Endothelial Cell Calcium Influx Mediates Trauma-induced Endothelial Permeability

Objective: We aimed to investigate if ex vivo plasma from injured patients causes endothelial calcium (Ca 2+ ) influx as a mechanism of trauma-induced endothelial permeability. Summary Background Data: Endothelial permeability after trauma contributes to post-injury organ dysfunction. While the mechanisms remain unclear, emerging evidence suggests intracellular Ca 2+ signaling may play a role. Methods: Ex vivo plasma from injured patients with “Low Injury/Low Shock” (injury severity score [ISS]<15, base excess [BE])≥-6mEq/L) and “High Injury/High Shock” (ISS≥15, BE<-6mEq/L) were used to treat endothelial cells. Experimental conditions included Ca 2+ removal from the extracellular buffer, cyclopiazonic acid pre-treatment to deplete intracellular Ca 2+ stores, and GSK2193874 pre-treatment to block the TRPV4 Ca 2+ channel. Live cell fluorescence microscopy and ECIS 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 (MLC) phosphorylation and actomyosin contraction. Results: Compared to Low Injury/Low Shock plasma, High Injury/High Shock induced greater cytosolic Ca 2+ increase. Cytosolic Ca 2+ increase, MLC 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. Conclusions: This study illuminates a novel mechanism of post-injury endotheliopathy involving Ca 2+ influx via 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.