The vascular endothelium is known to modulate the inflammatory response during surgery. Sevoflurane has been shown to protect against tumor necrosis factor alpha (TNF-α)-induced endothelial dysfunction, but the effects of other anesthetics or combinations with opioids on endothelial response are unclear.
Methods
In this in vitro study, we stimulated human umbilical vein endothelial cells with TNF-α (10 ng·mL−1) in triplicate in three independent experiments and treated them with sevoflurane (0.8%, 2.0%, and 4.0%), propofol (2, 5, and 10 μg·mL−1), remifentanil (2, 5, and 10 ng·mL−1) and fentanyl (0.5, 1.5, and 5 ng·mL−1) individually and in combinations. We evaluated the expression levels of endothelial adhesion molecules and proinflammatory cytokines using reverse transcription quantitative real-time polymerase chain reaction, Western blotting, and the enzyme-linked immunosorbent assay.
Results
Only sevoflurane significantly diminished the messenger ribonucleic acid (mRNA) and protein expression of adhesion molecules in the presence of TNF-α (E-selectin [sevoflurane 0.8%, P < 0.001; 2%, P = 0.03; 4%, P = 0.004], vascular cell adhesion molecule 1 [sevoflurane 0.8%, P < 0.001; 2%, P = 0.002; 4%, P < 0.001], and intercellular adhesion molecule 1 [sevoflurane 0.8%, P = 0.002; 2%, P = 0.007; 4%, P < 0.001]). Additionally, mRNA and protein expression of the proinflammatory cytokines interleukin [IL]-6 and IL-8 decreased after exposure to sevoflurane alone for (IL-6 mRNA: sevoflurane 0.8%, P = 0.004; 4%, P < 0.001; IL-8 mRNA: sevoflurane 4%, P = 0.02; IL-6 protein: sevoflurane 0.8%, P < 0.001; 2%, P = 0.003; 4%, P < 0.001; IL-8 protein: sevoflurane 0.8%, P = 0.03; 2%, P < 0.001; 4%, P = 0.008]). The addition of opioids did not change the expression in either of the adhesion molecules or inflammatory cytokines.
Conclusions
In this exploratory study, sevoflurane inhibited endothelial adhesion molecules and proinflammatory response in vitro, whereas propofol, remifentanil, or fentanyl did not possess the same effect. While the effects in vivo are unknown, these findings might highlight the potential impact of anesthetic choice on modulating the inflammatory response of endothelial cells.
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Surgical procedures are inevitably associated with a local and systemic inflammatory response. Although this evolutionary preserved response aims to limit further injury, restore homeostasis, and facilitate healing and repair, dysregulation can affect the recovery process of the patient and may lead to postoperative complications and organ failure.1,2
One of the first stages of response to surgical injury takes place at the inner lining of the microvasculature, the endothelial cells. Endothelial cells have been shown to play a key role in regulating barrier function, vascular tone, coagulation, and inflammation. Upon stress and injury, proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), are released in the blood and initiate increased expression of proinflammatory endothelial adhesion molecules (e.g., E-selectin, vascular cell adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1], and angiopoietin-2 [Ang2]), facilitating neutrophil migration and recruitment into underlying tissues, which contributes to organ injury and damage.3 Angiopoietin-2 is an antagonist of the tyrosine-protein kinase receptor TEK (Tie-2) receptor found on endothelial cells and is stored in Weibel–Palade bodies in the endothelial cell. Upon activation, Ang2 is released from the endothelium, destabilizing the endothelial barrier and causing vascular leakage.4
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Various strategies to modulate the surgically induced systemic inflammatory response, particularly by targeting endothelial function, have been studied using pharmacologic, immunomodulatory, and anesthetic interventions in both experimental models and clinical studies.5‐9 Previous studies have shown that several anesthetic agents have immune modulating effects and alter expression of endothelial adhesion molecules in ischemia reperfusion injury (IRI), which may ultimately affect immune cell responses and functions.10,11 From this perspective, modulating the surgical stress response through selecting anesthetics may be beneficial for the patient's recovery after surgery.
