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Mitochondrial arginase II constrains endothelial NOS-3 activity

Departments of ¹Anesthesiology and Critical Care Medicine, ²Medicine, and ³Biomedical Engineering, The Johns Hopkins Medical Institutions, Baltimore, Maryland; and ⁴Institute of Life Long Health and Department of Anesthesiology and Pain Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea

Submitted 15 June 2007 ; accepted in final form 3 September 2007

	ABSTRACT

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Emerging evidence supports the idea that arginase, expressed in the vascular endothelial cells of humans and other species, modulates endothelial nitric oxide (NO) synthase-3 (NOS-3) activity by regulating intracellular L-arginine bioavailability. Arginase II is thought to be expressed in the mitochondria of a variety of nonendothelial cells, whereas arginase I is known to be confined to the cytosol of hepatic and other cells. The isoforms that regulate NOS-3 and their subcellular distribution, however, remain incompletely characterized. We therefore tested the hypothesis that arginase II is confined to the mitochondria and that mitochondrial arginase II reciprocally regulates vascular endothelial NO production. Western blot analysis, immunocytochemistry with MitoTracker, and immunoelectron microscopy confirmed that arginase II is confined predominantly but not exclusively to the mitochondria. Arginase activity was significantly decreased, whereas NO production was significantly increased in the aorta and isolated endothelial cells from arginase II knockout (ArgII^–/–) mice compared with wild-type (WT) mice. The vasorelaxation response to acetylcholine (ACh) was markedly enhanced and the vasoconstrictor response to phenylephrine (PE) attenuated in ArgII^–/– in pressurized mouse carotid arteries. Furthermore, inhibition of NOS-3 by N^G-nitro-L-arginine methyl ester (L-NAME) impaired ACh response and restored the PE response to that observed in WT vessels. Vascular stiffness, as assessed by pulse wave velocity (PWV), was significantly decreased in ArgII^–/– compared with WT mice. On the other hand, 14 days of oral L-NAME treatment significantly increased PWV in both WT and ArgII^–/– mice, such that they were not significantly different from one another. These data suggest that arginase II is predominantly confined to the mitochondria and that this mitochondrial arginase II regulates NO production, vascular endothelial function, and vascular stiffness by modulating NOS-3 activity.

mitochondria; nitric oxide; vascular stiffness

Arginase shares L-arginine as a substrate with NOS-3 and hydrolyzes L-arginine to ornithine and urea as part of the urea cycle. It is increasingly recognized that arginase modulates NOS activity by regulating intracellular L-arginine bioavailability (2, 26). Thus the balance between arginase and NOS-3 activities, in part, regulates vascular endothelial NO production. Arginase activation/upregulation results in arginase/NOS imbalance and decreased NO production and has been demonstrated to contribute to endothelial dysfunction in a number of disease/pathophysiological processes, such as aging (2), diabetes (3, 6), hypertension (7, 12, 28), and atherosclerosis (18).

The two isoforms of mammalian arginase, arginase I and II, encoded by different genes (25), are expressed in different tissues. Furthermore, there appears to be significant species heterogeneity in isoform expression. Arginase I, located in the cytoplasm, is expressed most abundantly in the liver and is a critical enzyme in the urea cycle. Arginase II, on the other hand, is thought to be a mitochondrial enzyme and is expressed primarily in extrahepatic tissues, such as kidney (11), brain, small intestine, mammary gland, and macrophages. Accumulating evidence suggests that arginase II is the major isoenzyme in the endothelial cells (ECs) of humans and other species. The role of arginase II in ECs, however, remains incompletely understood, although there is now emerging evidence that it might be an important regulator of NO production (17, 18).

In cardiac myocytes, we recently demonstrated that arginase II, confined to the mitochondria, plays an important role in regulating neuronal NOS (NOS-1)-dependent myocardial contractile function (21). Considering that mitochondrial arginase II in myocytes regulates distinct and spatially confined pools of L-arginine and thus modulates NOS-1, we wished to determine whether arginase II is also confined to the mitochondria in vascular ECs and whether mitochondrial arginase II regulates vascular endothelial NO production and thereby endothelial function.

