined danger and pathogen associated molecular patterns. In an ideal case, hemoadsorption results in immediate recovery of microvascular endothelial cells’ (mEC) function and rapid recovery from catecholamine-dependency and septic shock. We here tested a single device, which consists of polystyrene-divinylbenzene core particles of 450 μm diameter with a high affinity for hydrophobic compounds. The current study aimed at the proof of concept that endothelial-specific damage mediators are adsorbed and can be recovered from hemoadsorption devices. Because of excellent clinical experience, we tested protein fractions released from a hemoadsorber in a novel endothelial bioassay. Video-based, long-term imaging of mEC proliferation and cell death were evaluated and combined with apoptosis and ATP measurements. Out of a total of 39 fractions recovered from column fractionation, we identified 3 fractions that caused i) inhibition of mEC proliferation, ii) increased cell death and iii) induction of apoptosis in mEC. When adding these 3 fractions to mEC, their ATP contents were reduced. These fractions contained proteins of approximately 15 kDa, and high amounts of nucleic acid, which was at least in part oxidized. The efficacy for endothelial cell damage prevention by hemoadsorption can be addressed by a novel endothelial bioassay and long-term video observation procedures. Protein fractionation of the hemoadsorption devices used is feasible to study and define endothelial damage ligands on a molecular level. The results suggest a significant effect by circulating nucleic acids – bound to an as yet undefined protein, which may constitute a major danger-associated molecular pattern (DAMP) in the exacerbation of inflammation when patients experience septic shock. Hemoadsorption devices may thus limit endothelial damage, through the binding of nucleic acid-bearing aggregates and thus contribute to improved endothelial barrier function.

Major endothelial damage markers identified from hemadsorption filters derived from treated patients with septic shock – endoplasmic reticulum stress and bikunin may play a role

“…An endothelial bioassay and protein chemistry of hemadsorbed materials were applied to identify the most detrimental compounds disrupting endothelial cells. The stepwise approach to first fractionate the hemadsorbed material from a panel of devices from 10 patients, as opposed to a single hemadsorber ( 7 ) and test the protein fractions on human endothelial cells rather than rat brain-derived microvascular endothelium as in Denzinger et al. ( 7 ), confirmed our previous notion, that a definable composition of molecular entities has the most detrimental effect on endothelial cell layers in vitro .…”

Adjuvant hemoadsorption therapy in patients with severe COVID-19 and related organ failure requiring CRRT or ECMO therapy: A case series

“…In addition to IL-6 removal and reduced vasopressor need, the third interesting finding of our study is the improvement in lung function. There is clear data from the field of experimental research, that hemoadsorption might also have a positive effect via protection of the vascular barrier function, 28 endothelial activation, and monocyte adhesion 29 as well as a limitation in endothelial damage, 30 resulting in less extravasation and better ventilation-perfusion matching. We observed improvements in oxygenation and also in lung mechanics during the course of hemoadsorption.…”

The role of hemoadsorption in cardiac surgery – a systematic review

“…The versatile nature of hemoadsorption may benefit postoperative patients in complex and severely impaired conditions, as CytoSorb ® not only removes cytokines, but also bilirubin, bile acids, myoglobin, some toxins, and various PAMPs and DAMPs [ 45 , 54 – 56 ]. As with any other blood purification technology, hemoadsorption carries a risk of inadvertent drug removal.…”