In a previous study, sevoflurane showed a protective effect against TNF-α-induced vascular endothelium dysfunction, which was found to be activated through the endothelial nitric oxide synthase (eNOS)/nitric oxide pathway and inhibition of nuclear factor kappa B (NF-κB).9 In this present study, we sought to test the hypothesis that sevoflurane and not propofol reduces the proinflammatory response of endothelial cells in TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). In addition, we aimed to explore the effects that the opioids remifentanil and fentanyl individually and in combination with sevoflurane and propofol have on the inflammatory response in endothelial cells in vitro.
Materials and methods
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 opioids
Propofol, 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.12‐16
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
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.
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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.
Results
Effects of sevoflurane, propofol, remifentanil, and fentanyl on endothelial adhesion molecule expression in tumor necrosis factor-alpha-stimulated human umbilical vein endothelial cells
Expression of platelet endothelial cell adhesion molecule (PECAM-1), also known as the endothelial integrity molecule CD31, did not change upon TNF-α stimulation compared with unstimulated cells (Fig. 2A, raw data and effect sizes [95% CI] can be found in ESM eTables 1 and 2). Nevertheless, we found a significant decrease in the presence of propofol (5 µg·mL−1) and sevoflurane (0.8% and 4.0%). In the case of 5 µg·mL−1 propofol, CD31 mRNA expression was ~1.23-fold lower (P = 0.03) than with TNF-α stimulation alone. For sevoflurane 0.8%, the difference was ~1.22-fold (P = 0.008) and ~1.28-fold lower (P = 0.007) in cells exposed to sevoflurane 4.0% .
Fig. 2
Effects of propofol, sevoflurane, remifentanil, and fentanyl exposure as single drugs on adhesion molecule expression in proinflammatory tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells. We exposed HUVECs to increasing concentrations of propofol (2, 5, and 10 µM), sevoflurane (0.8%, 2.0%, and 4.0%), remifentanil (2, 5, and 10 ng·mL−1), or fentanyl (0.5, 1.5, and 5.0 ng·mL−1) in the presence of TNF-α (10 ng·mL−1) for 4 hr. (A) Reverse transcription quantitative real-time polymerase chain reaction-determined mRNA levels of endothelial integrity molecule PECAM-1/CD31 and endothelial adhesion molecules E-selectin, VCAM-1, and ICAM-1 using GAPDH as housekeeping gene. We present data as fold change compared with control and analyzed them using one-way analysis of variance (Kruskal–Wallis) with post hoc correction using Dunn’s multiple comparisons tests. Bars show means (95% confidence intervals) of 3 independent experiments in triplicate. (B)–(E) Protein determination using Western blots of the endothelial adhesion molecules E-selectin, VCAM-1, and ICAM-1 and GAPDH as the protein-loading control in HUVEC lysates exposed to propofol (B), sevoflurane (C), remifentanil (D), or fentanyl (E). Per condition, n = 2 independent experiments
*P < 0.05 control vs TNF-α
#P < 0.05 TNF-α vs TNF-α + propofol, sevoflurane, remifentanil, or fentanyl
Tumor necrosis factor alpha induced an increase in mRNA (Fig. 2A, ESM eTable 1) and protein (Fig. 2B, ESM eFigs 1–4) expression of endothelial proinflammatory adhesion molecules E-selectin (P < 0.001), VCAM-1 (P = 0.02), and ICAM-1 (P < 0.001) in HUVECs compared with control cells. Increasing concentrations of propofol in the presence of TNF-α did not affect mRNA nor protein expression of E-selectin compared with TNF-α alone (Figs 2A and 2B, ESM eTables 1 and 2, eFigs 1–3).
In contrast, mRNA and protein expression of adhesion molecules E-selectin (sevoflurane 0.8%, P < 0.001; sevoflurane 2%, P = 0.03; sevoflurane 4%, P = 0.004), VCAM-1 (sevoflurane 0.8%, P < 0.001; sevoflurane 2%, P = 0.002; sevoflurane 4%, P < 0.001); and ICAM-1 (sevoflurane 0.8%, P = 0.002; sevoflurane 2%, P = 0.007; sevoflurane 4%, P < 0.001) were significantly decreased after exposure to sevoflurane in TNF-α-stimulated HUVECs, independent of the concentrations used (Figs 2A and 2B, ESM eTables 1 and 2, eFig. 2).
