Endothelial Trauma Depends on Surface Charge and Extracellular Calcium Levels
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We hypothesized that cationic histones bind to endothelial cell membranes through electrostatic interactions with negatively charged phospholipids, and consequently, that low Ca 2+ exacerbates toxicity by increasing binding and uptake of histones into cells. The role of the ubiquitous store-operated Ca 2+ entry (SOCE) in histone responses is also unknown. Here, studying endothelial cells in surgical preparations and in culture, we observed clinically relevant histone concentrations, produced fast plasma membrane movements including vesiculation, blebbing, ruffling, and cellular collapse. The cell membrane theatrics observed were markedly different from the uniform pattern of exocytosis and blebbing produced by calcium overload with ionomycin. Interestingly, membrane permeabilization produced by histones (and not ionomycin) was transient and a subset of cells recovered membrane integrity within 1 hour. A role for SOCE in histone responses was ruled out by genetic ablation of the ORAI1/2/3 channel trio. Removal of extracellular Ca 2+ prevented histone-induced intracellular Ca 2+ overload while surprisingly exacerbating plasma membrane deformation. Conversely, decreasing the density of the negative charge surface by adding calcium or multivalent cations gadolinium or increasing extracellular Ca 2+ levels effectively screened common membrane phospholipids from interactions with labeled histones and prevented endothelial damage in cells exposed to histones. Collectively, these results indicate that low extracellular Ca 2+ levels enhance interactions between histones and endothelial cell membrane phospholipids to increase cytotoxicity. Importantly, this supports the concept of aggressive Ca 2+ repletion during resuscitation to prevent hypocalcemia, stabilize the endothelial cell membranes and improve cardiovascular recovery from shock.
Significance
In acute critical illness, the rapid collapse of vascular endothelial functions drives aberrant blood clotting and organ failure through mechanisms that are not understood. The recent breakthrough in transfusion medicine showing that administration of donor plasma improves survival of trauma patients has transformed the massive transfusion protocols used in surgical settings, but the sodium citrate used to prevent coagulation often produces significant and severe hypocalcemia. Here, we demonstrate that cytotoxic trauma factors that are elevated in the blood during resuscitation bind endothelial cell phospholipids, and that low Ca 2+ exacerbates toxicity by increasing this interaction. These experiments lead to our surprising discovery of unexpected endothelial cell membrane theatrics that occur in response to injury, with direct visualization of fast plasma membrane movements and the release and reuptake of extracellular vesicles. These findings provide important insights into the nature of shock-induced endotheliopathy and highlight the potential cardiovascular risk associated with chelation-induced hypocalcemia during resuscitation.
