Helium alters the cytoskeleton and decreases permeability in endothelial cells cultured in vitro through a pathway involving Caveolin-1

Figure 1. Effect of helium on cellular actin filament alignment in HUVEC and HCAEC. Panel A: Immunofluorescent staining of actin (Rhodamin), Cav-1 and nuclei (Hoechst) in HUVEC. Panel B: Graph of the inner/border ratio of actin filaments following control gas or helium treatment in HUVEC. Panel C: Immunofluorescent staining of actin (Rhodamin), Cav-1 and nuclei (Hoechst) in HCAEC. Panel D: Graph of the inner/border ratio of actin filaments following control gas or helium treatment in HCAEC. Data are represented as mean ± SD, *p < 0.05. HUVEC = human umbilical vein endothelial cells. HCAEC = human coronary artery endothelial cells.

Figure 2. Effect of helium on Cav-1 levels in HUVEC and HCAEC. Panel A: Western blot results of the cellular ratio of Cav-1 compared to GAPDH loading controls from different time points following helium or control gas treatment in HUVEC. Panel B: Western blot results of the supernatant ratio of Cav-1 compared to albumin loading controls from different time points following helium or control gas treatment in HUVEC. Panel C: Western blot results of the cellular ratio of Cav-1 compared to GAPDH loading controls from different time points following helium or control gas treatment in HCAEC. Panel D: Western blot results of the supernatant ratio of Cav-1 compared to albumin loading controls from different time points following helium or control gas treatment in HCAEC. Cropped images of representative Western blot results are displayed below the respective graphs. Full-length blots are presented in Supplementary Figs S5–S8. Data are represented as mean ± SD, *p < 0.05. HUVEC = human umbilical vein endothelial cells. HCAEC = human coronary artery endothelial cells.

Figure 3. Effect of helium on VE-Cadherin and Cx43 levels in HUVEC and HCAEC. Panel A: Western blot results of the ratio VE-Cadherin compared to GAPDH loading controls in HUVEC at different time points following helium or control gas treatment. n = 14. Panel B: Western blot results of the ratio Cx43 compared to Tubulin loading controls levels in HUVEC at different time points following helium or control gas treatment. n = 11. Panel C: Western blot results of the ratio VE-Cadherin compared to GAPDH loading controls in HCAEC at different time points following helium or control gas treatment. n = 14. Panel D: Western blot results of the ratio of Cx43 compared to GAPDH loading controls in HCAEC at different time points following helium or control gas treatment. n = 14. Cropped images of representative Western blot results are displayed below the respective graphs. Full-length blots are presented in Supplementary Figs S9–S12. Data are represented as mean ± SD, *p < 0.05. HUVEC = human umbilical vein endothelial cells. HCAEC = human coronary artery endothelial cells.

Figure 4. Cav-1 siRNA transfection in HUVEC. Panel A: Western blot results of the ratio of Cav-1 compared to GAPDH loading control in HUVEC transfected with siRNA for Cav-1 and negative controls. Results of the transfected cells are shown as percentage of the negative control which was set at 100%. Cropped images of representative Western blot results are displayed below the respective graph. Full-length blots are presented in Supplementary Fig. S13. Panel B: Immunofluorescent images of HUVEC transfected with a red fluorescent protein (Block IT Alexa Flour). Cells were fixed 24 hours after transfection. Images of negative control are presented in Supplementary Fig. S4.

Figure 5. Effect of helium on permeability in HUVEC and Cav-1 siRNA transfected HUVEC. Panel A: Results of helium on permeability of a confluent monolayer of HUVEC, estimated by the transfer of FITC-BSA, at different time points compared to control gas. n = 7. Panel B: Results of helium on permeability of a confluent monolayer of Cav-1 siRNA transfected HUVEC, estimated by the transfer of FITC-BSA, at different time points compared to control gas. n = 8. Data are represented as mean ± SD, *p < 0.05. HUVEC = human umbilical vein endothelial cells.

Figure 6. Effect of helium on VE-Cadherin and Cx43 in Cav-1 siRNA transfected HUVEC. Panel A: Western blot results of the ratio VE-Cadherin compared to GAPDH loading controls in Cav-1 siRNA transfected HUVEC at different time points following helium or control gas treatment. n = 7. Panel B: Western blot results of the ratio Cx43 compared to Tubulin loading controls in Cav-1 siRNA transfected HUVEC at different time points following helium or control gas treatment. n = 7. Cropped images of representative Western blot results are displayed below the respective graphs. Full-length blots are presented in Supplementary Figs S14–S15. Data are represented as mean ± SD, *p < 0.05. HUVEC = human umbilical vein endothelial cells.

Figure 7. Supernatant analysis. Panel A: Effect of incubation with supernatant of helium and control gas treated cells on HUVEC Results of permeability of a confluent monolayer of HUVEC, estimated by the transfer of FITC-BSA, in HUVEC treated with supernatant of helium treated HUVEC at different time points. Controls were treated with supernatant of control gas treated HUVEC. n = 10. Panel B: Results of nanoparticle tracking analysis of supernatant after helium and control gas treated HUVEC. Results of nanoparticle tracking analysis of supernatant after helium and control gas treated HUVEC at different time points. n = 4. Data are represented as mean ± SD for panel A, and means for panel B, *p < 0.05. HUVEC = human umbilical vein endothelial cells.

Figure 8. Protocol outline. Confluent cells (HUVEC, Cav-1 siRNA transfected HUVEC and HCAEC) were exposed to either helium (70%) or control gas for 20 minutes in a specialised temperature controlled gas chamber. Cells were harvested either directly after treatment or after 2, 4, 6, 12 and 24 hours respectively. HUVEC = human umbilical vein endothelial cells. HCAEC = human coronary artery endothelial cells.