Endothelial Trauma Depends on Surface Charge and Extracellular Calcium Levels
We tested the hypothesis that the ubiquitous store-operated Ca2+ entry (SOCE) pathway contributes to histone-induced endothelial Ca2+ events. We also considered an alternate hypothesis: cationic electrostatic interactions between histones and negatively charged phospholipids deform endothelial membranes and thereby allow extracellular Ca2+ entry. A role for SOCE in histone responses was ruled out by genetic ablation of the ORAI1/2/3 channel trio; yet, histone effects were blocked by application of the multivalent cation gadolinium Gd3+. Using live cell video microscopy of endothelial cells labeled with membrane dye FM1-43, we recorded plasma membrane movements including vesiculation, blebbing, and ruffling of lamellipodia over 60 minutes following histone exposure. These cell membrane theatrics were markedly different from the uniform pattern of exocytosis and subsequent blebbing produced by calcium overload with ionomycin. The membrane permeabilization produced by histones, and not ionomycin, was transient and a subset of cells recovered membrane integrity within 1 hour. Removal of extracellular Ca2+ prevented histone-induced intracellular Ca2+ overload while surprisingly exacerbating plasma membrane deformation. Conversely, decreasing the density of the negative charge surface by adding calcium or or increasing extracellular Ca2+ 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 Ca2+ levels enhance interactions between histones and endothelial cell membrane phospholipids to increase cytotoxicity. Importantly, this supports the concept of aggressive Ca2+ repletion during resuscitation to prevent hypocalcemia, stabilize endothelial cell membranes and improve cardiovascular recovery from shock. SignificanceIn acute critical illness, the rapid collapse of vascular endothelial functions drives aberrant blood clotting and organ failure through mechanisms that are not understood. Emerging evidence that early administration of donor plasma improves survival of trauma patients has transformed the massive transfusion protocols used in surgical settings, but the sodium citrate included in transfused blood products to prevent coagulation often produces significant and severe hypocalcemia. Here, we demonstrate that cytotoxic trauma factors that are elevated in the blood during resuscitation interact electrostatically with endothelial cell phospholipids, and that low Ca2+ exacerbates toxicity by increasing this interaction. Using high speed video imaging, we demonstrate fast endothelial cell membrane movements in response to injury, including protrusion and ruffling of lamellipodia, release and reuptake of extracellular vesicles, and blebbing. 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.