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Stem Cell-Like Human Endothelial Progenitors Show Enhanced Colony-Forming Capacity After Brief Sevoflurane Exposure: Preconditioning of Angiogenic Cells by Volatile Anesthetics

Lucchinetti, Eliana, PhD*; Zeisberger, Steffen M., PhD; Baruscotti, Isabella, MS; Wacker, Johannes, MD§; Feng, Jianhua, MD, PhD*; Zaugg, Kathrin, MD, PhD; Dubey, Raghvendra, PhD; Zisch, Andreas H., PhD; Zaugg, Michael, MD¶#

doi: 10.1213/ANE.0b013e3181b5a277
Anesthetic Pharmacology: Research Reports
Free

BACKGROUND: Endothelial progenitor cells play a pivotal role in tissue repair, and thus are used for cell replacement therapies in “regenerative medicine.” We tested whether the anesthetic sevoflurane would modulate growth or mobilization of these angiogenic cells.

METHODS: In an in vitro model, mononuclear cells isolated from peripheral blood of healthy donors were preconditioned with sevoflurane (3 times 30 min at 2 vol% interspersed by 30 min of air). Colony-forming units were determined after 9 days in culture and compared with time-matched untreated control. Using magnetic cell sorting, CD133+/CD34+ endothelial progenitors were enriched from human umbilical cord blood, and vascular endothelial growth factor (VEGF), VEGFR2 (KDR), granulocyte colony-stimulating factor (G-CSF), STAT3, c-kit, and CXCR4 expressions were determined in sevoflurane-treated and untreated cells by real-time reverse transcriptase polymerase chain reaction. In a volunteer study with crossover design, we tested whether sevoflurane inhalation (<1 vol% end-tidal concentration) would mobilize endothelial progenitor cells from the bone marrow niche into the circulation using flow cytometry of peripheral blood samples. VEGF and G-CSF plasma levels were also measured.

RESULTS: In vitro sevoflurane exposure of mononuclear cells enhanced colony-forming capacity and increased VEGF mRNA levels in CD133+/CD34+ cord blood cells (P = 0.017). Sevoflurane inhalation in healthy volunteers did not alter the number of CD133+/CD34+ or KDR+/CD34+ endothelial progenitors in the circulation, but increased the number of colony-forming units (P = 0.034), whereas VEGF and G-CSF plasma levels remained unchanged.

CONCLUSIONS: Sevoflurane preconditioning promotes growth and proliferation of stem cell-like human endothelial progenitors. Hence, it may be used to promote perioperative vascular healing and to support cell replacement therapies.

From the *Department of Anesthesiology and Pain Medicine, University of Alberta, Edmonton, Alberta, Canada; †Department of Obstetrics and Gynecology, University Hospital Zurich; ‡Department of Obstetrics and Gynecology, Clinics for Reproductive Endocrinology, University Hospital Zurich; §Institute of Anesthesiology, University Hospital Zurich; ∥Department of Radiation Oncology, University Hospital Zurich, Zurich, Switzerland; ¶Department of Anesthesiology and Pain Medicine, University of Alberta; and #Perioperative Translational Medicine, Mazankowski Alberta Heart Institute, Edmonton, Alberta, Canada.

Accepted for publication June 5, 2009.

Supported by the 5th Frontiers in Anesthesia Research Award from the International Anesthesia Research Society, Cleveland, OH, USA (to MZ), grant no. 3200B0-103980/1 (to MZ) and no. 320000-117998 (to RD) from the Swiss National Science Foundation, Berne, Switzerland, a grant from the Mazankowski Alberta Heart Institute, Edmonton, Canada (to MZ), a grant from the EU commission LSHM-CT-2006-037261 “CRYSTAL” (to AHZ), and Oncosuisse grants OCS-02009-02-2007 (to KZ) and OCS-01551-08-2004 (to RD).

EL, SMZ, AHZ, and MZ equally contributed to this work.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.anesthesia-analgesia.org).

