Our analysis detected a number of up- and down-regulated transcripts already known to be involved in the protection of the heart or other vital organs (Fig. 3; please see Supplementary Table S3 available at www.anesthesia-analgesia.org). In particular, these included the up-regulated transcripts paraoxonase, 12-lipoxygenase, DnaJ (Hsp40), and the down-regulated transcripts chemokine ligand 5 (a potent neutrophil chemoattractant, also called epithelial neutrophil activating protein-78) and phosphodiesterase 5A. Control experiments with inhalation of 50% oxygen for 1 h did not show any transcriptional regulation of paraoxonase, 12-lipoxygenase, and acyl-CoA synthase (Fig. 4). Sevoflurane inhalation down-regulated transcripts involved in peroxisome proliferator-activated receptor (PPAR) regulation and fatty acid oxidation in blood cells. We have recently shown that sevoflurane anesthesia down-regulates PPARα and its coactivator protein, PGC-1α, which transcriptionally regulate metabolism and lead to a shift in fuel preference away from fatty acid oxidation in the heart (20). In the present study, GSEA confirmed the coordinate down-regulation of genes involved in fatty acid oxidation after sevoflurane inhalation (NES = −0.55, P = 0.00, FDR q-value = 0.08) in blood cells, which closely correlated with PPAR-related genes (PGC-1α pathway: NES = −1.22, P = 0.02, FDR q-value = 0.43) (Figs. 5A–C). Finally, a large number of regulated transcripts emerged with yet unknown function in the context of organ protection (please see Supplementary Table S3 available at www.anesthesia-analgesia.org).
To elucidate whether sevoflurane inhalation modulates protein expression consistent with a “second window” of preconditioning, we determined the expression of the important adherence molecules β2-integrin (CD11b) and L-selectin (CD62L) from cubital blood collected up to 72 h after sevoflurane application. Expression levels were determined separately on granulocytes (CD14 positive) and monocytes (CD15 positive). Sevoflurane administration significantly reduced (approximately 25%) the expression of L-selectin (CD62L) in granulocytes (P = 0.004), but had only a marginal effect on monocytes (Figs. 6A and B). These changes were most evident at 24 and 48 h after inhalation. In contrast, there was no decrease in β2-integrin (CD11b) expression in monocytes and granulocytes (data not shown). Importantly, granulocytes stimulated with the bacterial oligopeptide fMLP exhibited markedly reduced shedding of L-selectin (CD62L) (Fig. 6C and D, P = 0.009), indicating resistance to inflammatory stimulation. L-selectin (CD62L) shedding was maximally inhibited 24 to 48 h after sevoflurane exposure.
The salient findings of the present study are as follows. First, sevoflurane inhalation, even at low subanesthetic concentrations, rapidly altered the blood transcriptome on a genome-wide scale in healthy subjects, a prerequisite for late preconditioning, which depends on de novo protein synthesis. The observed transcriptional changes specifically involved genes with known biological significance in the context of late preconditioning or organ protection. In accordance with our previous findings in human hearts exposed to sevoflurane (20), genes involved in fatty acid oxidation, regulated by the PCG1α-pathway, were similarly down-regulated in the blood. Second, 24 to 48 h after sevoflurane exposure, i.e., consistent with the occurrence of a late or second window of preconditioning, the expression of L-selectin (CD62L), a key inflammatory adhesion molecule responsible for the tethering of leukocytes to the endothelium (17), was reduced by approximately 25% on granulocytes, which further exhibited an increased resistance to inflammatory stimulation. Taken together, using blood as a model system and standardized experimental conditions, we provide for the first time molecular evidence that the anesthetic gas, sevoflurane, can induce late preconditioning in humans. Clearly, our findings should be confirmed in the clinical setting with patients.
