Cell culture

We conducted a non-human-subject in vitro exploratory study; materials were purchased, and the research ethics committee was therefore not involved. The Endothelial Cell Facility of the University Medical Center Groningen (UMCG; Groningen, Netherlands) cultured HUVECs (CC2519, Lonza, Breda, Netherlands) in endothelial basal medium (EBM)-2 supplemented with endothelial cell growth medium-2 (EGM-2) microvascular (MV) SingleQuot kit supplements & growth factors (Lonza) at 37 °C and 5% CO2. Human umbilical vein endothelial cells were seeded one day before the experiment in 24-well plates (Corning Life Sciences, Amsterdam, Netherlands) and grown overnight to confluence and used the next morning. We used cells between passage 4–6. To induce an inflammatory response, we added 10 ng·mL−1 of TNF-α (Beromun®, Belpharma S.A., Luxembourg) to the HUVEC in culture, and after for 4 hr we harvested the cells. We executed all conditions in triplicate wells and performed all conditions in three independent experiments.

Endothelial cell exposure to anesthetics and opioidsPropofol, remifentanil, and fentanyl

We incubated HUVECs with clinically relevant concentrations of 2, 5, or 10 µg·mL−1 propofol (medium-chain triglyceride [MCT]/long-chain triglyceride [LCT] lipid emulsion [Fresenius SE & Co. KGaA, Bad Hoburg, Germany); 2, 5, or 10 ng·mL−1 remifentanil (Mylan, Pharmasolutions, Dublin, Ireland); or 0.5, 1.5, or 5.0 ng·mL−1 fentanyl (Bipharma, Hameln, Germany) in culture medium in the presence of 10 ng·mL−1 TNF-α.12,13,14,15,16

Sevoflurane

We placed HUVECs in an air-tight plastic bag that was connected to a mechanical ventilator (Dräger Zeus® ventilator, Drägerwerk AG & Co. KGaA, Lübeck, Germany) using a classical breathing circle system. We administered sevoflurane through a vaporizer and measured end-tidal sevoflurane concentrations. We exposed cells to clinically relevant concentrations of sevoflurane (0.8%, 2.0%, or 4.0%) in the presence of 10 ng·mL−1 TNF-α for 4 hr under a heat blanket of 38 °C. We also kept control cells under the same heat blanket but did not expose them to sevoflurane (Fig. 1).

Fig. 1

Diagram for the experimental paradigm showing the timeline of the experiments

ANGPT2 = angiopoietin-2; ELISA = enzyme-linked immunosorbent assay; HUVEC = human umbilical vein endothelial cell; ICAM-1 = intercellular adhesion molecule 1; IL = interleukin; mRNA = messenger RNA; RT-qPCR = reverse transcription quantitative real-time polymerase chain reaction; TNF-α = tumor necrosis factor alpha; VCAM-1 = vascular cell adhesion molecule 1

After treatment, we collected cell culture medium and stored it at  −20 °C until further analysis. We washed cells with cold phosphate buffered saline (Lonza) and lysed them for ribonucleic acid (RNA) in RLT-buffer containing 1% (v/v) β-mercapto ethanol or lysed them for protein analysis in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% [w/v] sodium deoxycholate, 0.1% [w/v] sodium dodecyl sulfate, 1% [v/v] IGEPAL) containing protease (Roche Diagnostics, Almere, Netherlands) and phosphatase inhibitors (Roche). We stored lysates for RNA analysis at −80 °C and lysates for protein analysis at −20 °C.