Address correspondence and reprint requests to Michael Zaugg, MD, DEAA, FRCPC, Department of Anesthesiology and Pain Medicine, University of Alberta, 8-120 Clinical Sciences Building, Edmonton Alberta, Canada T6G 2G3. Address e-mail to michael. zaugg@ualberta.ca.

Stem cell-like endothelial progenitor cells (EPCs) are characterized by 3 coexpressed cell surface markers, namely, CD34, CD133, and vascular endothelial growth factor (VEGF)R2 (KDR), and constitute an important endogenous pool of pluripotent regenerative cells responsible for vascular integrity and homeostasis.1–3 They derive from the bone marrow, circulate in the peripheral blood, and migrate to ischemic or injured tissue contributing to neovascularization and tissue repair.4,5 Although endothelial injury is primarily recovered by colonization of the injured site by these cells, current evidence suggests that EPCs also contribute to reendothelialization and neovascularization through paracrine secretion of angiogenic factors.6,7 The important role of EPCs in cardiovascular biology is supported by the fact that reduced numbers and function of circulating EPCs act as biomarkers predicting progression of atherosclerotic disease and the occurrence of future major cardiovascular complications.8,9

Human endothelium can be effectively protected against ischemia-reperfusion injury by volatile anesthetics, consistent with a pharmacologic preconditioning effect.10 On the other side, classic ischemic preconditioning mediates its cytoprotective effects through rapid recruitment of angiogenic EPCs to ischemic organs.11 Current experimental and clinical evidence supports a possible mechanistic link between organ protection by pharmacologic preconditioning elicited by volatile anesthetics and neovascularization and tissue regeneration by stem cell-like progenitor cells. First, gene expression profiling of human blood and heart tissue exposed to sevoflurane reveals upregulation of transcripts involved in stem cell activation including VEGF, STAT3, and granulocyte colony-stimulating factor (G-CSF) (Fig. 1).12,13 This is consistent with previous experimental findings showing that isoflurane preconditioning in rat hearts activates extracellular signal regulated kinase 1/2 and increases VEGF protein levels linking anesthetic preconditioning to proangiogenic activity.14 Second, we have demonstrated that sevoflurane inhalation modulates the expression of l-selectin and integrins in the blood of healthy volunteers.13 These molecules affect mobilization and homing of EPCs.15 Finally, Akt/PKB and endothelial nitric oxide synthase are essential components in activating and increasing angiogenesis, and volatile anesthetic-induced pre- and postconditioning profoundly enhance Akt/PKB and endothelial nitric oxide synthase activities in hearts.16,17

Figure 1.

Figure 1.

It is unclear whether anesthetics affect the biology or kinetics of human EPCs. Because these cells are rapidly mobilized perioperatively and accelerate vascularization and tissue repair,18 it is critical to know whether and how anesthetics and sedatives affect their biology. Moreover, an increasing number of cell replacement therapies in patients are conducted under anesthesia or sedation. Hence, we investigated the effects of the commonly used volatile anesthetic sevoflurane on colony-forming capacity, gene expression, and recruitment of human EPCs. Specifically, we hypothesized that sevoflurane exposure to EPCs would increase their growth capacity. In addition, we hypothesized that sevoflurane inhalation would affect the number of circulating EPCs, i.e., mobilize them from the bone marrow into the bloodstream, in an in vivo model of healthy volunteers.

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METHODS

Study Subjects and Human Umbilical Cord Blood Samples

The study was approved by the local ethics committee, and all subjects gave written informed consent (ClinicalTrials* registration number is NCT00526695). The research was performed in accordance with the Declaration of Helsinki (2000). Thirteen healthy male Caucasian volunteers (25–45 yr) participated in the study. Five subjects donated peripheral blood for the in vitro protocol, and 8 additional subjects participated in an in vivo study with crossover design, in which participants inhaled sevoflurane. All subjects were nonsmokers and refrained from drinks and food containing caffeine and dark chocolate for 24 h. Participants fasted overnight, and 20 mg of prophylactic esomeprazol was administered orally the night before sevoflurane inhalation. Umbilical cord blood samples were collected from placentas of 4 women aged 19–35 yr with no signs of infection and gestational ages between 38/0 and 41/5 wk immediately after elective cesarean delivery.