Effects of sevoflurane, propofol, remifentanil, and fentanyl on proinflammatory cytokine production in tumor necrosis factor-alpha-stimulated human umbilical vein endothelial cells
The proinflammatory cytokines IL-6 and IL-8, at both the mRNA (Fig. 3A, ESM eTables 1 and2) and protein (Fig. 3B, ESM eTables 3 and 4) levels, were increased upon TNF-α stimulation in HUVECs (IL-6 mRNA, P = 0.004; IL-8 mRNA, P < 0.001; IL-6 protein, P = 0.01; IL-8 protein, P < 0.001) Incubation of cells with propofol did not have any effects on concentrations of IL-6 and IL-8. Remifentanil similarly did not affect concentrations of IL-6 and IL-8. We found comparable results for the effects of fentanyl on IL-6 and IL-8 mRNA expression which when combined with TNF-α did not change at any of the concentrations used, compared with TNF-α alone (Fig. 3A, ESM eTables 1–4).
Fig. 3
Effects of propofol, sevoflurane, remifentanil, or fentanyl exposure as single drugs on proinflammatory cytokine expression in tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells. We exposed HUVECs to increasing concentrations of propofol (2, 5, and 10 µM), sevoflurane (0.8%, 2.0%, and 4.0%), remifentanil (2, 5, and 10 ng·mL−1), or fentanyl (0.5, 1.5, and 5.0 ng·mL−1) in the presence of TNF-α (10 ng·mL−1) for 4 hr. We analyzed data using one-way analysis of variance (Kruskal–Wallis) with post hoc correction using Dunn’s multiple comparisons tests. (A) Reverse transcription quantitative real-time polymerase chain reaction-determined mRNA levels of inflammatory cytokines IL-6, IL-8, and ANGPT2 using GAPDH as a housekeeping gene. We presented data as fold change to the control. Bars show means (95% confidence intervals) of 3 independent experiments in triplicate. (B) Protein concentration determination using ELISA of IL-6 and IL-8 in supernatant of HUVECs. Dots represent individual samples. Horizontal lines indicate the average value of three independent experiments in triplicate
*P < 0.05 control vs TNF-α
#P < 0.05 TNF-α vs TNF-α + propofol, sevoflurane, remifentanil, or fentanyl
Interestingly, sevoflurane exposure to TNF-α-stimulated HUVECs resulted in an overall ~3-fold decrease of IL-6 mRNA expression (sevoflurane 0.8%, P = 0.004; sevoflurane 2%, P = 0.06; sevoflurane 4%; P < 0.001) and a ~4.5-fold decrease in IL-8 (sevoflurane 0.8%, P = 0.05; sevoflurane 2%, P = 0.06; sevoflurane 4%, P = 0.02).
Concentrations of IL-6 protein (sevoflurane 0.8%, P < 0.001; sevoflurane 2%, P = 0.003; sevoflurane 4%, P < 0.001) and IL-8 protein (sevoflurane 0.8%, P = 0.003; sevoflurane 2%, P < 0.001; sevoflurane 4%, P = 0.008) also decreased when we exposed HUVECs to sevoflurane in presence of TNF-α (Figs 3A and 3B, eTables 3 and 4), independent of the concentrations used. Only exposure to the highest sevoflurane concentration (4%) resulted in a significant ~1.4-fold decrease of angiopoietin-2 (ANGPT2) mRNA (P = 0.01) in TNF-α-stimulated HUVECs compared with TNF-α stimulation alone (Fig. 3A, ESM eTables 1 and 2).
Mimicking balanced anesthesia in vitro: addition of the opioids remifentanil or fentanyl to propofol or sevoflurane on the endothelial response in tumor necrosis factor alpha-stimulated human umbilical vein endothelial cells
Addition of remifentanil to propofol in TNF-α-stimulated HUVECs had no effect on endothelial adhesion molecule expression compared with propofol alone. We found comparable results for the addition of fentanyl to propofol, in which TNF-α-stimulated HUVECs did not change compared with propofol alone (Figs 4A and 4B; ESM eTables 5 and 6, eFigs 5 and 6).
