Regulation of Vascular Tone, Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by dl-α-Lipoic Acid
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Hydrogen sulfide (H2S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H2S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H2S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H2S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H2S levels in rats. 3-MP (10 µmol/L to 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3–1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H2S production depended on enzymatic activity, although at higher concentrations (1–3 mmol/L) there was also evidence for an additional nonenzymatic H2S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants DL-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H2S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H2S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.
Recent studies have demonstrated that the endogenous biological mediator hydrogen sulfide (H2S) acts as a reducing agent and an antioxidant molecule (1, 2, 3, 4, 5, 6, 7). It exerts protective effects against hyperglycemic stress in the vascular endothelium and exhibits protective effects in animal models of diabetic complications (8, 9, 10, 11, 12, 13, 14, 15, 16).
The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is a newly identified endogenous source of hydrogen sulfide (H2S) in various cells and tissues, including vascular endothelial cells (17, 18, 19). We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may exert adverse effects on cellular homeostasis (20,21). Given the importance of H2S as a vasorelaxant (22, 23, 24, 25, 26, 27, 28, 29), proangiogenic agent (27,30, 31, 32, 33, 34, 35, 36) and mitochondrial/bioenergetic modulator (20,21,35,37, 38, 39, 40, 41, 42), here we examined whether the 3-MST/H2S system plays physiological regulatory roles in endothelial cells. Next, we examined whether a dysfunction of the 3-MST/H2S system develops in hyperglycemia/diabetes and tested whether this dysfunction can be corrected by dl-α-lipoic acid (LA), a known antioxidant and 3-MST cofactor (43, 44, 45).
Materials and Methods
The bEnd3 microvascular endothelial cell line was purchased from the American Type Culture Collection (Manassas, VA, USA), cultured at 37°C at 5% CO2, in a humidified chamber, with 5.5 mmol/L glucose containing DMEM (low glucose) or 40 mmol/L glucose (high glucose) with 10% FBS, 2 mmol/L glutamine, 100 IU/mL penicillin, 100µg/mL streptomycin, and 1% nonessential amino acids. In some experiments, the high glucose cell culture medium was supplemented with 100 µmol/L dihydrolipoic acid (DHLA).
shRNA-Mediated Silencing of 3-MST
bEnd3 cells were transduced at a multiplicity of infection (MOI) of 3 with a lentiviral vector containing shRNA sequences targeting 3-MST (SHCLNV, clone TRCN0000045359). A nontargeting control shRNA sequence (shNT) was used to control for off-target effects (SHC002V, MISSION shRNA; Sigma-Aldrich, St. Louis, MO, USA). Transduced cells were selected and maintained in DMEM media supplemented with puromycin (2µg/mL). Silencing of 3-MST was confirmed by Western blot analysis of the cell lysates.
Cell Proliferation Assay
bEnd3 cells were grown in full DMEM medium containing 5.5 mmol/L glucose (low-glucose control) or 40 mmol/L glucose (high glucose) for 14 d. Cells were then seeded in 12-well plates at a density of 6 × 103 cells/cm2. After overnight incubation, cells were exposed to vehicle, 3-MP or NaHS at various concentrations and allowed to proliferate for 48 h. Cells were then harvested and counted using a Neubauer hemocytometer. For real-time assessment of cell proliferation, the xCELLigence system (Roche Diagnostics Corporation, Indianapolis, IN, USA) was used as described (35). Briefly, wildtype, NT shRNA and 3-MST shRNA bEnd3 cells were cultured until approximately 70% confluence in complete medium. Cells were then detached by trypsin-EDTA and resuspended in fresh culture medium at 30,000 cells/mL; 200 µL of this cell suspension was added to each well to yield 6,000 cells/well on an E-Plate 96 (ACEA Biosciences, Inc., San Diego, CA, USA), a specially designed 96-well microtiter plate containing interdigitated microelectrodes to noninvasively monitor the cell proliferation by measuring the relative change in the electrical impedance of the cell monolayer, a unitless parameter named cell index (CI).
Wound Scratch Assay
bEnd3 cells were seeded at 5 × 104 cells/well into a 12-well plate and allowed to reach confluence. Following starvation for 8 h with DMEM containing 0.1% FBS, a scratch was made in a straight line across the diameter of each well by using a 200-µL sterile pipette tip. Cell monolayers were incubated in the presence or absence of 3-MP and NaHS at various concentrations. Images of the monolayers were taken at the start of the experiment and 48 h thereafter using an inverted phase microscope, and the wound sizes were determined by the NIS-Elements imaging software (Nikon, Tokyo, Japan).