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Study Protocols

Protocol A

These preliminary experiments served to evaluate whether our main hypothesis, i.e., whether sevoflurane would affect EPC growth, was indeed justified (Fig. 2A). Peripheral blood collected from healthy donors was used to isolate EPCs, as previously described.3 Briefly, the mononuclear cell (MNC) fraction was obtained through Ficoll density gradient centrifugation (Biocoll, Biochrom, Berlin, Germany) at 800g for 20 min. Cells were washed twice, suspended in endothelial growth medium EGM-2 (Clonetics, Basel, Switzerland) supplemented with 20% fetal bovine serum (FBS), and plated in fibronectin precoated 6-well plates at a density of 8 × 105/cm2. After 2 days, nonadherent MNCs were replated in a new fibronectin precoated 6-well plate and exposed to sevoflurane in air/5% CO2 (3 times 30 min at 2 vol% interspersed by 30 min of air/5% CO2) mimicking a sevoflurane preconditioning protocol (Fig. 2A). To determine the number of colony-forming units, cells were cultured for an additional 7 days, and EGM-2/FBS medium was changed every second day.

Figure 2.

Figure 2.

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Protocol B

To investigate mechanisms underlying sevoflurane- induced changes in EPC growth capacity, we enriched EPCs from human umbilical cord blood where EPCs are more prevalent (Fig. 2B).19 Human umbilical cord blood (55–95 mL) was collected from placentas immediately after cesarean delivery, and MNCs were isolated, as described earlier (Fig. 2B). Magnetic-activated cell sorting (MACS) was used to isolate EPCs by incubating MNCs with CD133/1-biotin monoclonal antibody and antibiotin antibody conjugated with superparamagnetic microbeads (clone AC133 Isolation Kit, Miltenyi Biotec, Bergisch-Gladbach, Germany). After washing, MNCs were processed through a MACS magnetic separation column (Miltenyi Biotec) to obtain purified CD133/1+ cells, as detailed in Figure S1 (see Supplemental Digital Content, http://links.lww.com/AA/A19 [Enrichment of EPCs by magnetic cell sorting. MNCs were incubated with CD133/1-biotin monoclonal antibody and antibiotin antibody conjugated with superparamagnetic microbeads and processed through a magnetic separation column to obtain purified CD133/1+. More than 90% of cells were positive for CD133 and CD34 after enrichment.]). Enriched CD133+ progenitors were washed twice, suspended in EGM-2/FBS, and cultured on fibronectin precoated 6-well plates at a density of 0.5 × 105 per well. Cells were rested overnight and exposed to sevoflurane in air/5% CO2 (3 times 30 min at 2 vol% interspersed by 30 min of air/5% CO2) mimicking a sevoflurane preconditioning protocol. Untreated cells served as time-matched control. Ninety minutes after the last exposure to sevoflurane, cells were rapidly frozen in liquid nitrogen and stored at −80°C for the determination of gene expression. An aliquot of the CD133/1+ cell fraction was incubated for 10 min in the dark at 4°C with phycoerythrin (PE)-conjugated anti-CD133/2 (clone 293C3, Miltenyi Biotec) and fluorescein isothiocyanate (FITC)-anti-CD34 (clone AC136, Miltenyi Biotec). Cells were washed in phosphate buffered saline containing 0.1% bovine serum albumin and centrifuged at 300g for 10 min. The purity of the cells was confirmed by single and two-color flow cytometry (Becton Dickinson, Basel, Switzerland) and Cell Quest software (Becton Dickinson). Isotype-matched mouse immunoglobulin served as control. A minimum number of 10,000 events was counted. After cell sorting, 90% ± 3% of gated cells were positive for the CD133 and CD34 surface markers.

