Helium induces Caveolin Secretion and decreases Permeability in Human Umbilical Vein Endothelial Cells

Abstract

Caveolins are structural proteins essential for the formation of caveolae, small lipid enriched membrane invaginations. Caveolins have been shown to be involved in anesthetic‐induced cardioprotection and play an important role in signal transduction in various cell types. The noble gas helium, a potent cardioprotectant, leads to decreased protein levels of caveolin‐1 and caveolin‐3 in mice hearts, whereas higher levels of these caveolins were found in the serum of mice. These data suggest that caveolins are secreted into the bloodstream after helium inhalation. As actin filaments are an integral part of the cytoskeleton and are located in close connection to caveolins in the plasma membrane we hypothesized that helium might affect the cytoskeleton and thereby induce secretion of caveolins. To investigate this hypothesis we used human umbilical vein endothelial cells (HUVEC), as the endothelium is considered the first target organ that gets in contact with circulating blood.HUVEC were isolated from fresh umbilical cords, pooled and grown upon a confluent monolayer in passage 3–5. Cells were exposed for 20 minutes to either helium (5% CO2, 25% O2, 70% He) or control gas (5% CO2, 25% O2, 70% N2) in a specialized gas chamber. Cells and supernatant were collected after 0 minutes, 2, 4, 6, 12 and 24 hours for Western blot analysis, RT‐PCR, immunofluorescence staining and permeability measurements.In the helium group, protein analysis showed a significantly lower expression of caveolin‐1 at 6 hours in cell lysate (13.35±1.30 in controls vs. 8.02±1.29 in helium treated cells, average light intensity (AVI), p<0.05) and a higher expression in supernatant starting at 6h (1.42±0.25 in controls vs. 8.26±4.5 in helium treated cells, AVI, p<0.05) Actin staining demonstrated a significant increase of the border/inner ratio of the filaments at 12 hours after helium exposure. Cell permeability measurement revealed a significantly lower flow‐through of FITC‐BSA at 6 (p<0.05) and 12 hours (p<0.05) in the helium group. RT‐PCR of Cav‐1 did not show significant differences.These findings suggest that caveolin‐1 is secreted after helium exposure in vitro and that the actin cytoskeleton filaments are regulated by helium. This mechanism might finally lead to a decreased permeability of the endothelium after helium inhalation.Support or Funding InformationThis project was in part funded by the IARS (International Anesthesia Research Society) and the SCA (Society of Cardiovascular Anesthesiologists).