Fig. 4
Addition of remifentanil or fentanyl to propofol or sevoflurane, respectively, does not change endothelial adhesion molecule expression in TNF-α-stimulated HUVECs. We exposed HUVECs to propofol (10 µg·mL−1) or sevoflurane (2.0%) in combination with remifentanil (10 ng·mL−1) or fentanyl (5 ng·mL−1) in the presence of TNF-α (10 ng·mL−1) for 4 hr. (A) Reverse transcription quantitative real-time polymerase chain reaction-determined mRNA levels of PECAM-1/CD31, E-selectin, VCAM-1, and ICAM-1 using GAPDH as housekeeping gene. We presented data as fold change to the control and analyzed them using one-way analysis of variance (Kruskal–Wallis) with post hoc correction using Dunn’s multiple comparisons tests. Bars show means (95% confidence intervals) of 3 independent experiments in triplicate. (B)–(E) Protein determination using Western blots of adhesion molecules E-selectin, VCAM-1, and ICAM-1 and GAPDH as a protein-loading control in HUVEC lysates exposed to propofol + remifentanil (B), propofol + fentanyl (C), sevoflurane + remifentanil (D), or sevoflurane + fentanyl (E). Per condition, n = 2 independent experiments
*P < 0.05 control vs TNF-α
#P < 0.05 TNF-α vs TNF-α + propofol, or sevoflurane
The combination of remifentanil with sevoflurane produced endothelial adhesion molecule expression results comparable with sevoflurane, which was also the case for fentanyl (Figs 4A and 4B, ESM eTables 5 and 6, eFigs 7 and 8).
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The mRNA and protein levels of IL-6 and IL-8 increased upon TNF-α-stimulation (P < 0.001) and did not alter when incubated with propofol in combination with remifentanil. The addition of fentanyl similarly did not affect mRNA and protein levels of IL-6 and IL-8 in TNF-α-stimulated HUVECs compared with propofol alone (Figs 5A and 5B, ESM eTables 5–8). We observed similar results when we added remifentanil to sevoflurane, as neither altered the inflammatory cytokine response when compared with sevoflurane alone.
Fig. 5
Addition of remifentanil or fentanyl to propofol or sevoflurane, respectively, did not change inflammatory cytokine expression in TNF-α-stimulated HUVECs. We exposed HUVECs to propofol (10 µg·mL−1) or sevoflurane (2.0%) in combination with remifentanil (10 ng·mL−1) or fentanyl (5 ng·mL−1) in the presence of TNF-α (10 ng·mL−1) for 4 hr. We analyzed data using one-way analysis of variance (Kruskal–Wallis) with post hoc correction using Dunn’s multiple comparisons tests. (A) Reverse transcription quantitative real-time polymerase chain reaction-determined mRNA levels of IL-6, IL-8, and ANGPT2 using GAPDH as a housekeeping gene. We present the data as fold change compared with control. Bars show means (95% confidence intervals) of 3 independent experiments in triplicate. (B) Protein concentration determination using ELISA of IL-6 and IL-8 in supernatant of HUVECs. Dots represent individual samples. Horizontal lines indicate the average value of 3 independent experiments in triplicate
*P < 0.05 control vs TNF-α
#P < 0.05 TNF-α vs sevoflurane + remifentanil or fentanyl
Lastly, the addition of remifentanil to propofol or sevoflurane in TNF-α-stimulated HUVECs did not affect ANGPT2 mRNA expression or fentanyl (Figs 5A and 5B, ESM eTables 5 and 6).
Discussion
In this exploratory study, we tested the hypothesis that sevoflurane and not propofol reduces the proinflammatory response of endothelial cells in an in vitro model of surgical stress using TNF-α-stimulated HUVECs. The results show that sevoflurane, at different clinically relevant concentrations though not in a dose-dependent manner, reduced the endothelial inflammatory response, whereas propofol, remifentanil, and fentanyl did not affect this response. Moreover, the combination of propofol or sevoflurane with remifentanil or fentanyl did not show an additive effect on the endothelial inflammatory response. The choice of anesthetic agents may therefore influence the inflammatory response of endothelial cells in the microvasculature during surgery.