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Protocol C

This volunteer study with a crossover design served to test whether sevoflurane inhalation would mobilize EPCs from the bone marrow niche into the circulation. Each participant underwent the same protocol with and without sevoflurane inhalation (Fig. 2C). Participants were randomly allocated to the protocols, which were at least 4 days apart (time between 2 inhalation periods). Experiments were performed in temperature-controlled (25°C) and quiet rooms. An IV line was placed into a cubital vein, and 50 mL 0.9% saline solution was infused during inhalation. Prophylactic ondansetron for nausea and vomiting (0.5 mg) was given IV. Sevoflurane in 50 vol% oxygen was inhaled for 1 h by the spontaneously breathing volunteers using a tight face mask connected to the common gas outlet of an anesthesia machine (Siemens Servo 900D ventilator, Siemens Life Support Systems, Sona, Sweden) to achieve an end-tidal concentration of 1.0 vol%. Control experiments consisted of 50 vol% oxygen inhalation for 1 h. Noninvasive arterial blood pressure, oxygen saturation, electrocardiogram, end-tidal carbon dioxide and sevoflurane concentrations (Draeger Infinity Delta XL, Draeger Medical Systems, Danvers, MA), and bispectral index (A2000 monitor® with 3 adhesive electrodes to the forehead, single channel: Fp1-Fpz, version 3.3; Aspect Medical Systems, Norwood, MA) were recorded. Blood samples for the various examinations (G-CSF and VEGF plasma levels, colony-forming unit assay of EPCs, and flow cytometry) were taken from the IV line at the indicated time points (Fig. 2C).

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Colony-Forming Unit Assay

Colony-forming units were counted by 2 independent investigators after 9 days in culture using phase contrast microscopy, as previously described.3 Only colonies with multiple thin flat cells emanating from a central cluster of at least 50 rounded cells were counted. The number of colony-forming units is given as colony-forming units per 106 MNCs.

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Real-Time Reverse Transcriptase Polymerase Chain Reaction for Gene Expression in CD133+ Enriched EPCs from Human Umbilical Cord Blood

Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for the determination of VEGF, VEGFR2 (KDR), G-CSF, STAT3, c-kit, and CXCR4 expression, as previously described.12,13 First strand cDNA was synthesized from 1 μg of total mRNA using Superscript II RT (Invitrogen, Basel, Switzerland) and oligo-dT as primer. RT-PCR quantification of the selected genes was performed on a Stratagene MX3000 real-time sequence detector instrument (Stratagene Europe, Amsterdam, The Netherlands) using Brilliant SYBR green QPCR Master Mix (Stratagene Europe) and the primers listed in Table 1. Amplification reactions were conducted with an initial step at 90°C for 3 min followed by 20–35 cycles. All PCR reactions were performed in triplicate, and ribosomal 18S (a constitutively expressed gene) was used as reference control. The predicted size of PCR products was confirmed by agarose gel electrophoresis.

Table 1

Table 1

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Circulating EPCs as Measured by Flow Cytometry

