Extracellular Histones Trigger Endothelial Calcium Signals that Paradoxically do not Cause Vasodilation, and Instead, Compromise Endothelial Function
Authors: Collier Daniel M. , Villalba Nuria M. , Sackheim Adrian , Bonev Adrian M. , Nelson Mark T. , Freeman Kalev 
Published: June 2021

Elevated levels of histone proteins are found in the circulation of patients following traumatic injury and are associated with vascular dysfunction, coagulopathy, sepsis, and poor patient outcome, yet the mechanism(s) involved are not known. One possible mechanism to affect vascular function is through alteration in endothelial cell (EC) calcium, as suggested by effects on cultured endothelial cells (Nat Med, 2009). We examined the spatial and temporal characteristics of histone‐induced EC Ca2+ signals in en face resistance‐sized mesenteric arteries (MAs) from endothelial cell‐specific genetically encoded Ca2+ indicator mice (cx40‐GCaMP5‐mCherry), using a spinning confocal microscope. Addition of histone protein to the bath solution (10 mg/ml, unfractionated histone protein mixture) induced large Ca2+ signals within seconds of application that spread within and between ECs. Histone‐induced signals are approximately twice as bright and spread 7 times farther than typical spontaneous Ca2+ signals in MA ECs. Contrary to the expectation that histone‐induced Ca2+ signaling would elicit vasodilation, luminal application of histones had no effect on diameter, and instead reduced dilations to endothelial‐dependent vasodilators. After 30 minutes of histone exposure, global cytosolic Ca2+ increased to greater than 500 nM, and appeared to cause cell death. To test this, we performed live cell imaging experiments in the presence of propidium iodide (PI, 100 ng/ml) as a real‐time marker of cell death. After 30 minutes of exposure to histone protein, 25% of EC nuclei per field were labeled with PI. To determine the route of Ca2+ increase, we used pharmacologic and genetic approaches to test known Ca2+ pathways such as IP3R mediated release, and TRPV4 and P2XR channel‐mediated influx. Removal of extracellular Ca2+, but not depletion of intracellular Ca2+ stores, prevented histone‐induced Ca2+ signals. Histone induced signals and cell death was not suppressed by inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels. However, Ca2+ events were suppressed by non‐selective P2X/YR antagonists (50 mM suramin and 10 mM PPADS), P2XR antagonists (1 mM TNPATP), and a P2X7R antagonist (100 mM amiloride). The response to histones was reduced, but not eliminated, in MA ECs from P2X7R‐knockouts, suggesting a possible involvement of P2X7Rs as well as an unknown pathway. However, P2X7R is sufficient to generate histone induced cation current in Xenopus ooctyes. The data demonstrate that histones are robust activators Ca2+ signaling and EC death in native resistance‐sized arteries.Support or Funding InformationDMC is supported by NIH (5K99HL133451‐02); KF by NIH (1R01GM123010‐01); and MTN by NIH (5R01HL131181‐02, 5R01HL121706‐04, 5R37DK053832‐19, 7UM1HL120877‐04), The Fondation Leducq, Horizon 2020, and the Totman Medical Research Trust.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.