	METHODS

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Animals. Ten-week-old male wild-type (WT; C57BL/6J) mice from Jackson Laboratories were used as a control and fed a normal diet for 6 wk. Arginase II knockout (ArgII^–/–) mice were a gift from Dr. O'Brien, Baylor College of Medicine, and were bred and housed in our Institution. Male 10-wk-old ArgII^–/– mice were also fed a normal diet for 6 wk. All procedures and protocols were approved by the Institutional Animal Care and Use Committee of The Johns Hopkins University School of Medicine.

Aorta preparation. Heparin was administered 1 h before death. The animals were anesthetized with ketamine-acepromazine intraperitoneally, and the thoracic aorta, from distal aortic arch to the diaphragmatic level, was dissected, removed, and immersed in Krebs solution containing (in mM) 118 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 2.5 CaCl₂, 1.2 MgSO₄, 25 NaHCO₃, and 11.1 glucose. The vessels were carefully cleaned of connective tissue and cut into 2- to 3-mm rings. Aortic rings were immediately frozen in liquid nitrogen and stored at –80°C until assayed.

Isolation of mouse ECs. ECs were isolated from WT and ArgII^–/– mice vessel rings, as previously described (13). In brief, the dissected aorta from heparinized mice was immersed in heparin-containing 20% FBS-DMEM and washed with serum-free DMEM. After the aorta was filled with collagenase type II (2 mg/ml in serum-free DMEM, Sigma) and incubated for 45 min at 37°C, ECs were removed from the aorta by flushing with 5 ml of 20% FBS-DMEM. After centrifugation of this solution to harvest the ECs, they were cultured in a collagen type I-coated dish. To remove smooth muscle cells, the cells were washed with warmed PBS, and complete medium G containing 20% FBS, 100 U/ml penicillin-G, 100 µg/ml streptomycin, 2 mM L-glutamine, 1x nonessential amino acids, 1x sodium pyruvate, 25 mM HEPES (pH 7.0–7.6), 100 µg/ml heparin, 100 µg/ml EC growth supplements, and DMEM was added. All experiments were performed on second-passage number mouse aortic ECs.

Arginase activity. Arginase activity was determined by quantitating urea production using the spectrophotometric method with -isonitrosopropiophenone as described previously (21). Briefly, cell lysates were incubated with 75 µl manganese chloride solution (50 mM Tris·Cl, pH 7.5) at 60°C for 10 min and further reacted with 50 µl substrate L-arginine (0.5 mol/l, pH 9.7) at 37°C for 1 h. After the reaction was stopped by adding 400 µl of the acid solution mixture, -isonitrosopropiophenone (25 µl, 9% in absolute ethanol) was added. The optical density of the mixture heated at 100°C for 45 min was then measured spectrophotometrically at 550 nm.

Nitrate and nitrite or nitrogen oxide measurement. NO production from tissue lysates was evaluated by measuring nitrite levels using a NO assay kit (Calbiochem) by Griess reaction as previously described (21).

Estimation of NO generation with fluorescence probes, 4,5-diaminofluoroscein. ECs isolated from aorta of WT and ArgII^–/– mice were labeled with a fluorescent probe to NO [4,5-diaminofluoroscein (DAF), 5 µmol/l, 30 min] at room temperature and protected from light. After being washed with PBS, cells were then fixed with paraformaldehyde (3%) for 20 min. Images were acquired using a Nikon TE-200 epifluorescence microscope (with a x60 objective) and collected using Openlab software (Improvision) and an internally cooled 12-bit charge-coupled device (CCD) camera (CoolsnapHQ, Photometrics).

Immunofluorescence microscopy. To stain mitochondria, cells cultured on fibronectin (Invitrogen)-coated slides were incubated with 25 nM MitoTracker Red CMXRos (Molecular Probes). Cells were fixed and permeabilized with 3% paraformaldehyde and 0.5% Triton X-100 in PBS, rinsed with PBS, and incubated with polyclonal antibody against arginase II (Santa Cruz Biotechnol) and with Cy5-conjugated anti-rabbit IgG antibody. Images were acquired using epifluorescence microscopy (Eclipse, Nikon) with a x60 objective. Epifluorescence images were collected using Openlab software (Improvision, Lexington, MA) and an internally cooled 12-bit CCD camera (CoolsnapHQ, Photometrics, Tucson, AZ).