To increase the sensitivity and specificity of fluorescent-activated cell sorting (FACS) analysis, MNCs from peripheral blood samples were first isolated by density gradient centrifugation.20 Cells were suspended in endothelial basal medium/20% fetal calf serum (FCS)/10% dimethyl sulfoxide and frozen at −80°C until analysis. Thawed cells were washed with phosphate buffered saline/0.5% bovine albumin/2 mM EDTA and suspended in FACS buffer. FcR (cell surface receptors targeting the crystallizable fragment domain of immunoglobulin [Ig]G or IgE antibodies) blocking reagent (Miltenyi Biotec) was added and incubated for 30 min at 4°C in the dark. Afterward, a 50-μL cell suspension (1 × 106 cells) was added to a 5-mL Falcon tube and incubated for 30 min at 4°C in the dark with 2 μL of antibody solution as follows: 1) MNCs alone without antibody (control for autofluorescence), 2) MNCs + IgG1-PE (BD Biosciences, San José, CA) (isotype control for single staining), 3) MNCs + IgG1-APC (BD Biosciences) (isotype control for single staining), 4) MNCs + IgG2a-FITC (BD Biosciences) (isotype control for single staining), 5) MNCs + KDR (VEGFR)-PE (R&D Systems, Minneapolis, MN) (single staining), 6) MNCs + CD133/1-APC (Miltenyi Biotec) (single staining), 7) MNCs + CD34-FITC (Miltenyi Biotec) (single staining), 8) MNCs + KDR-PE + CD34-FITC (double staining), 9) MNCs + CD133/1-APC + CD34-FITC (double staining), 10) MNCs + IgG1-APC + IgG2a-FITC (isotype control for double staining), and 11) MNCs + IgG1-PE + IgG2a-FITC (isotype control for double staining). After incubation, MNCs were washed twice with FACS buffer, centrifuged at 300g for 5 min at 4°C, and resuspended in 0.5 mL FACS buffer. Cell fluorescence was measured immediately after staining using a 2 laser FACSCalibur instrument (Becton Dickinson). The enumeration strategy for EPCs was as follows. First, MNCs were distinguished from each other by typical physical characteristics, resulting in well-delineated cellular subpopulations that are easily identified on forward (cell size) and side-scatter (granularity) plots (lymphocyte-like versus monocyte-like cells). Next, CD133/1-APC versus side scatter of all events was displayed. A gate was set around the CD133/1-positive cells, which was subsequently used to enumerate single- and double-stained cells. A minimum of 30,000 events was counted. Results are given as mean fluorescence intensity and percent double-staining CD133+/CD34+ and KDR+/CD34+ EPCs per 106 MNCs. Data were analyzed using CellQuest software (Becton Dickinson).

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Enzyme-Linked Immunosorbent Assay to Determine VEGF and G-CSF Plasma Levels

Plasma levels of VEGF and G-CSF were determined in the healthy volunteers inhaling sevoflurane using commercially available enzyme-linked immunosorbent assay kits according to the manufacturer's guidelines (Quantikine Human VEGF and G-CSF Immunoassay, R&D Systems). Blood was collected into EDTA plasma tubes, immediately centrifuged for 10 min at 10,000g, aliquoted, and stored at −80°C before analysis. The lower detection limit for VEGF was 5 pg/mL and for G-CSF 1 pg/mL. The mean coefficient of variation was <5% for both assays.

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Statistical Analysis

The Kolmogorov-Smirnov test was used to test for normality of the underlying data distribution. Data are presented as mean ± sd and bar charts or median (quartiles) and box plots, respectively, depending on the underlying data distribution. Paired t-tests or Wilcoxon's signed rank test were used for comparisons between sevoflurane and control samples depending on the underlying data distribution. For VEGF/G-CSF plasma levels and circulating EPCs, as measured with flow cytometry, two-way analysis of variance for repeated measures followed by appropriate multiple comparison procedures, was used to evaluate differences over time between groups. If data were not normally distributed, a log transformation was applied before analysis of variance. Based on a mean of 0.10% CD133+/CD34+ cells in MNCs and an sd of 0.07%, we estimated that 8 subjects would be necessary to detect a doubling of these cells with a power of 80% and an α of 0.05. P < 0.05 was considered significant. Analyses were performed using SigmaStat Version 2 (SPSS, Chicago, IL).

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RESULTS

Brief In Vitro Exposure of MNCs to Sevoflurane Increases the Colony-Forming Capacity of Stem Cell-Like EPCs

In the first set of in vitro experiments, we tested whether sevoflurane would change the colony-forming capacity of EPCs isolated from peripheral venous blood samples of healthy blood donors. MNCs from each individual were preconditioned with 2 vol% sevoflurane (Fig. 2A). Untreated cells from the same blood collection served as time-matched control. After 9 days in culture, colony-forming units of EPCs with their typical morphology, represented by a “nidus” of rounded cells surrounded by spindle-shaped cells, were counted. In all 5 experiments, sevoflurane-treated MNCs exhibited a higher number of colony-forming units compared with untreated MNCs (P = 0.065) (Fig. 3).