Cell Migration Assay
A modified Boyden chamber cell migration assay was used as described (35) in the presence of various concentrations of 3-MP or NaHS. In some experiments, the cell culture medium was also supplemented with 100 µmol/L DHLA. Migration was microscopically quantified at 200x magnification.
Aortic Ring Angiogenesis Assay
The thin gel rat/mouse aortic ring assay was conducted as described (27). Vessel sprouting was stimulated with various concentrations of 3-MP or NaHS. The angiogenic response was measured in the live cultures by counting the number of neovessels.
Western Blot Analysis
bEnd3 cells were lysed in NP-450 lysis buffer (Invitrogen [Thermo Fisher Scientific Inc., Waltham, MA, USA]), diluted in NuPAGE LDS Sample Buffer (Invitrogen [Thermo Fisher Scientific]), sonicated, and boiled. Lysates were resolved on 4%–12% NuPage Bis-Tris acrylamide gels (Invitrogen [Thermo Fisher Scientific]) and transferred to PVDF membranes. Membranes were blocked with Starting Block T20 (Thermo Scientific [Thermo Fisher Scientific]) and then probed overnight with primary antibodies against 3-MST (Atlas Antibodies, Stockholm, Sweden), CSE (Proteintech Group, Inc., Chicago, USA), CBS (Abnova, Taipei City, Taiwan), phosphorylated (Ser473) or total Akt (Cell Signaling Technology, Beverly, MA, USA) and phosphorylated (Ser239) or total VASP (Cell Signaling Technology). Anti-rabbit horseradish peroxidase (HRP) conjugate secondary antibody (Cell Signaling Technology) was applied for 2 h at room temperature. Enhanced chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA) was used to detect the signal on high-sensitivity film (Kodak, Rochester, NY, USA). HRP-conjugated antibody against actin (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. The intensity of Western blot signals was quantified by densitometry using the ImageJ software (NIH, Bethesda, MD, USA; https://doi.org/rsb.info.nih.gov/ij/).
Measurement of H2S Production bEnd3 Cells (Live Cells and Cell Homogenates)
To test the effect of hyperglycemia on 3-MST activity H2S production in live cells, bEnd3 cells were grown in full DMEM media containing 5.5. mmol/L glucose (low glucose) or 40 mmol/L glucose (high glucose) for 14 d. Next, 30,000 bEnd3 cells were seeded in Lab-Tek II chamber coverglass system and incubated overnight. Cells were loaded with 10 µmol/L of the fluorescent H2S probe 7-azido-4-methylcoumarin (AzMC) for 30 min as described (40). 3-MP was added at various concentrations and cells were further incubated for 1 h. Cells were washed three times with phosphate-buffered saline (PBS), and the specific fluorescence of the dye was visualized using Nikon Eclipse 80i inverted microscope and NIS-Elements software. In an additional set of studies, 3-MP-induced H2S production was measured in endothelial cell homogenates using AzMC (10 µmol/L) at 37°C for 1 h, in a 96-well plate format (150µg total protein per well in 200 µL volume). Control experiments included measurement of 3-MP-induced H2S production in cell homogenates that have been heat-inactivated (5 min, 100°C) to inhibit enzymatic activity (since 3-MP enzymatic inhibitors are not available). Additional controls included measurement of H2S production in PBS, in the absence of cell homogenate. Since at higher concentrations, 3-MP induced an increase in AzMC fluorescence, additional studies were conducted where 3-MP solutions (1 mmol/L) were made in PBS in vacutainer tubes and incubated at 37°C for 20 min. In one group of samples, a needle was inserted through the cup to reach the gas phase and connected to a vacuum pump. In the other group of samples, the tubes remained closed. After 20 min, H2S levels in the solution were measured.
Male Sprague Dawley rats (∼300 g of weight) and male C57BL/6 mice (8 wks of age) were obtained from Charles River Laboratories (Wilmington, MA, USA) or Jackson Laboratories (Bar Harbor, ME, USA) respectively. Animals were housed in an air-conditioned environment (22 ± 1°C, 50 ± 5% relative humidity, 12-h light-dark cycle) with free access to standard chow diet. Animals were allowed five days to acclimatize before the experiments.