Different pathways, including the transcription factor NF-κB, regulate the expression of the adhesion molecules E-selectin, VCAM-1, and ICAM-1 in endothelial cells.17 Inhibitor of nuclear factor kappa B (IκB) kinase proteins upstream regulate the activation of NF-κB, of which the majority consists of the p50/p65 heterodimer, and this is involved in cytokine production and cell survival. Among other pathways, stimulation with the proinflammatory cytokine TNF-α induces downstream phosphorylation and degradation of IκB, facilitating nuclear translocation of NF-κB and transcription of target genes of adhesion molecules E-selectin, VCAM-1, and ICAM-1 in the nucleus.18 Surgery-induced stress is not only TNF-α mediated: the pattern of proinflammatory gene expression in HUVECs differs when different proinflammatory cytokines stimulate endothelial cells. For instance, Kuldo et al. showed that IL-1β mainly induced IL-6, IL-8, and cyclooxygenase-2 gene expression in HUVECs, whereas TNF-α more profoundly affected the expression of cell adhesion molecules (E-selectin, VCAM-1, and ICAM-1).19
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In our study, TNF-α-stimulated HUVECs exposed to sevoflurane showed reduced expression of adhesion molecules, suggesting that sevoflurane-based anesthesia plays a role in the NF-κB pathway in endothelial cells. These findings corroborate the study of Yu et al.; they showed that sevoflurane pretreatment inhibited the TNF-α-induced phosphorylation and degradation of IκBa, and thus the translocation of NF-κB p65 to the nucleus.20 Another study has shown that sevoflurane suppressed both NF-κB and MAPK signalling pathways in lipopolysaccharide (LPS)-activated microglia.21 In addition to the abovementioned results, we also examined the NF-κB pathway but did not observe an inhibitory effect on NF-κB signalling in HUVEC lysates exposed to sevoflurane in presence of TNF-α (ESM eFig. 9). In addition to NF-κB, other pathways may also be involved in activation through eNOS or upregulation of retinoid-related orphan receptors alpha, which have recently been shown to bind to sevoflurane and inhibit endoplasmic reticulum stress in LPS-treated HUVECs.22,23 Unravelling the exact signal transduction pathway of sevoflurane-induced endothelial protection upon proinflammatory activation is most likely multifactorial and cannot be explained by a single pathway.
Unlike sevoflurane, propofol had almost no effect on endothelial adhesion molecules or proinflammatory cytokines in this in vitro exploratory study. Nevertheless, from other studies, propofol is known to suppress the expression of ICAM-1 and VCAM-1. In a study where HUVECs were treated with tumor-conditioned medium (HUH7) following propofol administration at different concentrations, propofol inhibited the expression of adhesion molecules (E-selectin, ICAM-1, and VCAM-1).24 Furthermore, propofol improved endothelial function in septic rats and propofol had a protective effect on activated endothelial cell barrier dysfunction through inhibition of NF-κB and activation of eNOS.25 In these studies, the effects of propofol on adhesion molecules could be explained by the use of different pathways/study designs, measuring different intervals, working with different incubation times, or using different concentrations, although our study showed no difference with increasing concentrations either. Moreover, we also did not observe an inhibiting or stimulating effect of the opioids remifentanil and fentanyl compared with propofol or sevoflurane alone. This is in line with another study performed in HUVECs, in which both opioids did not have influence on the monocyte–endothelial adherence, while sufentanil increased U937-HUVEC adhesion.26
The extrapolation of results from in vitro studies to clinical settings with human patients is associated with well-known limitations. Our results may therefore differ compared with in vivo animal experiments and heterogenous human patient populations under general anesthesia. In vitro experiments lack interaction with immune cells and supporting cells, blood flow and nervous innervation, and microvascular differences in milieu interieur. Second, although we do not consider it plausible and precautionary measures have been taken, it cannot be determined with certainty whether one or more of the anesthetics bound to a protein in the medium that we used. In other words, the actual concentration of the anesthetic in the medium is not exactly known. Additionally, we were unable to graph a classical sigmoidal dose–response relationship since sevoflurane abolished the proinflammatory response after TNF-α at our lowest tested concentration (0.8%).