Figure 3.

Figure 3.

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Sevoflurane Increases VEGF mRNA Levels in CD133+/CD34+ EPCs Enriched from Human Umbilical Cord Blood

We then sought to determine whether sevoflurane exposure would change the gene expression of EPCs, which could explain their enhanced growth capacity. Because of the low number of EPCs in the peripheral blood of healthy volunteers, we used human umbilical cord blood and MACS to obtain more than 90% pure CD133+/CD34+ cells for RT-PCR analysis (Fig. 2B). Enriched CD133+/CD34+ cells were exposed to sevoflurane preconditioning. The RT-PCR analysis shows that sevoflurane selectively increased VEGF mRNA levels (P = 0.017) in CD133+/CD34+ EPCs (Fig. 4). These experiments provide evidence that sevoflurane, similar to its well-known hypoxic preconditioning,21 promotes growth in EPCs.

Figure 4.

Figure 4.

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Sevoflurane Inhalation Does Not Alter the Number of Circulating EPCs or Change the VEGF and G-CSF Plasma Levels but Increases Colony-Forming Capacity of EPCs Collected from Blood of Healthy Volunteers

In this study with volunteers inhaling low-dose (<1 vol% end-tidal) sevoflurane for 1 h, we tested whether sevoflurane would affect the number of EPCs in the peripheral blood, i.e., mobilize EPCs from the bone marrow into the circulation. Also, this study should test whether the in vitro effect of sevoflurane on colony-forming capacity would be observed after in vivo exposure. Each study participant was undergoing the same protocol with and without sevoflurane treatment (Fig. 2C). The numbers of EPCs, as defined by established cell surface markers, were determined in the peripheral blood of the volunteers before sevoflurane inhalation (baseline) and 7 and 24 h later, using flow cytometry. Systemic plasma levels of VEGF and G-CSF, 2 important endogenous stem cell mobilizing factors,22 were measured at baseline, 4, 7, and 24 h after sevoflurane inhalation. The changes of mean fluorescence intensity from baseline for CD34, CD133, and KDR markers, and the numbers of double-positive staining cells KDR+/CD34+ and CD133+/ CD34+ were compared between the treated (sevoflurane inhalation) and untreated protocol. Our results show that none of the measured cell subpopulations significantly changed over time or between protocols suggesting that sevoflurane at the concentration we used does not mobilize EPCs from the bone marrow into the circulation (Fig. 5). This observation was further supported by the lack of significant alterations in VEGF and G-CSF plasma levels (Fig. 6). Colony-forming capacity was further determined from blood samples of the volunteers at baseline and 24 h later in the 2 protocols with and without sevoflurane inhalation. For 6 of the 8 volunteers, all 4 cultures for determining colony-forming units (2 in each study arm) were of good quality and could be used for comparison between the 2 protocols. Interestingly, the difference in the number of colony-forming units per 106 MNCs between baseline and the 24-h samples was increased after brief sevoflurane inhalation when compared with control (P = 0.034) (Fig. 7), suggesting that our in vitro observations may also be relevant to the in vivo situation.

Figure 5.

Figure 5.

Figure 6.

Figure 6.

Figure 7.

Figure 7.