The potential of late preconditioning to provide enduring protection (lasting 30-fold longer compared to early preconditioning) is of potential clinical importance. However, it was elusive whether late preconditioning, i.e., protection developing 24 to 48 h after the application of the preconditioning stimulus, would also occur in humans in response to volatile anesthetics. Animal studies indicate that volatile anesthetics offer protection from ischemia-reperfusion injury in heart (12) and brain (26) tissue lasting for up to 48 h. Tonkovic-Capin et al. (13) first reported successful late preconditioning of the heart in a rabbit model using isoflurane at 1% end-tidal concentration. Subsequent studies confirmed these findings in rats (10–12) and mice (9). Interestingly, emulsified formulations of volatile anesthetics equivalent to only 0.2 MAC, and with no apparent sedation markedly reduced infarct size in rabbit hearts when administered IV 24 h before test ischemia (15). Signaling pathways involved in volatile anesthetic-induced late preconditioning are reminiscent of those observed in other types of late preconditioning (4,5), including reactive oxygen and nitrogen species as triggers, and 12-lipoxygenase, cycloxygenase-2, and inducible nitric oxide synthase (iNOS) as mediators or effectors, and the magnitude of the afforded protection is similar. In accordance with these animal studies, the present study now provides, for the first time, evidence of a late protective phenotype after sevoflurane inhalation in humans.
Late preconditioning directly depends on transient transcriptional or translational changes (for h), and the delayed and prolonged protection is consistent with altered protein expression (for days). To test our hypothesis, we used the easily accessible blood compartment as a human model system, and determined the time course of transcriptional changes after sevoflurane inhalation in healthy volunteers. No transcriptional changes were measured after inhalation of 50% oxygen alone. However, genes encoding key protective proteins were significantly regulated after sevoflurane inhalation within only a short period, consistent with results obtained in humans after remote ischemic preconditioning of the limb (27). The observed changes in gene expression were only transient. Peak transcriptional changes occurred within 1 h of sevoflurane inhalation, but most of the changes disappeared after 24 h. This time course is characteristic for the transcriptional changes underlying the second window of protection in the heart. Salloum et al. reported peak iNOS mRNA expression in mice already 1–2 h after a single dose of sildenafil, a late preconditioning-inducing drug (4). Subsequently, the increase in iNOS mRNA completely vanished, but changes in cardioprotective iNOS protein expression were not measured before 24 h after the application of the triggering drug (4). This contention is consistent with our findings of an early transient transcriptional response (within hours) followed by late more persistent changes in protein expression (24–48 h later). Of note, changes in mRNA and protein expression were only modest (30%–40% compared to baseline) similar to our human study, but had marked effects on infarct size (from 30% to 6% of the area at risk). Interestingly, 12-lipoxygenase, which was recently implicated in delayed cardiac protection in mice (9), was upregulated. Conversely, phosphodiesterase 5A was down-regulated by sevoflurane. Inhibition of phosphodiesterase 5A by sildenafil was previously shown to induce late preconditioning in mice through the iNOS pathway (4). Additional potentially protective transcripts that were regulated in the blood transcriptome included the antioxidant paraoxonase (28), heat shock protein 40, known to prevent cytochrome c release from mitochondria, and a number of transcripts with less well-established function in the context of late preconditioning, such as EGR1, a master switch coordinating upregulation of stress genes, or NR1D2, a nuclear hormone receptor acting as a link between metabolism and inflammation (29). Collectively, our data show that sevoflurane inhalation reprograms blood transcriptome toward a defensive phenotype in humans.