In Vivo Angiogenesis Assay
All procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch in accordance with the U.S. National Institutes of Health-approved Guide for the Care and Use of Laboratory Animals (46). We performed a 30% total body surface area burn injury in Sprague Dawley rats, as described previously (27). In a first set of experiments, normoglycemic (nondiabetic control) rats were randomly divided into two groups and a full-thickness scald burn was created on their backs under deep anesthesia. Starting at d 1 and for the following 28 d, rats received daily subcutaneous (s.c.) injections of either saline or 3-MP (300µg/kg per day) in the volume of 100 µl per injection at four equally spaced sites in the transition zone between burn site and healthy tissue. Images of the lesions at d 28 were obtained on a transparent sheet and the areas were quantified by NIS-Elements imaging software. In a subsequent set of experiments, diabetes was induced by streptozotocin (STZ) administered in ice-cold citrate buffer (pH = 4.5) and injected intraperitoneally (IP) at a dose of 60 mg/kg to animals fasted overnight (10). Four days later, development of diabetes was assessed by measuring blood glucose using a glucometer. Seven days after STZ injection, burn wounds were created and wound areas at d 28 of injury was evaluated, as described above for normoglycemic rats. A group of rats received daily LA (100 mg/kg, IP) starting d 1 of burn injury. LA was dissolved as described by Cameron and colleagues (44). The powder was mixed with sterile saline, and NaOH was added until the suspension dissolved. The pH was then brought to pH 7.4 with HCl. Daily s.c. injections of either saline, 3-MP (300µg/kg/day) or NaHS (300µg/kg/day) were performed. For the Matrigel plug assay (Corning Matrigel Plug; Corning, Tewskbury, MA, USA), C57BL/6J mice were injected s.c. with 500 µL of Matrigel. 3-MP (300-1000µg/kg/day) was injected s.c. in proximity of the Matrigel plugs twice a day for 10 d. The Matrigel plugs were recovered by dissection, digested by Drabkin reagent, and angiogenesis was assessed by hemoglobin measurements, as described (27).
Mesenteric Bed Relaxation
The mesenteric bed preparation was performed according to Warner (47). Rats were anesthetized with ketamine/xylazine cocktail. The superior mesenteric artery was cannulated and whole vascular bed perfused for 5 min at 2 mL/min with heparinized (10 IU/mL) and oxygenated (95% O2, 5% CO2) Krebs buffer composed of 115.3 mmol/L NaCl, 4.9 mmol/L KCl, 1.46 mmol/L CaCl2, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 25.0 mmol/L NaHCO3, and 11.1 mmol/L glucose. The mesenteric bed was then isolated, connected to a disposable pressure transducer and changes in perfusion pressure were measured by PowerLab data acquisition software (ADInstruments Inc., Colorado Springs, CO, USA). After approximately 20 min of equilibration, the adrenergic agonist methoxamine was added to the Krebs solution, and relaxation responses to bolus injections of 3-MP (1–100 ng, in 100 µL) were evaluated.
Measurement of Microvascular Blood Flow
Skin microvascular blood flow was measured using a PeriFlux 5000 laser-Doppler flow meter (Perimed Inc., Ardmore, PA, USA). Rats were anesthetized by ketamine/xylazine (IP). The laser-Doppler probe was placed on the shaved dorsal skin of the animals and stable basal signal was recorded. Changes in the s.c. microcirculatory perfusion were detected following intracutaneous injections of either vehicle (saline), 3-MP (0.3–3 mg/kg) or NaHS (0.1–1 mg/kg).
Arterial Blood Pressure Measurement
Rats were anaesthetized by IP injection of ketamine/xylazine cocktail. The left carotid artery was cannulated and connected to a pressure transducer (ADInstruments Inc.). The femoral vein was also cannulated. A heating pad was used to keep the rat’s body temperature stable at 37°C. After 60 min of equilibration, bolus injections of increasing doses of 3-MP (0.3–3 mg/kg) or NaHS (0.1–1 mg/kg) were made into the femoral vein with 15 min intervals between injections. Data acquisition and analysis were accomplished by the PowerLab system.
Plasma H2S Measurement
3-MP or NaHS were given IP at a dose of 1 mg/kg. Whole blood was collected 30 min later in K2EDTA blood collection tubes and centrifuged at 4°C for 15 min at 2000g. Plasma was isolated and H2S levels were measured with the fluorescent H2S probe 7-azido-4-methylcoumarin (AzMC) (40). After incubation of 200 µL of plasma with 10 µmol/L AzMC, the fluorescence was measured using SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA.) at ex = 365, em = 450 nm. H2S levels were calculated against a NaHS standard curve.
