Extracellular Histones Induce Propagating Ca2+ Influx, Ca2+ Overload, and Endothelial Cell Death in Resistance‐sized Mouse Mesenteric Arteries
Authors: Collier Daniel M , Villalba Nuria M , Sackheim Adrian M , Freeman Kalev , Nelson Mark T 
Published: November 2023

Elevated levels of histone proteins are found in the circulation of patients following traumatic injury (healthy: 2.3 μg/ml, 4 hrs post trauma: 10–230 μg/ml, Abrams et al. 2013. Am. J. Respir. Crit. Care. Med.) and are associated with coagulopathy, sepsis, and poor patient outcome. The mechanism and specific ion channels involved have not been identified. We used resistance‐sized mouse mesenteric arteries (MA) to study the effects of extracellular histones on native tissue. To study the spatial and temporal characteristics of histone induced endothelial cell (EC) Ca2+ signals, we utilized spinning disk confocal microscopy of en face MAs from endothelial cell‐specific genetically encoded Ca2+ indicator mice (cx40‐GCaMP5‐mCherry). Addition of histone protein to the bath solution (10 μg/ml, unfractionated histone protein mixture of H1, H2a, H2b, H3, and H4) induced large Ca2+ events within seconds of application that propagate within and between ECs. Histone induced events are approximately twice as bright and spread 7 times farther than typical spontaneous Ca2+ events in MAs, suggesting a role for histone protein in EC Ca2+ overload and death. We performed imaging experiments in the presence of propidium iodide (PI, 100 ng/ml) as a marker of cell death. After 30 minutes of exposure to histone protein, 25% of EC nuclei (7 of 28 cells) per field were labeled with PI. PI labeling is reduced when Ca2+ is omitted from the bath solution. Consistent with EC death, NS309 (SK/IK agonist) dilations are reduced after exposing the lumen to histone proteins. In cells labeled with PI, cytosolic Ca2+ levels increased from 220 nM to 400 nM after 5 minutes of exposure, and 790 nM after 30 minutes of histone exposure, demonstrating that Ca2+ is increased before cell death. To determine the route of Ca2+ entry, we used pharmacologic and genetic approaches to test known Ca2+ pathways such as TRPV4 and P2XRs. Histone induced propagating events were present after depletion of intracellular stores with CPA, confirming Ca2+ influx. Activation of TRPV4 with 100 nM GSK10167901 caused EC Ca2+ overload and cell death, however histone induced events were not prevented by inhibition (100 nM GSK2193874) or genetic ablation of TRPV4. Propagating events were suppressed by non‐selective P2X/YR (50 μM suramin) and P2XR antagonists (1 μM TNPATP), and a P2X7R selective antagonist (100 μM amiloride), suggesting a role for ionotropic purinergic receptor mediated Ca2+ influx in ECs. The data demonstrate that histones are robust activators Ca2+ signaling in the vascular endothelium through a mechanism that does not require IP3Rs and TRPV4 channels.Support or Funding InformationDMC is supported by NIH (1K99HL133451‐01) and the AHA (16SDG27030005); KF by NIH (1K08GM098795‐05), UVM Dept. of Surgery, U.S. Army (W911NF‐10‐1‐0376), Luoxis Corp., and The Beckman Foundation; and MTN by NIH (1R01HL131181‐01, 5R01HL121706‐03, 4R37DK053832‐18, 7UM1HL120877‐04), The Fondation Leducq, Horizon 2020, and the Totman Medical Research Trust.