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DISCUSSION

The major findings of this study are as follows. First, brief exposure of human MNCs to sevoflurane in vitro increases colony-forming units of stem cell-like EPCs after 9 days in culture. Second, CD133+/CD34+ EPCs enriched from human umbilical cord blood by MACS markedly increased VEGF mRNA levels after exposure to sevoflurane preconditioning indicating that sevoflurane activates a proangiogenic gene program in these pluripotent cells. Finally, and in contrast to our initial hypothesis, sevoflurane inhalation at the concentrations we used (<1 vol% end-tidal) did not increase the number of circulating EPCs in volunteers, i.e., recruit EPCs from the bone marrow into the circulation, or change the systemic plasma levels of VEGF and G-CSF, 2 potent EPC mobilizing factors.22 Nonetheless, consistent with our in vitro studies on EPC growth, the volunteer study also showed an increase in colony-forming capacity of EPCs after in vivo inhalation of sevoflurane, raising the possibility that our in vitro findings may also be relevant to the in vivo situation. Together, the anesthetic sevoflurane at even low concentrations affects stem cell biology by enhancing growth capacity of angiogenic EPCs. Based on the observed changes in mRNA levels and the delayed occurrence, this phenomenon is consistent with a “late preconditioning” effect.

The identification of factors and mechanisms modulating EPC number and function is currently under intensive investigation.23 Reduced and/or dysfunctional EPCs were found in patients with coronary artery disease, heart failure, hypertension, diabetes, advanced age, sedentary life style, and cigarette smoking. In contrast, physical activity, young age, and multiple drugs and hematopoietic cytokines including statins, estrogens, PPARγ agonists, G-CSF, and erythropoietin were found to increase the number and function of EPCs.24 Our study now provides evidence that sevoflurane, and probably other volatile anesthetics, favorably modulate EPC growth. One possible mechanism of increased growth capacity after sevoflurane exposure is the increased expression of VEGF observed in these cells. VEGF is a potent angiogenesis molecule and endothelial mitogen.25 In the presence of VEGF, CD34+ progenitors develop into endothelial-like cells by decreasing the expression of CD1a and CD83 molecules, leading to mature endothelial cells.26 Because CD34+ cells express the high-affinity receptor VEGFR2 (Flk-1, KDR), it is likely that the observed increased VEGF expression also activated antiapoptotic STAT3 protein via a paracrine pathway.27

Our previous studies in coronary artery bypass graft (CABG) patients and healthy volunteers also suggested that sevoflurane inhalation could potentially mobilize EPCs from the bone marrow niche and increase the number of circulating EPCs.12,13 The use of sevoflurane in off-pump CABG surgery increased the G-CSF signaling pathway compared with propofol anesthesia.12 G-CSF induces the release of proteinases cleaving adhesive integrins on stem cells.28 Moreover, in a protocol with healthy volunteers inhaling sevoflurane at an end-tidal concentration of 0.5–1.0 vol%, sevoflurane reduced l-selectin (CD62L) expression on leukocytes and increased their resistance to inflammatory activation at 24–48 h after exposure.13 Importantly, l-selectin was previously reported to be responsible for the adhesion of stem cells in the bone marrow microenvironment, and the use of selectin ligands has been proposed for effective stem cell mobilization.29 Despite these strong rationales, we were unable to detect changes (more than twofold) in circulating EPCs after sevoflurane inhalation in the peripheral blood. However, we cannot exclude that smaller changes may have occurred.

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Preconditioning of Proangiogenic Stem Cells: A Novel Strategy for Improving Organ Protection and Tissue Repair

Improvement in organ function subsequent to stem cell transplantation is directly related to the number of surviving cells and can be increased by antecedent in vitro or in vivo preconditioning.23 A number of experimental studies showed that anoxic preconditioning of stem cells makes them more resistant to lethal ischemic injury or stress.21,30,31 Obviously, preconditioning initiates a “primed state” in stem cells allowing them to better cope with the harsh microenvironment of injured tissue. Importantly, this optimization of engraftment conditions and cell survival by preconditioning lasts for several days.32 The addition of VEGF to EPC cultures was previously shown to activate Akt/PKB and improve EPC survival, and transplantation of hypoxic stem cells exhibiting increased VEGF expression resulted in increased angiogenesis, as well as enhanced morphometric and functional benefits of stem cell therapy.21,30 Similarly, in vitro33 and/or in vivo34 sevoflurane preconditioning entailing increased VEGF mRNA levels in stem cells may serve as an alternative to hypoxic preconditioning and be a novel elegant strategy to enhance donor cell survival. Because the heart also has its own resident (“endogenous”) stem cell populations, sevoflurane inhalation further opens the possibility to promote the heart's self-repairing mechanisms by stimulating these cells.35