Mitochondrial Isolation and Bioenergetic Analysis
The XF24 Extracellular Flux Analyzer (Seahorse Biosciences, North Billerica, MA, USA) was used to measure mitochondrial bioenergetic function, as described (38). Mitochondria from rat livers (control, diabetic, either treated with vehicle or with LA, as described above) were isolated by differential centrifugation and used for bioenergetic analysis. In the “coupling assay,” respiration by the mitochondria (15µg/well) was sequentially measured in a coupled mitochondrial state with the addition of succinate, as a complex II substrate into the assay media. The detection of basal respiration, state 2 was followed by state 3 (phosphorylating respiration, in the presence of ADP and substrate), state 4 (nonphosphorylating or resting respiration) following conversion of ADP to ATP, finally state 4o was induced with the addition of oligomycin. Next, maximal uncoupler-stimulated respiration (state 3u) was detected by the administration of the uncoupling agent, FCCP. At the end of the experiment the complex III inhibitor, antimycin A was applied to completely shut down the mitochondrial respiration. The coupling assay examines the degree of coupling between the electron transport chain (ETC), and the oxidative phosphorylation (OXPHOS), and can distinguish between ETC and OXPHOS with respect to mitochondrial function/dysfunction. In a separate set of studies, “electron flow” experiments were also conducted. This analysis, which is conducted in mitochondria uncoupled with FCCP (4 µmol/L), allows the functional assessment of selected mitochondrial complexes together in the same time frame. Mitochondrial electron transport was stimulated by the addition of pyruvate and malate (10 mmol/L and 2 mmol/L, respectively, to enable the activity of all complexes), with succinate (10 mmol/L, in the presence of the complex I inhibitor rotenone, 2 µmol/L, to direct the electron flow exclusively through complexes II, III and IV) or with the artificial substrates ascorbate and TMPD (10 mmol/L and 100 µmol/L, respectively, in the presence of the complex III inhibitor antimycin at 4 µmol/L, to selectively activate complex IV). In one set of experiments, the bioenergetic responses from the four groups (control, diabetic, either treated with vehicle or with LA) were analyzed for basal bioenergetic parameters. In another set of experiments (coupling assay), mitochondria were treated with the 3-MST substrate 3-MP (10 µmol/L) in vitro, followed by bioenergetic analysis. In an additional set of experiments (coupling assay), mitochondria were treated with DHLA (100 nmol/L) prior to the addition of 3-MP (10 µmol/L), followed by bioenergetic analysis.
Data are shown as mean ± SEM. One-way and two-way ANOVA with Bonferroni multiple comparison test were used to detect differences between groups. Statistical calculations were performed using GraphPad Prism 5 analysis software (GraphPad Software Inc., La Jolla, CA, USA).
3-MP is a Microvascular Relaxant
InVivo Administration of 3-MP Increases Circulating H2S Levels
IP injection of 3-MP to rats at a dose of 1 mg/kg caused an approximately threefold increase in circulating H2S levels at 30 min following treatment, as detected in the plasma using a fluorescent probe. NaHS (1 mg/kg), which was used as a positive control, increased circulating H2S levels as well (Figure 1D).
The Proangiogenic Effect of 3-MP In Vitro Is Associated with the Activation of Akt and Protein Kinase G (PKG)
Contribution of Enzymatic and Nonenzymatic H2S Production to the Effects of 3-MP
3-MP Exerts Proangiogenic Effects In Vivo
Hyperglycemia Impairs the 3-MP/3-MST/H2S Pathway
Lipoic Acid Restores the Proangiogenic Effect of 3-MP in Hyperglycemia and Diabetes
LA, a naturally occurring dithiol compound, is known as an antioxidant and an essential cofactor of mitochondrial enzymes (44,45). LA directly terminates free radicals and chelates transition metal ions. Recent studies showed that LA acts as a stimulator/cofactor of 3-MST (43). To test whether LA may improve or restore the proangiogenic effect of 3-MP in hyperglycemia, bEnd3 cells were grown in hyperglycemia (40 mmol/L glucose) for 14 d in presence or absence of 100 µmol/L of the reduced form of LA (DHLA). DHLA restored the effect of 3-MP to stimulate bEnd3 endothelial cell migration in hyperglycemia (see Figure 5E). To test the effect of lipoic acid in vivo, STZ-diabetic rats were subjected to the burn wound healing model. Rats also received either vehicle or LA administrations (100 mg/kg, IP daily), starting 7 d following STZ injection and continuing for an additional 28-d period. 3-MP or NaHS was given locally in a fashion identical to the studies discussed in Figure 3. The ability of 3-MP to enhance the wound closure was restored by LA treatment in STZ-diabetic rats (see Figure 5F).