Ribonucleic acid isolation and gene expression analysis by quantitative real-time polymerase chain reaction

We isolated total RNA using the RNeasy Plus Mini Kit (QUIAGEN, Hilden, Germany) according to the manufacturer's instructions. We determined the integrity of RNA using gel electrophoresis, while we measured yield (OD260) and purity (OD260/OD280 ratio) with a Nanodrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies LLC, Wilmington, DE, USA). We reverse transcribed samples to complementary deoxyribonucleic acid (cDNA) using random hexamer primers (Promega Corporation, Leiden, Netherlands) and SuperScript III (Invitrogen, Breda, Netherlands). We used 10 ng cDNA to perform quantitative polymerase chain reaction (PCR) analysis using assay-on-demand primer/probe sets (Taqman Gene Expression, Thermo Fisher Scientific, Inc., Breda, Netherlands) (Electronic Supplementary Material [ESM] eTable 1) on a ViiA7 Real-Time PCR System (Thermo Fisher). We averaged the obtained duplicate cycle threshold (CT) values for each sample. Gene expression was normalized to the expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in ΔCT value. We calculated the average messenger RNA (mRNA) levels relative to GAPDH using 2−ΔCT.

Western blot determination of endothelial adhesion molecules

We determined total protein concentrations in cell lysates using the Detergent Compatible (DC) Protein Assay (Bio-Rad Laboratories B.V., Veenendaal, Netherlands) according to the manufacturer’s instructions. We loaded proteins and separated them in a 10% polyacrylamide gel (Bio-Rad) and transferred them to nitrocellulose membrane (Bio-Rad). We confirmed transfer of proteins using 0.1% (w/v) Ponceau S (Thermo Fisher) staining in 5% (v/v) acetic acid, whereafter membranes were horizontally cut through a 70-kD band of PageRuler™ Prestained Protein Ladder (#26616, Thermo Fisher). We blocked membranes with 5% (w/v) skimmed milk (ELK; Campina®, Zaltbommel, Netherlands) in Tris-buffered saline (TBS; 20 mM Tris [w/v], 0.15 M NaCl [w/v] pH 7.5) supplemented with 0.1% (v/v) Tween 20 (Sigma-Aldrich, Merck Life Science N.V., Amsterdam, Netherlands) for at least 1 hr at room temperature. We probed membranes overnight at 4 °C with primary antibody (ESM eTable 2) in 5% ELK in TBS with Tween 20 (TBS-T) 0.1%. The next day, we washed the membranes extensively with TBS-T 0.1% and incubated them with a horseradish peroxidase (HRP)-labelled secondary antibody in 5% ELK in TBS-T 0.1% for 1 hr at room temperature. We used Gel Doc XR imaging system (Bio-Rad) to visualize bands with chemiluminescence (Millipore). We used GAPDH as a protein-loading control.

Protein quantification of interleukin-6 and interleukin-8 in cell culture medium by enzyme-linked immunosorbent assay

We determined secreted interleukin (IL)-6 and IL-8 concentrations in cell culture medium using ELISA MAX™ Standard Set Human IL-6 (BioLegend, London, UK; catalogue No. 430501) and IL-8 (BioLegend; catalogue No. 431501) according to the manufacturer’s instructions.

Statistical analysis

For mRNA, we calculated the fold change relative to the control for each sample per experiment and constructed bar charts displaying means with their corresponding 95% confidence intervals (CIs), of at least three independent experiments in triplicate. We calculated statistical analyses using one-way analysis of variance (Kruskal–Wallis) with post hoc correction using Dunn’s multiple comparisons tests. For each measurement (anesthetics/opioids alone or in combination), we tested whether TNFα stimulation was different than control. Thereafter, we tested whether a single anesthetic/opioid or combinations in different concentrations in the presence of TNFα was different than TNFα-stimulated conditions.

For IL-6 and IL-8 protein concentrations, we plotted individual absolute levels per mL in scatter plots. We carried out statistical analyses using one-way analysis of variance (Kruskal–Wallis) with post hoc correction using Dunn’s multiple comparisons tests. For each measurement (anesthetics/opioids alone or in combination), we tested whether TNFα stimulation was different than the control. Thereafter, we tested whether a single anesthetic/opioid or combinations in different concentrations in the presence of TNFα were different than TNFα-stimulated conditions. In this exploratory study, we did not calculate a sample size but used common sample size numbers for in vitro studies (n = 3 replicates at each data point). We performed all statistical analyses using Prism 9.5 (GraphPad Software, San Diego, CA, USA). We regarded P values of less than 0.05 to be statistically different.