DISTINCT PATTERNS OF ENDOTHELIAL CELL ACTIVATION PRODUCED BY EXTRACELLULAR HISTONES AND BACTERIAL LIPOPOLYSACCHARIDE.

Abstract: Objective : Vascular endothelial cells (ECs) sense and respond to both trauma factors (histone proteins) and sepsis signals (bacterial lipopolysaccharide, LPS) with elevations in calcium (Ca 2+ ), but it is not clear if the patterns of activation are similar or different. We hypothesized that within seconds of exposure, histones but not LPS would produce a large EC Ca 2+ response. We also hypothesized that histones would produce different spatio-temporal patterns of Ca 2+ events in veins than in arteries. Methods : We studied cultured ECs (EA.hy926) and native endothelial cells from surgically opened murine blood vessels. High-speed live cell imaging of Ca 2+ events were acquired for 5 min before and after stimulation of cultured ECs with histones or LPS alone or in combination. Histone-induced EC Ca 2+ events were also compared in native endothelial cells from resistance-sized arteries and veins. Ca 2+ activity was quantified as "Ca 2+ prevalence" using custom spatiotemporal analysis. Additionally, cultured ECs were collected after 6 h of exposure to histones or LPS for RNA sequencing. Results : ECs-both in culture and in blood vessels-rapidly increased Ca 2+ activity within seconds of histone exposure. In contrast, LPS exposure produced only a slight increase in Ca 2+ activity in cultured ECs and no effect on blood vessels over 5-min recording periods. Histones evoked large aberrant Ca 2+ events (>30 s in duration) in both veins and arteries, but with different spatio-temporal patterns. Ca 2+ activity in arterial ECs often appeared as "rosettes", with Ca 2+ events that propagated from one cell to all adjacent surrounding cells. In veins, ECs responded individually without spreading. Surprisingly, exposure of cultured ECs to LPS for 5 min before histones potentiated EC Ca 2+ activity by an order of magnitude. Exposure of ECs to histones or LPS both increased gene expression, but different mRNAs were induced. Conclusions : LPS and histones activate ECs through mechanisms that are distinct and additive; only histones produce large aberrant Ca 2+ events. ECs in arteries and veins display different patterns of Ca 2+ responses to histones.