Diabetes Elicits Marked Alterations in Mitochondrial Function
The Mitochondrial Activity of the 3-MST/H2S Pathway Is Reduced in Diabetes with Restoration by Lipoic Acid
The main novel results of the present report are the following: (a) the 3-MP/3-MST/H2S pathway regulates vascular function (acts as a microvascular vasorelaxant); (b) the 3-MP/3-MST/H2S pathway acts as a physiological stimulator of angiogenesis; (c) the 3-MP/3-MST/H2S pathway develops a functional impairment in hyperglycemia (suppression of the angiogenic and bioenergetic responses); and (d) lipoic acid improves/restores the function of the 3-MP/3-MST/H2S pathway in hyperglycemia. On the basis of these findings, we speculate that the impairment of the 3-MP/3-MST/H2S pathway may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also speculate that pharmacological supplementation of H2S and/or the restoration of the 3-MST system may be a potential future experimental therapeutic approach. It is conceivable that some of the previously noted beneficial and therapeutic effects of lipoic acid in diabetic complications (including vascular disease and diabetic neuropathy) (44,45,52,53) may be, at least in part, related to the lipoic acid-induced enhancement of the H2S pathway in diabetic animals or diabetic patients.
The 3-MP/3-MST pathway is receiving increasing attention as a novel enzymatic system involved in physiological H2S generation (with CSE and CBS being the other two physiological H2S-generating enzymes, which have been studied for longer times and are currently much better characterized). Although the presence of 3-MST and the metabolism of 3-MP by 3-MST in various tissues has been known for over three decades (54), the production of H2S by this pathway has only been realized in the last few years. There are now several reports demonstrating 3-MP/3-MST induced H2S production in the central and peripheral nervous system (55,56) and in the vascular endothelium (17), as well as in various other cells and tissues (20,38,43). The oxidative-stress-sensitive nature of 3-MST (57,58) and the functional consequence of this property (in terms of reduced H2S production and impaired cellular bioenergetics) (20) have also been demonstrated in recent years. Moreover, the molecular structure and biochemical profile of the human 3-MST enzyme has recently been characterized in significant detail (59). However, the physiological and especially the pathophysiological relevance of the 3-MP/3-MST/H2S pathway remains largely unexplored. A study by Shibuya and colleagues that demonstrated H2S production by 3-MP via 3-MST in endothelial cells focused on the localization of the enzyme within the endothelium and the biochemical aspects of H2S production, but did not explore the functional consequences of the H2S produced by 3-MST. The results of the present study, demonstrating that 3-MP induces microvascular relaxation, angiogenesis and in vivo H2S production extend the prior findings and suggest that 3-MST-derived H2S, in addition to CBS- and CSE-derived H2S plays physiological regulatory roles in the cardiovascular system as a vasodilator and proangiogenic local hormone. The reduced proliferative capacity of the 3-MST-silenced endothelial cells, shown in the current study, indicates a significant role of the 3-MP/3-MST/H2S system in the physiological maintenance of angiogenesis. This effect may be due to a combination of the stimulation of H2S-mediated signaling pathways (such as Akt and PKG, as well as, potentially, many other pathways) and H2S- mediated stimulatory bioenergetic effects (to contribute to the generation of ATP, the biological “fuel” needed for cell proliferation, movement and division). It should be emphasized that, similar to the case with the other two H2S-producing enzymes CSE and CBS, there may be marked differences in the expression and activity of 3-MST in different vascular beds, blood vessels of different sizes and different species. Therefore, additional studies (including studies in 3-MST deficient mice) are needed to extend the physiological importance and the overall applicability of the current findings.
The current results further support the view (67) that authentic H2S donors have future therapeutic potential in conditions associated with hyperglycemia. While the current report utilized an STZ model of diabetes, hyperglycemia is also important in the pathophysiological context of critical illness (for example, acute hyperglycemia post-surgery) (68). Likewise, the wound healing studies reported here were discussed in the context of diabetes, but wound healing is also highly relevant in the context of critical illness (burn injury) (69,70). Therefore, some of the conclusions and implications of the current report may also well be applicable for the pathophysiology and experimental therapy of various forms of critical illness. With respect to the therapeutic potential of the 3-MST substrate 3-MP, based on the current results, we conclude that the highest efficacy will be achieved if it is simultaneously applied with lipoic acid (and/or possibly other antioxidants).
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by the American Diabetes Association (7-12-BS-184) and the National Institutes of Health (R01GM107846) to C Szabo. C Coletta was supported by a fellowship by the American Heart Association. K Módis was supported by the James W. McLaughlin Fellowship Fund of the University of Texas. The editorial assistance of Ms. Li Li Szabo is appreciated.
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