Distinct Patterns of Endothelial Cell Activation Produced by Extracellular Histones and Bacterial Lipopolysaccharide

Objective: Vascular endothelial cells (ECs) sense and respond to both trauma factors (histone proteins) and sepsis signals (bacterial lipopolysaccharide, LPS) with elevations in calcium (Ca2+), 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 Ca2+ response. We also hypothesized that histones would produce different spatio-temporal patterns of Ca2+ 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 Ca2+ events were acquired for 5 minutes before and after stimulation of cultured ECs with histones or LPS alone or in combination. Histone-induced EC Ca2+ events were also compared in native endothelial cells from resistance-sized arteries and veins. Ca2+ activity was quantified as "Ca2+ prevalence" using custom spatiotemporal analysis. Additionally, cultured ECs were collected after 6 hours of exposure to histones or LPS for RNA sequencing.

Results: ECs - both in culture and in blood vessels - rapidly increased Ca2+ activity within seconds of histone exposure. In contrast, LPS exposure produced only a slight increase in Ca2+ activity in cultured ECs and no effect on blood vessels over 5-minute recording periods. Histones evoked large aberrant Ca2+ events (>30 seconds in duration) in both veins and arteries, but with different spatio-temporal patterns. Ca2+ activity in arterial ECs appeared as "rosettes", with Ca2+ events that propagated from one cell to all adjacent surrounding cells. In veins, ECs responsed individually without spreading. Suprisingly, exposure of cultured ECs to LPS for 5-minutes before histones potentiated EC Ca2+ 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 Ca2+ events. ECs in arteries and veins display different patterns of Ca2+ responses to histones.