Mitochondria fractionation and Western blot analysis. The cells were washed twice with PBS, scraped, and centrifuged at 600 g for 5 min. The pellet was resuspended in 1 ml of cold mitochondria isolation buffer containing 0.3 M sucrose, 1 mM EGTA, 5 mM MOPS, 5 mM KH₂PO₄, and 0.1% BSA (pH 7.4) and then homogenized. Disrupted cells were centrifuged at 2,600 g for 5 min, and the supernatant was further centrifuged at 15,000 g for 10 min at 4°C to obtain the crude mitochondrial and cytosol fraction. The resulting supernatant was centrifuged at 100,000 g for 1 h to separate the microsomal fractions. The cytosol and mitochondria fractions were mixed with 2x SDS sample buffer containing 125 mM Tris (pH 6.8), 4% SDS, and 20% glycerol and then sonicated for 5 s. Each sample was resolved by 10% SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad), analyzed with antibodies according to the supplier's protocol, and visualized with peroxidase and an enhanced chemiluminescence system (Pierce).

Normalization was performed using anti-β-tubulin antibody (BD Bioscience, 1:1,000). Densitometry analysis of bands was performed with NIH ImageJ software.

Immunoelectron microscopy. Immunoelectron microscopy was performed as follows. Human aortic ECs were fixed in 4% paraformaldehyde in PBS (pH 7.4) at room temperature for 1 h. The buffer was replaced with 8% paraformaldehyde-PBS solution, and the cells were postfixed overnight at 4°C. Cells were embedded in gelatin, and ultrathin sections were cut from the cell pellet and collected in PBS, followed by incubation in the primary antibodies (rabbit anti-Arg II; 1:50 dilution) for 24 h at 4°C. After the sections were washed, the secondary antibodies labeled with 6-nm gold particles were applied. The cell sections were examined with a transmission electron microscope (Hitachi 7600 TEM), and images were digitally acquired.

In vitro vascular reactivity in mouse aorta. Mice were terminally anesthetized using ketamine-acepromazine (100 and 10 mg/kg ip, respectively), following which the thoracic aorta was dissected, removed, and immersed in cold oxygenated Krebs-Ringer bicarbonate solution (95% O₂-5% CO₂, pH 7.4, 37°C). The vessel was carefully cleaned and cut into 1.5-mm rings and suspended for isometric tension recording in organ chambers, as previously described (27). Protocols were performed on rings beginning at their optimum resting tone, previously determined to be 500 mg for mouse aorta. This resting tone was reached by stretching rings in 100-mg increments separated by 10-min intervals. Data were collected using a MacLab system and analyzed using Dose Response Software (AD Instruments). Vessel rings were preconstricted with phenylephrine (PE, 1 µM), and their vasorelaxant dose response to ACh (1 nM to 10 µM) were recorded. After washout and return to resting tension, vessels were again preconstricted with PE and their response to sodium nitroprusside (SNP) was determined (1 nM to 10 µM).

In vitro vascular reactivity in carotid arteries. Carotid arteries were dissected free from connective tissue in Krebs solution under a microscope and placed in a vessel chamber. Both ends of the carotid arteries were cannulated and sutured to two glass micropipettes. The chamber was filled with oxygenated Krebs solution (95% O₂-5% CO₂, pH 7.4), which circulated from a 50-ml reservoir at a flow rate of 50 ml/min, and maintained at 37°C. Intraluminal pressure of 50 mmHg was maintained throughout the experiment. The artery was equilibrated for 30 min before each experiment. The external diameter of the carotid arteries was measured using video-microscopic techniques. Vasoconstrictor responses to cumulative doses of PE (1 nM to 10 µM) were measured. Vasorelaxant responses to ACh (1 nM to 100 nM) and SNP (1 nM to 10 µM) were tested in PE (1 µM)-preconstricted carotid arteries. Furthermore, vasoactive response to PE and ACh were assessed after a 30-min preincubation with the NOS-3 inhibitor N^G-nitro-L-arginine methyl ester (L-NAME, 100 µM). Vasorelaxation is expressed as percent relaxation from the PE-induced preconstriction condition using vessel external diameter.