To take the present findings from bench to bedside, future studies should investigate the effects of anesthetics on both systemic inflammatory response and localized endothelial response in specific organs. Additionally, the impact of subclinical concentrations of sevoflurane on the inflammatory response in vivo should be investigated. We plan to perform studies applying surgical stress to mice under different anesthesia regimens to investigate organ-specific differences in endothelial adhesion molecule expression. While such investigations align with previous work conducted by our group, they are beyond the scope of the current research project.27
In summary, our study shows that in vitro sevoflurane, but not propofol, inhibited the endothelial proinflammatory response to TNF-α at clinically relevant concentrations. Notably, the addition of opioids to either sevoflurane or propofol did not alter this effect.
Author contributions
Rozemarijn S. Tuinhout contributed to writing up the first draft of the paper. Rianne M. Jongman contributed to data collection, laboratory measurements, and made revisions. Gertrude J. Nieuwenhuijs-Moeke and Michel M. R. F. Struys contributed to study design and made corrections in the first draft of the paper. Wayel H. Abdulahad contributed to laboratory measurements and made corrections in the first draft of the paper. Matijs van Meurs contributed to the study design, writing of the paper, and made revisions. Dirk J. Bosch contributed to data analysis, the writing of the first draft of the paper, and made revisions.
Acknowledgements
We would like to thank Timara Kuiper from the UMCG Endothelial Cell Facility (Groningen, Netherlands) for providing endothelial cells for our experiments.
Disclosures
Gertrude J. Nieuwenhuijs-Moeke reports having no conflicts of interest related to this investigation. She received (over the last 3 years) a research grant of Sedana Medical (Danderyd, Sweden). Matijs M. R. F. Struys reports having no conflicts of interest related to this investigation. His research group/department received (over the last 3 years) research grants and consultancy fees from Masimo (Irvine, CA, USA), Becton Dickinson (Eysins, Switzerland), Fresenius (Bad Homburg, Germany), Dräger (Lübeck, Germany), Paion (Aachen, Germany), Medcaptain Europe (Andelst, the Netherlands), Baxter (Chicago, IL, USA), and HanaPharm (Seoul, Republic of Korea). He receives royalties on intellectual property from Demed Medical (Sinaai, Belgium) and Ghent University (Gent, Belgium).
Funding statement
No external funding.
Prior conference presentations
This research was presented at Euroanaesthesia 2022 (4–6 June, Milan, Italy) and the Dutch Science Day for Anesthesiologists (29 September 2023, Utrecht, the Netherlands).
Editorial responsibility
This submission was handled by Dr. Stephan K. W. Schwarz, Editor-in-Chief, Canadian Journal of Anesthesia/Journal canadien d’anesthésie.
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Effects of sevoflurane, propofol, remifentanil, and fentanyl on the endothelial proinflammatory response: an in vitro exploratory study
Verfasst von
Rozemarijn S. Tuinhout, BSc
Rianne M. Jongman, PhD
Gertrude J. Nieuwenhuijs-Moeke, MD, PhD
Wayel H. Abdulahad, PhD
Michel M. R. F. Struys, MD, PhD
Matijs van Meurs, MD, PhD
Dirk J. Bosch, MD, PhD
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In diesem CME-Kurs können Sie Ihr Wissen zur EKG-Befundung anhand von zwölf Video-Tutorials auffrischen und 10 CME-Punkte sammeln.
Praxisnah, relevant und mit vielen Tipps & Tricks vom Profi.
Wie gut die Chancen von Schlaganfallpatienten stehen, von einer endovaskulären Thrombektomie zu profitieren, lässt sich offenbar bereits am CT ohne Kontrast bei Klinikaufnahme abschätzen. Entscheidend scheint die Wasseraufnahme im Infarktgebiet zu sein.
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Chronische Schmerzen und therapieresistent: Botulinumtoxin A könnte dafür eine Lösung sein. Wie der Wirkstoff in der Orthopädie eingesetzt wird, welche Evidenz dafür spricht und wie es um die Kostenübernahme steht, erklärte Dr. Stephan Grüner auf dem Kongress für Orthopädie und Unfallchirurgie.
