Plasma from Volunteers Breathing Helium Reduces Hypoxia-Induced Cell Damage in Human Endothelial Cells-Mechanisms of Remote Protection Against Hypoxia by Helium

Abstract

Purpose: Remote ischemic preconditioning protects peripheral organs against prolonged ischemia/reperfusion injury via circulating protective factors. Preconditioning with helium protected healthy volunteers against postischemic endothelial dysfunction. We investigated whether plasma from helium-treated volunteers can protect human umbilical vein endothelial cells (HUVECs) against hypoxia in vitro through release of circulating of factors.

Methods: Healthy male volunteers inhaled heliox (79% helium, 21% oxygen) or air for 30 min. Plasma was collected at baseline, directly after inhalation, 6 h and 24 h after start of the experiment. HUVECs were incubated with either 5% or 10% of the plasma for 1 or 2 h and subjected to enzymatically induced hypoxia. Cell damage was measured by LDH content. Furthermore, caveolin 1 (Cav-1), hypoxia-inducible factor (HIF1α), extracellular signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription (STAT3) and endothelial nitric oxide synthase (eNOS) were determined.

Results: Prehypoxic exposure to 10% plasma obtained 6 h after helium inhalation decreased hypoxia-induced cell damage in HUVEC. Cav-1 knockdown in HUVEC abolished this effect.

Conclusions: Plasma of healthy volunteers breathing helium protects HUVEC against hypoxic cell damage, possibly involving circulating Cav-1.

Keywords: Caveolin-1; Endothelial conditioning; Helium; Ischemic preconditioning; Remote.

Fig 1

Protocol outline. Healthy volunteers received either helium or control gas via face mask in 2 cycles (separated by 2 weeks). Blood samples were taken at baseline (T1) and 30 min (T2), 6 h (T3) and 24 h (T4) after treatment (b). HUVECs were incubated with plasma and subjected to 24 h of hypoxia, after which cells and supernatant were harvested for analysis

Fig. 2

Effect of remote helium-conditioned plasma on hypoxia-induced damage in Huvecs. Quantification of lactate dehydrogenase (LDH) activity as marker of hypoxia-induced cell damage in the supernatant of Huvecs, following 10% plasma incubation for 2 h prior to start of 24-h hypoxia. a Relative LDH activity after helium inhalation, baseline (T0) = 1. b Relative LDH activity after control gas inhalation, baseline (T0) = 1. Columns represent means (± 95% confidence interval (CI)). *p < 0.05 compared with baseline

Fig. 3

Effect of remote helium conditioning on caveolin-1 levels in HUVEC and plasma from volunteers. a ELISA results of caveolin-1 in plasma from volunteers at different time points. b Summarised western blot results of caveolin-1 in cell lysate of Huvecs following 24 h of hypoxia. c Summarised western blot results of caveolin-1 in supernatant of Huvecs subjected to 24 h of hypoxia. Columns represent means (± 95% CI). *p < 0.05 to baseline

Fig. 4

Cav-1 transfection abolishes remote helium-conditioned plasma protection in Huvecs exposed to hypoxia. a Levels of cellular Cav-1 in Huvecs transfected with Cav-1 siRNA (16%) and negative control siRNA (100%). Columns represent means (± 95% CI). b Absolute LDH activity of Cav-1-transfected Huvecs incubated with plasma from baseline and 6 h after helium treatment. Columns represent means (± 95% confidence interval (CI)). *p < 0.05 compared with baseline. Huvecs human umbilical vein endothelial cells

Fig. 5

Effect of remote helium conditioning on signal transduction kinases in Huvecs. a Western blot results of the ratio of HIF1α compared with GAPDH loading controls in cell lysate of Huvecs following 24 h of hypoxia. b Western blot results of the ratio of ERK1/2 and loading control GAPDH in cell lysate of Huvecs following 24 h of hypoxia. c Western blot results of activated (phosphorylated to total STAT3) STAT3 in cell lysate of Huvecs following 24 h of hypoxia. Columns represent means (± 95% CI). *p < 0.05 to baseline

Fig. 6

The role of eNOS in remote helium conditioning. a Western blot results of the ratio of activated (phosphorylated to total) eNOS in Huvecs after prehypoxic exposure to plasma. b Results of ELISA measurements of eNOS in the supernatant of Huvecs after prehypoxic exposure to plasma. Columns represent means (± 95% CI). *p < 0.05 to baseline