Endothelial Cell Calcium Influx Mediates Trauma-induced Endothelial Permeability.

Abstract

To investigate whether ex vivo plasma from injured patients causes endothelial calcium (Ca 2+ ) influx as a mechanism of trauma-induced endothelial permeability. Endothelial permeability after trauma contributes to postinjury organ dysfunction. While the mechanisms remain unclear, emerging evidence suggests intracellular Ca 2+ signaling may play a role. Ex vivo plasma from injured patients with "low injury/low shock" (injury severity score <15, base excess ≥-6 mEq/L) and "high injury/high shock" (injury severity score ≥15, base excess <-6 mEq/L) were used to treat endothelial cells. Experimental conditions included Ca 2+ removal from the extracellular buffer, cyclopiazonic acid pretreatment to deplete intracellular Ca 2+ stores, and GSK2193874 pretreatment to block the transient receptor potential vanilloid 4 (TRPV4) Ca 2+ channel. Live cell fluorescence microscopy and electrical cell-substrate impedance sensing were used to assess cytosolic Ca 2+ increases and permeability, respectively. Western blot and live cell actin staining were used to assess myosin light chain phosphorylation and actomyosin contraction. Compared with low injury/low shock plasma, high injury/high shock induced greater cytosolic Ca 2+ increase. Cytosolic Ca 2+ increase, myosin light chain phosphorylation, and actin cytoskeletal contraction were lower without extracellular Ca 2+ present. High injury/high shock plasma did not induce endothelial permeability without extracellular Ca 2+ present. TRPV4 inhibition lowered trauma plasma-induced endothelial Ca 2+ influx and permeability. This study illuminates a novel mechanism of postinjury endotheliopathy involving Ca 2+ influx through the TRPV4 channel. TRPV4 inhibition mitigates trauma-induced endothelial permeability. Moreover, widespread endothelial Ca 2+ influx may contribute to trauma-induced hypocalcemia. This study provides the mechanistic basis for the development of Ca 2+ -targeted therapies and interventions in the care of severely injured patients.