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Clinical Implications

First, during surgical interventions, the vessel wall is exposed to thrombo-inflammatory processes, which may enhance the progression of atherosclerosis, or destabilize plaques and induce myocardial infarction and stroke. On the other side, EPCs rapidly mobilize during CABG surgery, contributing to accelerated vascularization and improved postoperative cardiac function.18 Given the fact that endothelial and EPC function further predict cardiovascular outcome,8,9 we reason that a potentially increased colony-forming capacity activity after sevoflurane exposure in surgical patients might be beneficial in the perioperative period, when vascular injury and wound healing are of particular concern. Second, the concept behind the use of stem cells in “regenerative medicine” is based on the potential of distinct progenitors for vessel and tissue repair. Stem cells were first administered into the periinfarct area during CABG surgery, but new routes of delivery have recently emerged including IV, over-the-wire intracoronary or transendocardial administration.24,36 Notably, most of these procedures are conducted under sedation. Our data now suggest that sevoflurane may be used to support cell replacement therapies in regenerative medicine. Finally, our findings also raise the concern that prolonged subclinical doses of sevoflurane might promote tumor growth and/or exert teratogenicity. Although current literature does not support these concerns for humans,37 our results are compatible with observations that the use of general anesthetics might promote metastatic cancer growth compared with neuraxial anesthesia.38 Hence, additional research is warranted.

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Specific Remarks and Study Limitations

Despite the use of control groups for nonspecific binding of antibodies and correction for background autofluorescence, difficulties in defining and enumerating EPCs remain. For example, there is no internationally accepted standardized set of criteria for the definition of EPCs. The cell culture definition of EPCs centers on colonies of spindle-shaped cells derived from MNCs, but some studies argue that, instead, these cells derive from hematopoietic lineage.39 Also, flow cytometry studies on rare events risk nonspecificity and the inability to discriminate between signal and noise. In this study, we exclusively investigated growth and mobilization of human EPCs after sevoflurane exposure. Future studies should also examine the effects of sevoflurane on migration and homing of these critical cells. In our volunteer study, we rather observed a decrease in colony-forming capacity in the control arm (baseline–24 h), which was offset by sevoflurane inhalation. Because serotonin promotes colony formation in hematopoietic stem cells,40 and serotonin receptors including 5-HT3 receptors were identified on various blood cells, we speculate that the use of ondansetron, a 5-HT3-receptor antagonist, might be responsible for this.41 Therefore, future studies should also clarify the impact of commonly used perioperative drugs on EPC biology. Whereas we conducted the in vitro cell experiments with 2 vol% sevoflurane, the volunteers inhaled sevoflurane at <1 vol% end-tidal concentration. However, the amount of effectively dissolved and biologically active sevoflurane in the 2 conditions were comparable, because the in vitro experiments were conducted in medium with low protein content and thus lower sevoflurane solubility.42 Finally, we only determined VEGF mRNA levels and not protein levels in CD133+/CD34+ EPCs enriched from umbilical cord blood. However, our results are supported by the previous findings by Wang et al.14 showing a rapid increase in VEGF protein levels in rat heart tissue exposed to isoflurane.

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CONCLUSIONS

We here report for the first time that brief sevoflurane exposure increases VEGF mRNA levels in human EPCs and enhances their colony-forming capacity. Pharmacologic preconditioning of angiogenic cells may promote perioperative vascular healing in wounds and injured vital organs. Clearly, the relevance of our findings now needs to be evaluated in additional experimental studies and in the clinical arena with patients who have multiple comorbidities.

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ACKNOWLEDGMENTS

The authors thank Dr. Marina Jamnicki (Resident in Anesthesiology at the University Hospital Zurich, Switzerland) for blood sample collection, as well as the laboratory technicians and postanesthesia care unit nurses, colleagues, and all volunteers who participated in this study.

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