Extracellular histones induce calcium signals in the endothelium of resistance-sized mesenteric arteries and cause loss of endothelium-dependent dilation.

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca but not depletion of intracellular Ca stores prevented histone-induced Ca signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca signaling. We found that histones induce an influx of Ca in ECs that does not cause vasodilation but instead causes Ca overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.