Pulse wave velocity. Vascular stiffness was determined pre- and posttreatment by measuring pulse wave velocity (PWV) using an ECG-triggered 10-MHz Doppler probe (Indus Instruments) at thoracic and abdominal aorta locations. The animals were anesthetized and maintained with 1% to 1.5% isoflurane. Animals were positioned supine with legs and arms taped to ECG electrodes incorporated into a temperature-controlled printed circuit board (THM100, Indus Instruments, Houston, TX). Rectal temperature was monitored with a probe (Physitemp, Clifton, NJ) and maintained at 37°C throughout the procedure. Both thoracic and abdominal aortic flows were acquired at a depth of 2 to 4 and 5 to 6 mm, respectively, with a 2-mm diameter, 10-MHz Doppler probe (Indus Instruments). These sites of measurement were marked upon image acquisition, and the separation distance between them was measured. PWV (in m/s) was calculated as the quotient of separation distance and the time difference between pulse arrivals, as measured from R-peaks of the ECG. Data analysis of Doppler and ECG signals was performed off-line using DSPW software from Indus Instruments.

Statistics. All data are represented as means ± SE. Statistical significance was determined by t-test or two-way ANOVA with Bonferroni posttests (Graphad Prism 4 software). A values of P < 0.05 was considered significant.

	RESULTS

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Arginase II is present predominantly in the mitochondria. Mitochondria were isolated from the cytoplasm using a previously described technique (21). To determine the subcellular localization of arginase II, Western blot analysis was performed with a mitochondrial-specific [voltage-dependent anion channel (VDAC)] and a cytoplasm-specific protein (β-tubulin) in addition to arginase II and NOS-3. As demonstrated in Fig. 1A, there is adequate isolation/purification of mitochondria since the mitochondrial protein, VDAC, is found exclusively in the mitochondrial fraction, whereas β-tubulin is found only in the cytoplasmic fraction. Importantly, arginase II is found predominantly, though not exclusively, in the mitochondrial fraction. To further determine the subcellular localization of arginase II, we costained ECs from WT and ArgII^–/– mice with the mitochondrial-specific dye MitoTracker (red) and an arginase II-specific dye (green). As illustrated in Fig. 1B, arginase II is expressed in ECs from WT but not ArgII^–/– mice. Furthermore, arginase II appears to be located in the mitochondria, as is demonstrated in the merged image showing both arginase II and MitoTracker. To further confirm the compartment-specific site of arginase II, we used immunoelectron microscopy. As demonstrated in Fig. 1C, in the isolated human ECs, the preponderance of immunogold staining is confined to the mitochondria.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Subcellular localization of arginase II. A: in Western blot, voltage-dependent anion channel (VDAC) and β-tubulin are used as mitochondrial and cytoplasmic marker proteins, respectively. Arginase II is found in mitochondrial fraction (M), but little is present in the cytoplasmic fraction (C) in mouse (MAECs) and human (HAECs) aortic endothelial cells (n = 3 experiments). B: endothelial cells from wild-type (WT) and arginase II knockout (ArgII–/–) mice were stained with a mitochondrial specific dye MitoTracker (red) and costained with arginase II (green). Arginase II appears to colocalize with the mitochondria in endothelial cells from WT mice but not expressed in ArgII–/– mice (n = 3 experiments). C: some gold particles are seen in the cytoplasm in immunoelectron microscopy, but gold particles are predominantly confined to the mitochondria (arrows) of the isolated HAECs. Comb, combined; NOS, nitric oxide (NO) synthase.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Effect of arginase II deficiency on arginase activity and NO production. A: arginase activity was significantly reduced in aorta from ArgII–/– compared with WT mice (*P < 0.01; n = 8 experiments from 4 animals). B: ArgII–/– mice had a significant increase in NO production compared with WT mice (*P < 0.01; n = 8 experiments from 4 animals). C: phenotype of isolated cells was confirmed by using endothelial cell-specific protein NOS-3 (red). NO production in isolated endothelial cells from ArgII–/– and WT mice was measured by NO-sensitive dye, 4,5-diaminofluoroscein (DAF; green). DAF fluorescence was significantly increased in ArgII–/– compared with WT mice (n = 3 experiments). D: Western blot and cumulative quantitative data (E) demonstrating the absence of arginase II in ArgII–/– mice (K, kidney, positive control). Lack of expression of arginase I in either WT or ArgII–/– mice (L, liver, positive control). Downregulation of NOS-3 in ArgII–/– mice (*P < 0.05, #P < 0.01, n = 4 experiments). AU, arbitrary units.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Effect of arginase II deficiency and the NOS-3 inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on vascular endothelial function in mouse aorta and carotid. A: endothelium-dependent relaxation responses to acetylcholine (ACh) in aortic rings were markedly enhanced in ArgII–/– compared with WT mice (*P < 0.01, WT vs. ArgII–/–, n = 6 experiments). B: responses to sodium nitroprusside (SNP) were no different. C: similarly endothelium-dependent responses to ACh were enhanced in carotid arteries of ArgII–/– mice (*P < 0.01, WT vs. ArgII–/–, n = 4 experiments). Although ACh responses after preincubation with L-NAME were significantly impaired in the carotid arteries of both WT and ArgII–/– mice, the responses were no different between the groups. D: vasodilatory responses to the endothelial-independent vasodilator SNP were identical in ArgII–/– and WT mice (n = 4 experiments). E: contractile response to phenylephrine (PE) was significantly attenuated in carotid rings from ArgII–/– compared with WT mice. After preincubation with L-NAME, the pressor response to PE of ArgII–/– was restored and identical to that of WT mice (*P < 0.01 vs. WT, #P = not significant vs. WT + L-NAME, n = 4 experiments).