Leukocytes have a central role in the pathogenesis of ischemia-reperfusion damage. In the present study, we show that inhalation of sevoflurane decreases the expression of L-selectin (CD62L) in granulocytes and attenuates their responsiveness to inflammatory stimulation 24 to 48 h after exposure, consistent with a second window of protection. Extravasation of leukocytes is mediated by adhesion molecules, and exacerbates tissue injury after restoration of blood supply to the site of ischemia (30,31). In the dynamic interaction between leukocytes and endothelium, the cell surface receptor L-selectin (CD62L) and its proteolytic shedding mediate the initial steps of leukocyte tethering and rolling, while β2-integrin (CD11b) facilitates subsequent firm adhesion and transmigration (17). L-selectin (CD62L) is constitutively expressed on the surface of leukocytes. Inflammatory stimulation via G-protein coupled receptors, as mimicked in our study by the chemoattractant fMLP, rapidly activates L-selectin (CD62L) by phosphorylation and subsequent cleavage of its ectodomain, increasing the binding activity to the endothelium (17). Thus, our findings suggest that sevoflurane induces a resistance of leukocytes against noxious stimulation. Blockade of selectin-mediated leukocyte adhesion improves post-ischemic function in the heart (32), while genetically engineered mutations that prevent shedding of cell surface receptors markedly diminish the deleterious inflammatory response (33). In contrast to remote ischemic preconditioning of the limb, in our study small-dose sevoflurane did not decrease β2-integrin (CD11b) expression or its shedding in response to the chemoattractant fMLP. This is in agreement with previous in vitro experiments showing that 0.5 MAC isoflurane inhibited shedding of L-selectin (CD62L), but not β2-integrin (CD11b), in response to chemoattractants, whereas 1 MAC isoflurane affected both adhesion molecules (34). Although L-selectin (CD62L) was not among the differentially regulated transcripts in our human experiments, calmodulin, a calcium regulatory protein, which co-precipitates with L-selectin (CD62L) and tightly regulates its surface expression and function (35), was markedly down-regulated after sevoflurane inhalation (Fig. 5). This notion raises the interesting possibility that delayed alterations in L-selectin (CD62L) expression and function might be indirectly caused by reduced calmodulin expression. It has been traditionally thought that the biological effects of fast-acting anesthetic gases with low blood-gas solubility dissipate rapidly after exhalation. In contrast to this belief, our study clearly demonstrates that these gases continue to exert their potentially protective actions long after their physical clearance from the human body, consistent with published results in patients undergoing coronary artery bypass grafting surgery (36).
We have recently shown that IV and volatile anesthetics differentially regulate the transcriptional response to cardiac surgery in human hearts (20). In particular, sevoflurane shifted the metabolic fuel preference away from fatty acid oxidation, which closely correlated with improved postoperative cardiac function. Myocardial substrate metabolism critically affects cardiac function (37), and switching metabolic fuel preference away from fatty acid oxidation improves recovery after ischemia and even affects long-term outcome (38). The present study confirms similar metabolic changes in the blood after subanesthetic sevoflurane inhalation, and further suggests that the transcriptional changes in fatty acid oxidation may be due to down-regulation of the PGC-1α pathway. This nuclear receptor coactivator critically controls cellular energy metabolism, and thus determines oxygen consumption (39). Moreover, inhibition of the PGC-1α pathway was recently shown to reduce uptake of oxidized low-density lipoprotein into macrophages and to prevent their retention in atherosclerotic vessel walls, thereby stabilizing vulnerable plaques (40). Hence, modulation of the PGC-1α pathway, as observed after sevoflurane administration, may be a novel antiischemic and plaque-stabilizing strategy in perioperative medicine.
The sample size of this study is small. However, a highly standardized protocol was used in healthy volunteers to overcome confounding variables. In this study, we used ranitidine to protect the participants from the potential hazard of pulmonary aspiration, and some previous reports suggest a role of histamine receptors in modifying expression of adhesion molecules (41,42). However, there was no transcriptional regulation in our control experiments after inhalation of 50% oxygen and administration of ranitidine alone. Also, changes in L-selectin expression exhibited the maximum at 24–48 h after sevoflurane inhalation. At this time, a single dose of ranitidine is cleared from the body, making a relevant effect on adhesion molecules unlikely. Moreover, the H2 receptor antagonist, ranitidine, did not affect histamine-induced expression of adherence molecules (42), nor did it prevent histamine-induced leukocyte rolling (41). Future studies should help to precisely delineate the underlying signaling components. Whether the detected molecular evidence of late preconditioning by sevoflurane successfully translates into clinical benefits requires additional randomized controlled trials.
This study provides molecular evidence of late preconditioning after sevoflurane inhalation in healthy volunteers. Because the late phase lasts significantly longer than the early phase, its protective potential merits further investigation in the clinical arena.
The authors thank the PACU nurses, colleagues, and volunteers, who participated in this study.
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