View larger version (7K): [in this window] [in a new window]   Fig. 4. Vascular stiffness using pulse wave velocity (PWV). PWV was measured before and after treatment with the NOS inhibitor L-NAME for 14 days in both WT and ArgII–/– mice. The baseline PWV was significantly decreased in ArgII–/– compared with WT mice. After L-NAME treatment, PWV was significantly increased in both WT and ArgII–/– mice (*P < 0.01, **P < 0.001, n = 6 experiments).

	DISCUSSION

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We have demonstrated for the first time that arginase II, the predominant isoform in ECs, is confined mainly to the mitochondria. Moreover, arginase II regulates endothelial NO production since ECs from ArgII^–/– mice have increased endothelial NO production, enhanced NO-mediated endothelial-dependent vasorelaxation, and increased arterial compliance.

There is accumulating evidence in human ECs that arginase II is the predominantly expressed isoform (17, 18). Our previous data (18) demonstrated that arginase II is the predominant isoform present in human and mouse ECs. In nonendothelial tissue, it is well established that arginase II is confined predominantly but not exclusively to the mitochondria (kidney, etc.) (15). However, this subcellular distribution has never previously been demonstrated in ECs. Our group has also previously determined that the arginase II isoform confined to the mitochondria in cardiac myocytes is capable of regulating NOS-1. This is due to the close juxtaposition of the mitochondria and the sarcoplasmic reticulum-containing NOS-1 and likely reflects dual enzymatic competition for their shared substrate L-arginine in specific subcellular compartments/pools. Using both immunoprecipitation and Western blotting techniques in subcellular fractions and immunoelectron microscopy, we have now demonstrated this phenomenon in ECs. Topal et al. (23) demonstrated that depletion of freely exchangeable L-arginine pools using extracellular L-lysine did not modulate the influence of arginase on EC NO release. This suggests the presence of different L-arginine pools, at least one of which is accessible to NOS and arginase, but is not exchangeable with extracellular L-arginine. Moreover, these observations are consistent with those of Simon et al. (19), who have characterized three pools of L-arginine, one of which is exchangeable with extracellular L-arginine and two which are not. One of these latter appears to be that which is accessible to arginase.

Previous studies resulted in the conventional understanding of NOS-3 regulation, which suggested that NOS-3 was restricted primarily to the caveoli and confined to a signaling domain with surface receptors and the cationic amino acids transport (16). This is at odds with the concept that arginase II, a mitochondrially located enzyme, could reciprocally regulate NOS-3. It is now recognized, however, that dynamic trafficking of NOS-3 (in lipid rafts from the surface to the inside of the cell) may be critical in its regulation. Furthermore, it appears that there are a number of different NOS-3 pools that might be active and respond to different physiological and pharmacological stimuli (9, 20, 22). Recent work by Gao et al. (10), demonstrating copurification of NOS-3 with mitochondria in human umbilical vein endothelial cells, suggests a potential mechanism underlying reciprocal NOS-3 regulation by mitochondrial arginase II. Moreover, this group also demonstrated that NOS-3 was docked to the outer mitochondrial membrane by a novel anchoring mechanism that was sensitive to protease cleavage. The data support the idea that NOS-3 may indeed be colocalized with arginase II in a functional domain that could share a common L-arginine pool. This is completely consistent with our findings regarding the subcellular distribution of NOS-3 and arginase II.

Our findings could have important implications for our understanding of pathophysiological disease processes. A recent review of mitochondria and endothelial function by Davidson and Duchen (5) highlights the role of endothelial mitochondria in the pathogenesis of atherosclerosis and diabetic vascular disease. For example, endothelial mitochondria-mediated increases in reactive oxygen species (ROS) production in response to oxidized LDL exacerbate endothelial dysfunction (29). Furthermore, high EC turnover in atherosclerosis and the associated necessary activation of apoptotic pathways regulated by mitochondria also implicate these organelles in this process (4). Our group has recently demonstrated that oxidized LDL activates arginase by a mechanism that involves a release of its constraint by the microtubular structure (17). Furthermore, we demonstrated that inhibition of arginase in an atherogenic mouse model results in a significant decrease in endothelial ROS production, increase in NO production (restoration of the impaired nitros-redox balance), improvement in endothelial function, decrease in vascular stiffness, and reduction in plaque load (1). Thus the spatial confinement of arginase II to the mitochondria, its reciprocal regulation of NOS-3-dependent NO, and most likely ROS production make it a potential target for therapy in atherosclerosis.

It is now well recognized that large-artery vascular stiffness, as measured by PWV and the augmentation index, is an important characteristic of vascular health, and an increase in vascular stiffness represents an independent risk factor for adverse cardiovascular events. We have demonstrated that PWV is a sensitive indicator of endothelial dysfunction in that even small changes in endothelial function with resultant effects on the underlying vascular smooth muscle cause significant changes in PWV. In isolated vascular endothelial function studies, we demonstrated enhanced NO-dependent vasorelaxant responses in ArgII^–/– mice. Interestingly, this translated to a significant basal decrease in PWV. Furthermore, acute treatment with the NOS inhibitor L-NAME (not long enough to induce profound vascular remodeling; see Ref. 12a) increased vascular stiffness in both WT and ArgII^–/– mice, such that vascular stiffness was similar in both groups. The data support two important concepts: 1) that the basal decrease in PWV in ArgII^–/– mice is most likely a function of an increase in basal NO, and 2) that endothelial NO is a significant modulator of vascular stiffness and that the latter may be a sensitive in vivo indicator of vascular endothelial function/dysfunction.

In summary, we have demonstrated that endothelial arginase II is confined predominantly to the mitochondria where it regulates NO production and vascular stiffness. Its spatial confinement has important implications for diseases, for example, atherosclerosis and diabetes, in which endothelial dysfunction is in part modulated by mitochondria.

	GRANTS

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This work was supported by the National Institute on Aging Grant R01-AG-021523, National Aeronautics and Space Administration (NASA) Grant NNJ05HF03G, and National Space Biomedical Research Institute Grant through NASA CA00405.

	ACKNOWLEDGMENTS

 
We thank Dr. O Brien, Baylor College of Medicine, Houston, TX, for the generous gift of the Arg II^–/– mice.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. Berkowitz, Anesthesia, Tower 711, The Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287 (e-mail: dberkowi{at}bme.jhu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* H. Kyo Lim, H. Kyoung Lim, and S. Ryoo contributed equally to this work.

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